Primitive Erythroblasts Research Articles

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Circulating primitive murine erythroblasts undergo complex proteomic and metabolomic changes during terminal maturation

AbstractPrimitive erythropoiesis is a critical component of the fetal cardiovascular network and is essential for the growth and survival of the mammalian embryo. The need to rapidly establish a functional cardiovascular system is met, in part, by the intravascular circulation of primitive erythroid precursors that mature as a single semisynchronous cohort. To better understand the processes that regulate erythroid precursor maturation, we analyzed the proteome, metabolome, and lipidome of primitive erythroblasts isolated from embryonic day (E) 10.5 and E12.5 of mouse gestation, representing their transition from basophilic erythroblast to orthochromatic erythroblast (OrthoE) stages of maturation. Previous transcriptional and biomechanical characterizations of these precursors have highlighted a transition toward the expression of protein elements characteristic of mature red blood cell structure and function. Our analysis confirmed a loss of organelle-specific protein components involved in messenger RNA processing, proteostasis, and metabolism. In parallel, we observed metabolic rewiring toward the pentose phosphate pathway, glycolysis, and the Rapoport-Luebering shunt. Activation of the pentose phosphate pathway in particular may have stemmed from increased expression of hemoglobin chains and band 3, which together control oxygen-dependent metabolic modulation. Increased expression of several antioxidant enzymes also indicated modification to redox homeostasis. In addition, accumulation of oxylipins and cholesteryl esters in primitive OrthoE cells was paralleled by increased transcript levels of the p53-regulated cholesterol transporter (ABCA1) and decreased transcript levels of cholesterol synthetic enzymes. The present study characterizes the extensive metabolic rewiring that occurs in primary embryonic erythroid precursors as they prepare to enucleate and continue circulating without internal organelles.

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.

1001 – MAKING BLOOD BEFORE A BLOOD STEM CELL

Erythroid cells comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the mammalian embryo. In the beginning of the last century, it was recognized that distinct “primitive” and “definitive” red cell populations circulate in the embryo. Building on pioneering studies by Metcalf mapping myeloid progenitors in embryonic time and space, we determined that overlapping waves of primitive (EryP-CFC) and definitive (BFU-E) erythroid progenitors emerge in the yolk sac of pre-circulation murine embryos. EryP-CFC generate a synchronous wave of cells that comprises ∼40% of all cells in the mouse embryo, which undergo rapid hemoglobin accumulation, maturational globin switching, complex metabolic reprogramming, and ultimately enucleation, indicating that primitive erythropoiesis is indeed ‘mammalian’ in character. While EPO regulates the terminal maturation of primitive erythroblasts, it has remained unclear what factors regulate their initial emergence. Our recent studies indicate that Stat3 signaling, activated independently of EPO, may serve this function. Unlike EryP-CFC, the BFU-E that arise in the yolk sac are clonally associated with multiple myeloid lineages, and go on to seed the early fetal liver to serve as a critical bridge between primitive hematopoiesis and HSC-derived blood cell production. Interestingly, definitive, but not primitive, erythroblasts can self-renew when cultured ex vivo, through a Bmi1-regulated process. Overexpression of BMI1 in adult murine and human erythroblasts increases their in vitro self-renewal capacity, potentially providing a means of generating sufficient cultured red cells to meet future blood banking and transfusion needs. In summary, survival and growth of the mammalian embryo relies upon a complex and highly regulated process of robust red cell production from 2 distinct erythroid lineages that are initiated independently of HSCs soon after the start of gastrulation. Erythroid cells comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the mammalian embryo. In the beginning of the last century, it was recognized that distinct “primitive” and “definitive” red cell populations circulate in the embryo. Building on pioneering studies by Metcalf mapping myeloid progenitors in embryonic time and space, we determined that overlapping waves of primitive (EryP-CFC) and definitive (BFU-E) erythroid progenitors emerge in the yolk sac of pre-circulation murine embryos. EryP-CFC generate a synchronous wave of cells that comprises ∼40% of all cells in the mouse embryo, which undergo rapid hemoglobin accumulation, maturational globin switching, complex metabolic reprogramming, and ultimately enucleation, indicating that primitive erythropoiesis is indeed ‘mammalian’ in character. While EPO regulates the terminal maturation of primitive erythroblasts, it has remained unclear what factors regulate their initial emergence. Our recent studies indicate that Stat3 signaling, activated independently of EPO, may serve this function. Unlike EryP-CFC, the BFU-E that arise in the yolk sac are clonally associated with multiple myeloid lineages, and go on to seed the early fetal liver to serve as a critical bridge between primitive hematopoiesis and HSC-derived blood cell production. Interestingly, definitive, but not primitive, erythroblasts can self-renew when cultured ex vivo, through a Bmi1-regulated process. Overexpression of BMI1 in adult murine and human erythroblasts increases their in vitro self-renewal capacity, potentially providing a means of generating sufficient cultured red cells to meet future blood banking and transfusion needs. In summary, survival and growth of the mammalian embryo relies upon a complex and highly regulated process of robust red cell production from 2 distinct erythroid lineages that are initiated independently of HSCs soon after the start of gastrulation.

Circulating Primitive Erythroblasts in the Murine Embryo Undergo Complex Proteomic and Metabolomic Changes during Terminal Maturation

Primitive erythropoiesis is a critical component of the fetal cardiovascular network and is essential for growth and survival of the mammalian embryo. The need to rapidly establish a functional cardiovascular system in the post-implantation murine embryo is met, in part, by the intravascular circulation of primitive erythroid precursors that mature as a single semi-synchronous cohort. To better understand the processes that regulate erythroid precursor maturation, we analyzed the proteome, metabolome and lipidome of primitive erythroblasts isolated from embryonic day (E)10.5 and E12.5 of mouse gestation, which represents their transition from basophilic erythroblast (BasoE) to orthochromatic erythroblast (OrthoE) stages of maturation. Previous transcriptional and biomechanical characterization of these precursors has highlighted a transition towards the expression of protein elements characteristic of mature red blood cell structure and function, including initial formation of a functional erythroid-specific cytoskeleton. Our analysis confirmed a loss of organelle-specific protein components - from mRNA maturation to translation, from metabolism to protein degradation - including ribosome, spliceosome, mitochondria and proteasomes. In parallel, we observed a metabolic rewiring towards the pentose phosphate pathway, glycolysis and the Rapoport-Luebering shunt, which is consistent with the progressively decreasing steady state levels of carboxylic acids, acylcarnitines and free fatty acids along with decreased mitochondrial content. In addition, activation of the pentose phosphate pathway in particular may stem from increases in the expression of mature red blood cell components including hemoglobin chains and band 3 that control oxygen-dependent metabolic modulation. Furthermore, this metabolic reprogamming may reflect increased control of oxidative stress in line with increased expression of several antioxidant enzymes, including catalase, peroxiredoxins and glutathione peroxidase 4. On the other hand, accumulation of oxylipins and cholesteryl esters in primitive OrthoE cells was paralleled by increased transcript levels of the p53-regulated cholesterol transporter (ABCA1) and decreased transcript levels of cholesterol synthetic enzymes including mevalonate kinase (MVK) and phosphomevalonate kinase (PVMK), highlighting transcriptional control of metabolic reprogramming during this developmental process. The present study characterizes the extensive metabolic rewiring that occurs in primary embryonic erythroid precursors as they prepare to enucleate and continue circulating without internal organelles. DisclosuresNemkov: Omix Thecnologies: Other: Co-founder. Hansen: Omix Technologies: Other: Co-founder; ScalmiBio: Membership on an entity's Board of Directors or advisory committees. Palis: Rubius Therapeutics: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

ERK2 Substrate Binding Domains Perform Opposing Roles in Pathogenesis of a JAK2V617F-Driven Myeloproliferative Neoplasm

The interaction between ERK2 and its substrates is critically mediated by two domains, the common docking (CD) D-domain and DEF-binding pocket (DBP) domain. Previous studies have suggested that ERK2 is not only necessary to drive hematopoietic and myelo-erythroid development, but it is also important for the pathogenesis of hematological cancers, as revealed by recurrent ERK2 somatic mutations in many types of lymphoma and leukemia. Here we show that the activation of ERK2 in JAK2V617F-driven myeloproliferative neoplasm (MPN) enhance polycythemia vera (PV) progression from erythrocytosis to myelofibrosis when ERK2-DBP binding capacity is disabled. Conversely, targeting the ERK2 D-domain preserves ERK2 catalytic function while reducing outgrowth and proliferation of human and murine MPN cell lines.To determine whether ERK2 binding domains play a role in the pathogenesis of JAK2V617F-driven MPN, we generated an ERK2-Y261A (Erk2 Y261A) mutant knockin mouse model in which the ERK2-DBP domain was inactivated, but ERK2 kinase activity and D-domain function was preserved. We observed splenomegaly in Erk2 Y261A/Y261A, characterized by a 10-fold expansion of CD44+/Ter119+ primitive erythroblasts and Ter119+CD44+/ FSC hi immature erythroid progenitors. We then transplanted Jak2 V617F-expressing ERK2 wildtype (WT), knockout (KO) or DBP hematopoietic progenitors (HSPCs) into irradiated, immunodeficient mice (Rag2 −/−Il2rg −/−) and observed disease resembling human PV, with elevated RBC counts, hematocrit, and hemoglobin levels. ERK2-DBP mutant recipients exhibited mild but sustained erythrocytosis with a prevalence of circulating GFP+ monocytes and neutrophils. At 12-weeks post-transplantation, ERK2-DBP mutant recipients had significant splenic burden and decreased bone marrow cellularity relative to WT and KO recipients. Reticulin staining confirmed progression towards myelofibrosis with interstitial infiltration in both the BM and the spleen of ERK2 DBP mutant recipients.To understand the molecular basis of ERK-DBP domain in JAK2V617F-driven MPN, we performed in vitro colony forming unit assay to assess the clonogenic potential of JAK2V617F-expressing HSPCs in cytokine supplemented-methylcellulose supporting myeloid or erythroid progenitor development. Ectopic JAK2V617F expression markedly reduced colony formation by both ERK2 wildtype and knockout HSPCs. On the other hand, JAK2V617F expression did not reduce colony formation by ERK2-DBP HSPCs. These differences were not evident in methylcellulose cultures that supported erythroid development, suggesting that disabling ERK-DBP binding capacity primarily affects the myeloid lineage. Furthermore, we found that JAK2V617F increased senescence associated β-galactosidase (SA-βGa) activity in ERK2-WT HSPCs. SA-βGal activity was modestly affected by ERK deficiency but was strongly attenuated by the ERK2-DBP mutation, supporting the notion that ERK2-DBP domain promotes oncogene-induced senescence (OIS). Previously, we identified transcription factor, Early growth response 1 (Egr1) which interacts with ERK2-DBP domain and plays a critical role as a tumor suppressor in myeloid neoplasms. We observed that JAK2V617F expression failed to suppress colony formation and senescence by Egr1-deficient HSPCs. The ability of EGR1 to restore these effects is abrogated by inactivating the DEF motif (EGR1-Y252A). Together, this data demonstrates that the interaction between ERK2-DBP and Egr1 is required for JAK2V617F-mediated colony suppression and senescence induction.Finally, to determine whether ERK-D domain acts to promote progression by targeting substrates distinct from those that bind to the DBP domain, we developed an inhibitor (#76) of the ERK-D domain. Indeed, 76 spared ERK phosphorylation and EGR1 induction but impaired RSK phosphorylation, a critical D-domain substrate involved in cancer progression. Moreover, 76 suppressed colony formation by JAK2V61F-expressing HSPCs akin to the active site MEK/ERK inhibitor, U0126. By altering the 4-ethoxybenylidene moiety of 76, we were able to enhance anti-proliferative effects of the parent compound against human MPN cell lines, SET-2, UKE-1, and Ku812. Our findings identify ERK2-domain specific roles in the pathogenesis of JAK2V617F driven MPN and supports a novel therapeutic approach to targeting JAK2 and MAPK dependent MPN. DisclosuresLevine: Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Lilly: Honoraria; Janssen: Consultancy; Astellas: Consultancy; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees. Wiest: Janssen Pharmaceuticals: Research Funding.

Cdan1 Is Essential for Primitive Erythropoiesis.

Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disease characterized by moderate to severe macrocytic anemia and pathognomonic morphologic abnormalities of the erythroid precursors, including spongy heterochromatin. The disease is mainly caused by mutations in CDAN1 (encoding for Codanin-1). No patients with homozygous null type mutations have been described, and mouse null mutants die during early embryogenesis prior to the initiation of erythropoiesis. The cellular functions of Codanin-1 and the erythroid specificity of the phenotype remain elusive. To investigate the role of Codanin-1 in erythropoiesis, we crossed mice carrying the Cdan1 floxed allele (Cdanfl/fl) with mice expressing Cre-recombinase under regulation of the erythropoietin receptor promoter (ErGFPcre). The resulting CdanΔEry transgenic embryos died at mid-gestation (E12.5–E13.5) from severe anemia, with very low numbers of circulating erythroblast. Transmission electron microscopy studies of primitive erythroblasts (E9.5) revealed the pathognomonic spongy heterochromatin. The morphology of CdanΔEry primitive erythroblasts demonstrated progressive development of dyserythropoiesis. Annexin V staining showed increases in both early and late-apoptotic erythroblasts compared to controls. Flow cytometry studies using the erythroid-specific cell-surface markers CD71 and Ter119 demonstrated that CdanΔEry erythroid progenitors do not undergo the semi-synchronous maturation characteristic of primitive erythroblasts. Gene expression studies aimed to evaluate the effect of Cdan1 depletion on erythropoiesis revealed a delay of ζ to α globin switch compared to controls. We also found increased expression of Gata2, Pu.1, and Runx1, which are known to inhibit terminal erythroid differentiation. Consistent with this data, our zebrafish model showed increased gata2 expression upon cdan1 knockdown. In summary, we demonstrated for the first time that Cdan1 is required for primitive erythropoiesis, while providing two experimental models for studying the role of Codanin-1 in erythropoiesis and in the pathogenesis of CDA type I.

Cellular Basis of Embryonic Hematopoiesis and Its Implications in Prenatal Erythropoiesis.

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

Different effects of an N-phenylimide herbicide on heme biosynthesis between human and rat erythroid cells

Rat developmental toxicity including embryolethality and teratogenicity (mainly ventricular septal defects and wavy ribs) were produced by S-53482, an N-phenylimide herbicide that inhibits protoporphyrinogen oxidase (PPO) common to chlorophyll and heme biosynthesis. The sequence of key biological events in the mode of action has been elucidated as follows: inhibition of PPO interferes with normal heme synthesis, which causes loss of blood cells leading to fetal anemia, embryolethality and the development of malformations. In this study we investigated whether the rat is a relevant model for the assessment of the human hazard of the herbicide. To study effects on heme biosynthesis, human erythroleukemia, human cord blood, and rat erythroleukemia cells were treated with the herbicide during red cell differentiation. Protoporphyrin IX, a marker of PPO inhibition, and heme were determined. We investigated whether synchronous maturation of primitive erythropoiesis, which can contribute to massive losses of embryonic blood, occurs in rats. The population of primitive erythroblasts was observed on gestational days 11 through 14. Heme production was suppressed in rat erythroid cells. In contrast, heme reduction was not seen in both human erythroid cells when PPO was inhibited. Rats underwent synchronous maturation in primitive erythropoiesis. Our results combined with epidemiological findings that patients with deficient PPO are not anemic led us to conclude that human erythroblasts are resistant to the herbicide. It is suggested that the rat would be an inappropriate model for assessing the developmental toxicity of S-53482 in humans as rats are specifically sensitive to PPO inhibition by the herbicide.

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.

The Condensin II Subunit, NcapH2, Is Required for Proper Embryonic Erythroid Terminal Maturation and Embryonic Viability in a Primary Mouse Model

The process of nuclear condensation is an integral part of erythroid differentiation. We have previously shown that loss of the histone methyltransferase, Setd8, results in severe embryonic (primitive) erythroid anemia due to loss of chromatin condensation and proper erythroid terminal maturation (Malik 2017). Setd8 mediated H4K20 mono-methylation is an important mediator of higher order chromatin condensation and can recruit members of the Condensin II complex (Lau 2015). ondensin I and II complexes are five protein, large complexes responsible for DNA organization and packaging during mitosis. In eukaryotes, the complexes are composed of conserved SMC components, with each Condensin having a unique kleisin bridge protein and two HEAT subunits. Subunits of Condensin II are highly expressed in erythroid cells, suggesting an erythroid specific function. Overexpression of the Condensin II HEAT subunit, Cap-G2, accelerates erythroid differentiation in MEL cells (Xu 2006). To interrogate the role of Condensin II in a primary model, we mated endogenous EpoR driven cre expressing mice with mice containing a floxed allele around Condensin II kleisin subunit, NcapH2. Embryos homozygous for EpoR specific excision of NcapH2 suffered from severe primitive erythroid anemia with visible anemia beginning at embryonic day (E11.5) and lethality occurring at approximately E13.5. Peripheral blood analysis at E11.5 was significant for erythroblasts with enlarged cell size and nuclear size, as well erythroblasts with dysplastic appearing nuclei. Knockout embryos at E12.5 exhibited peripheral blood anemia, with an increase in the number of dysplastic appearing cells, and decreased fetal liver coloration suggesting a failure of both primitive and fetal definitive erythropoiesis. Failure of the embryonic erythroid lineage to expand was confirmed in a primary cell culture system in which loss of proliferation was limited to mid to late stage precursors, suggesting that Condensin II function is critical beyond the proerythroblast stage of maturation when nuclear condensation is occurring and mass spectrometry demonstrates that H4K20 mono-methylation accumulates. Characterization of primitive erythroblasts from knockout embryos showed increased cell size, increased scatter in the size of the nucleus, and a high frequency of cells with punctate nuclear staining. In addition, the subset of erythroid cells with punctate nuclei did not appear to have a nuclear envelope, suggesting a pre-mitotic error, likely during G2 of the cell cycle. Indeed, knockout cells showed a buildup of cells in G0-G1 of the cell cycle, suggesting arrest following a defective cell division. Together, our data suggests that Condensin II is required during erythroid maturation for proper cell cycle progression and chromatin organization, and that chromatin modifications, such H4K20me1, may be a required mechanism to control the higher-level organization of chromatin. This control is required in red cell precursors, as they undergo rapid expansion, maturation, and nuclear condensation simultaneously. Understanding these mechanisms will give valuable insight into the unique sensitivity of red cells to chromatin perturbations observed in bone marrow failure and myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

The Histone Methyltransferase Setd8 Alters the Chromatin Landscape and Regulates the Expression of Key Transcription Factors during Erythroid Differentiation

Setd8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 mRNA is expressed ~10-fold higher in erythroid cells than any other cell type (biogps.org) and Setd8 protein levels increase in concert with GATA1 levels during erythroid differentiation of CD34+ HSPCs, suggesting Setd8 may have a role regulating the erythroid transcriptome. Consistent with this hypothesis, erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia and global transcriptomic analyses of sorted populations of E10.5 Sed8 null and control erythroblasts demonstrated a profound defect in transcriptional repression, with 340/345 differentially expressed genes (DEG) expressed at higher levels in the Setd8 null cells than controls (Malik Cell Reports 2017).Primitive erythroblasts mature and enucleate in a semi-synchronous manner in circulation. To better understand the function of Setd8 in regulating the erythroid transcriptome, we extended our transcriptomic analyses by performing RNA-seq in sorted E9.5 Sed8 null (EpoRCre+; Setd8 Δ/Δ) and control (EpoRCre+; Setd8 Δ/+) erythroblasts. The Setd8 null cells failed to repress 20/137 (15%) of the genes that are down regulated in control cells from E9.5 to E10.5. Although relatively few genes were impacted, those genes were enriched for the pathway “Oxidative Stress” (adjusted p-value 0.009) suggesting that Setd8 may regulate specific functions during terminal erythroid maturation.We next compared the DEG in Setd8 null erythroblasts to transcriptomic changes that occur as a cell transcends the hematopoietic hierarchy, gaining lineage specificity while suppressing the multi-lineage transcriptome (GSE14833). A large fraction, 105/345 (~30%), of genes up-regulated in Setd8 null erythroblasts, are also up-regulated in multipotent progenitors compared to proerythroblasts. In contrast, only 16/345 (5%) were also up-regulated in granulocyte-monocyte progenitors suggesting that Setd8 does not repress other lineage restricted signatures. Together, these results suggest that Setd8 regulates repression of the multi-lineage transcriptome during erythroid differentiation from multipotent progenitor cells.To gain insights into how Setd8 regulates the erythroid transcriptome, we performed ATAC-seq (Buenrostro Nature Methods 2013) on sorted populations of erythroblasts from E10.5 Sed8 null and control embryos. Cell number for the Setd8 null samples was limited due to anemia, with ~1000 cells used for each replicate. Setd8 and control replicates were aggregated and accessible regions were identified using MACS2. Regions more accessible in Setd8 null cells were identified by computing a log2 ratio between Setd8 null and control samples using deepTools bamCompare. In addition, we utilized ChIPmentation (Schmidl Nature Methods 2015) to assay H3K27me3 occupancy across the genome of WT E10.5 erythroblasts to identify regions of heterochromatin in maturing erythroblasts. Two replicates were performed using 2.5-5x105 cells per assay, and peak called was done using MACS2.A total of 157 genes were identified that had more accessible chromatin in Setd8 null cells and contained an enrichment for H3K27me3 in WT cells suggesting that these genes should be repressed during normal erythropoiesis. Among these were several DEG that were up-regulated in the Setd8 null cells including Hhex, Cd63, and Gata2. Genomic data integration also identified several additional transcriptional regulators that are active in earlier hematopoietic progenitors but typically silenced during erythroid differentiation including Notch1 and Cebpa. Pathway analysis of the 157 genes identified several stemness-related pathways including “Transcriptional regulation of pluripotent stem cells” and “OCT4, SOX2, NANOG repress genes related to differentiation” (adjusted p-values 0.005 and 0.008, respectively). The chromatin regions that were more accessible in the Setd8 null cells were enriched for the DNA binding motifs of the transcription factor ERG (p-value 1-257), SCL (p-value 1e-193), and NRF1 (p-value 1e-101). Taken together, these data suggest that Setd8 works in concert with erythroid transcription factors to repress the transcriptional network in stem and progenitor cells and establish appropriate patterns of gene expression during erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo

AbstractThe transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebrafish, prRBCs express 2 of 3 zebrafish Gfi1/1b paralogs, Gfi1aa and Gfi1b. The recently identified zebrafish gfi1aa gene trap allele qmc551 drives erythroid green fluorescent protein (GFP) instead of Gfi1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of Gfi1b in gfi1aaqmc551 homozygous embryos. To study the transcriptome of the Gfi1aa/1b double-depleted cells, we performed an RNA-Seq experiment on GFP-positive prRBCs sorted from 20-hour-old embryos that were heterozygous or homozygous for gfi1aaqmc551, as well as wt or morphant for gfi1b. We subsequently confirmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combined, the data showed that in the absence of Gfi1aa, the synchronously developing prRBCs were delayed in activating late erythroid differentiation, as they struggled to suppress early erythroid and endothelial transcription programs. The latter highlighted the bipotent nature of the progenitors from which prRBCs arise. In the absence of Gfi1aa, Gfi1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed Gfi1aa-depleted prRBCs even further, showing that Gfi1aa and Gfi1b together set the pace for prRBC differentiation from hemangioblasts.

The phenotypic and functional properties of mouse yolk-sac-derived embryonic macrophages

Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn et al., 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath et al., 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Wynn et al., 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells.The goal of the present study was to isolate and expand progenitors of yolk-sac-derived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP+ populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP+/tg mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis.

Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis. Cells were obtained from first trimester human placental tissues. Biological properties including surface antigen composition, differentiation, proliferation, enucleation, and degeneration were studied as gestation progressed. A microdroplet culture system was developed to observe the behavior of these cells in vitro. Histology showed that primitive erythroblasts undergo maturation from polychromatic to orthochromatic erythroblasts and can differentiate spontaneously in vitro. Cell surface markers and nuclear gene expression suggest that the cells do not possess stemness properties, despite being primitive in nature. They have limited proliferative activity and highly deacetylated chromatin, but a microdroplet culture system can prolong their viability under normoxic conditions. No apoptosis was seen by 11weeks' gestation, and there was no enucleation in vitro. These properties confirm that viable cells with intact nuclei can be obtained at very early gestation for genetic analysis.

Functional Analysis of Erythroid Progenitors by Colony-Forming Assays.

The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize. It is these terminal cells that comprise the mature colonies of erythroid cells derived from the progenitors cultured in semisolid media. The in vitro generation of erythroid colonies requires cytokine support, most notably erythropoietin (EPO), which is critical for CFU-E survival and for promoting erythroblast maturation.During mouse embryogenesis, a transient population of primitive erythroid colony-forming progenitors (EryP-CFC) emerges in the yolk sac and gives rise to a wave of maturing primitive erythroblasts in the fetal bloodstream. This wave of EryP-CFC is followed closely by a wave of BFU-E in the yolk sac that enter the bloodstream and seed the fetal liver to generate the first definitive red cells in the fetus. BFU-E in the fetal liver, unlike those in the adult bone marrow, can give rise to colonies in vitro when cultured with EPO alone and also are more sensitive to EPO levels. Here, we describe methods for the in vitro culture of murine embryonic (primitive) and fetal/adult (definitive) erythroid progenitors in semisolid media.

The Ets2 Repressor Factor (Erf) Is Required for Effective Primitive and Definitive Hematopoiesis.

Erf is a gene for a ubiquitously expressed Ets DNA-binding domain-containing transcriptional repressor. Erf haploinsufficiency causes craniosynostosis in humans and mice, while its absence in mice leads to failed chorioallantoic fusion and death at embryonic day 10.5 (E10.5). In this study, we show that Erf is required in all three waves of embryonic hematopoiesis. Mice lacking Erf in the embryo proper exhibited severe anemia and died around embryonic day 14.5. Erf epiblast-specific knockout embryos had reduced numbers of circulating blood cells from E9.5 onwards, with the development of severe anemia by E14.5. Elimination of Erf resulted in both reduced and more immature primitive erythroblasts at E9.5 to E10.5. Reduced definitive erythroid colony-forming activity was found in the bloodstream of E10.5 embryos and in the fetal liver at E11.5 to E13.5. Finally, elimination of Erf resulted in impaired repopulation ability, indicating that Erf is necessary for hematopoietic stem cell maintenance or differentiation. We conclude that Erf is required for both primitive and erythromyeloid progenitor waves of hematopoietic stem cell (HSC)-independent hematopoiesis as well as for the normal function of HSCs.

Microfluidic assay of the deformability of primitive erythroblasts.

Primitive erythroblasts (precursors of red blood cells) enter vascular circulation during the embryonic period and mature while circulating. As a result, primitive erythroblasts constantly experience significant hemodynamic shear stress. Shear-induced deformation of primitive erythroblasts however, is poorly studied. In this work, we examined the deformability of primitive erythroblasts at physiologically relevant flow conditions in microfluidic channels and identified the regulatory roles of the maturation stage of primitive erythroblasts and cytoskeletal protein 4.1 R in shear-induced cell deformation. The results showed that the maturation stage affected the deformability of primitive erythroblasts significantly and that primitive erythroblasts at later maturational stages exhibited a better deformability due to a matured cytoskeletal structure in the cell membrane.

Primitive erythrocytes are generated from hemogenic endothelial cells

Primitive erythroblasts are the first blood cells generated during embryonic hematopoiesis. Tracking their emergence both in vivo and in vitro has remained challenging due to the lack of specific cell surface markers. To selectively investigate primitive erythropoiesis, we have engineered a new transgenic embryonic stem (ES) cell line, where eGFP expression is driven by the regulatory sequences of the embryonic βH1 hemoglobin gene expressed specifically in primitive erythroid cells. Using this ES cell line, we observed that the first primitive erythroblasts are detected in vitro around day 1.5 of blast colony differentiation, within the cell population positive for the early hematopoietic progenitor marker CD41. Moreover, we establish that these eGFP+ cells emerge from a hemogenic endothelial cell population similarly to their definitive hematopoietic counterparts. We further generated a corresponding βH1-eGFP transgenic mouse model and demonstrated the presence of a primitive erythroid primed hemogenic endothelial cell population in the developing embryo. Taken together, our findings demonstrate that both in vivo and in vitro primitive erythrocytes are generated from hemogenic endothelial cells.

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.