Definitive Red Blood Cells Research Articles

Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells

AbstractDuring development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FL-derived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FL- and iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FL- and iPSC-derived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

Definitive Erythropoiesis from Pluripotent Stem Cells: Recent Advances and Perspectives.

Derivation of functional and mature red blood cells (RBCs) with adult globin expression from renewable source such as induced pluripotent stem cells (iPSCs) is of importance from the clinical point of view. Definitive RBC generation can only be succeeded through production of true hematopoietic stem cells (HSCs). There has been a great effort to obtain definitive engraftable HSCs from iPSCs but the results were mostly unsatisfactory due to low, short-term and linage-biased engraftment in mouse models. Moreover, ex vivo differentiation approaches ended up with RBCs with mostly embryonic and fetal globin expression. To establish reliable, standardized and effective laboratory protocols, we need to expand our knowledge about developmental hematopoiesis/erythropoiesis and identify critical regulatory signaling pathways and transcription factors. Once we meet these challenges, we could establish differentiation protocols for massive RBC production for transfusion purposes in the clinical setting, performing drug screening and disease modeling in ex vivo conditions, and investigating the embryological cascade of erythropoiesis. More interestingly, with the introduction of relatively efficient and facile genome editing tools, genetic correction for inherited RBC disorders such as sickle cell disease (SCD) would become possible through iPSCs that can subsequently generate definitive HSCs, which then give rise to definitive RBCs producing β-globin after transplantation.

Unravelling pathways downstream Sox6 induction in K562 erythroid cells by proteomic analysis

The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6−/− red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562 cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation. To obtain an overview on processes downstream to Sox6 expression, we performed a differential proteomic analysis on human erythroid K562 cells overexpressing Sox6. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24 A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these proteins during erythropoiesis.

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells.

: We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs. As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.

Advances in understanding erythropoiesis: evolving perspectives.

Red blood cells (RBCs) are generated from haematopoietic stem and progenitor cells (HSPCs) through the step-wise process of differentiation known as erythropoiesis. In this review, we discuss our current understanding of erythropoiesis and highlight recent advances in this field. During embryonic development, erythropoiesis occurs in three distinct waves comprising first, the yolk sac-derived primitive RBCs, followed sequentially by the erythro-myeloid progenitor (EMP) and HSPC-derived definitive RBCs. Recent work has highlighted the complexity and variability that may exist in the hierarchical arrangement of progenitors responsible for erythropoiesis. Using recently defined cell surface markers, it is now possible to enrich for erythroid progenitors and precursors to a much greater extent than has been possible before. While a great deal of knowledge has been gained on erythropoiesis from model organisms, our understanding of this process is currently being refined through human genetic studies. Genes mutated in erythroid disorders can now be identified more rapidly by the use of next-generation sequencing techniques. Genome-wide association studies on erythroid traits in healthy populations have also revealed new modulators of erythropoiesis. All of these recent developments have significant promise not only for increasing our understanding of erythropoiesis, but also for improving our ability to intervene when RBC production is perturbed in disease.

Primitive and definitive erythropoiesis in mammals

Red blood cells (RBCs), which constitute the most abundant cell type in the body, come in two distinct flavors- primitive and definitive. Definitive RBCs in mammals circulate as smaller, anucleate cells during fetal and postnatal life, while primitive RBCs circulate transiently in the early embryo as large, nucleated cells before ultimately enucleating. Both cell types are formed from lineage-committed progenitors that generate a series of morphologically identifiable precursors that enucleate to form mature RBCs. While definitive erythroid precursors mature extravascularly in the fetal liver and postnatal marrow in association with macrophage cells, primitive erythroid precursors mature as a semi-synchronous cohort in the embryonic bloodstream. While the cytoskeletal network is critical for the maintenance of cell shape and the deformability of definitive RBCs, little is known about the components and function of the cytoskeleton in primitive erythroblasts. Erythropoietin (EPO) is a critical regulator of late-stage definitive, but not primitive, erythroid progenitor survival. However, recent studies indicate that EPO regulates multiple aspects of terminal maturation of primitive murine and human erythroid precursors, including cell survival, proliferation, and the rate of terminal maturation. Primitive and definitive erythropoiesis share central transcriptional regulators, including Gata1 and Klf1, but are also characterized by the differential expression and function of other regulators, including myb, Sox6, and Bcl11A. Flow cytometry-based methodologies, developed to purify murine and human stage-specific erythroid precursors, have enabled comparative global gene expression studies and are providing new insights into the biology of erythroid maturation.

Tropomodulin3-Null Mice Are Embryonic Lethal with Anemia and Impaired Definitive Erythropoiesis in the Fetal Liver

Abstract 1036Tropomodulin1 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 compensated, sphero-elliptocytic anemia with fragile RBCs, due to mis-regulation of actin filament lengths and a disrupted membrane skeleton lattice (Moyer et al. 2010. Blood 116:2590–2599). The mild phenotype may be explained by the appearance of Tmod3, which is not found in wild-type RBCs but is in Tmod1-null RBCs, albeit at only 1/5th of normal Tmod1 levels. Tmod3 is ubiquitously expressed, including in erythroblasts, and thus the presence of Tmod3 in Tmod1-null RBCs is likely due to aberrant persistence of Tmod3 during terminal differentiation and maturation. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) in which intron 1 of the Tmod3 locus was disrupted by retroviral-mediated insertion of a gene-trap vector. The insertion creates a novel fusion transcript joining sequences from exons 5’ to the insertion site to the beta-galactosidase marker. Tmod3+/− mice are viable and fertile, but Tmod3−/− animals are embryonic lethal, with 94% of nulls dying between E13.5 – E15.5 (175 total embryos, E12.5-E18.5). Tmod3−/− embryos are pale and anemic starting at E13.5, with much smaller livers. Tmod3 mRNA and protein are reduced in Tmod3+/− and absent in Tmod3−/−, demonstrating that this is a true null. Tmod1 mRNA and protein are also reduced, consistent with anemia, fewer erythroblasts and RBCs, indicating that Tmod1 expression does not compensate for absence of Tmod3. The peripheral blood of wild-type E13.5-E15.5 embryos contains abundant large primitive and smaller definitive RBCs, but few or no definitive enucleated RBCs are observed in Tmod3-null embryos. Fetal livers from Tmod3−/− embryos were about ½ the size of wild-type livers, and histology showed altered cellularity. However, the proliferative potential of erythroid progenitors appears not to be impaired based on similar numbers of BFU-E and CFU-E colonies from Tmod3+/+ and Tmod3−/− fetal livers. Cytospins of dissociated fetal liver cells from E13.5-E14.5 embryos reveal the presence of erythroblast/macrophage islands in both genotypes, but with some macrophages containing abundant ingested cellular debris in the Tmod3−/− cytospins. High magnification images show increased erythroblast blebbing in absence of Tmod3, suggesting a weaker cortex, and/or apoptosis of erythroblasts. Flow cytometry of E14.5 fetal liver erythroblasts labeled with Ter119 and CD71 indicate that the late stage R3 population is reduced by about 40–50% in absence of Tmod3, while R1-R2 populations are somewhat increased (possibly as a compensatory response to fetal anemia). In addition, Annexin V staining shows a 2-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. To explore the role of macrophages in the Tmod3−/− phenotype, we performed immunofluorescence staining and confocal microscopy of fetal liver cryosections stained with F4/80 (macrophages), Ter119 (erythroblasts) and Hoechst (nuclei). Macrophages displayed strikingly less dendritic morphologies in the Tmod3−/−, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Our data show that definitive erythropoiesis is impaired during terminal differentiation in absence of Tmod3, and we speculate that this is due to defective erythroblast-macrophage interactions during terminal differentiation. Experiments are in progress to determine whether Tmod3 function is required in erythroblasts or macrophages, and to identify the molecular pathway in which Tmod3 functions in erythropoiesis. Supported by NIH/NHLBI grant R01HL083464 to V.M.F. Disclosures:No relevant conflicts of interest to declare.

Definitive Hematopoiesis In the Mammalian Embryo Prior to HSC Formation.

AbstractAbstract 1599The ontogeny of hematopoiesis in mammalian embryos is complicated by the requirement for functional blood cells prior to the emergence of hematopoietic stem cells or the bone marrow microenvironment. In the murine embryo, transplantable HSC are first evident at embryonic day (E) 10.5 and the first few HSC are found in the fetal liver hematopoietic environment by E12.5. However, two overlapping waves of hematopoietic potential arise in the yolk sac before E10.5. The first “primitive” wave produces progenitors from E7.25 to E8.5 with primitive erythroid, megakaryocyte and macrophage potentials. The resulting primitive erythroid cells mature within the circulation and support embryonic growth past E9.5. At E8.5, a second wave of hematopoiesis begins in the yolk sac and generates definitive erythroid and multiple myeloid progenitors that are the proposed source of the hematopoietic progenitors seeding the fetal liver before HSC colonization. We have identified a cell population displaying a unique cell surface immunophenotype in the E9.5 yolk sac that contains the potential to form definitive erythroid cells, megakaryocytes, macrophages and all forms of granulocytes within days of in vitro culture. Furthermore, all definitive hematopoietic colony-forming cells (BFU-E, CFC-myeloid and HPP-CFC) in the E9.5 yolk sac have this immunophenotype. These erythro-myeloid progenitors (EMP) are lineage-negative and co-express ckit, CD41, CD16/32 and Endoglin. Interestingly, this is not an immunophenotype evident in the adult bone marrow. Other markers that have been associated with HSC formation (AA4.1, ScaI) or with lymphoid potential (IL7R, Flt3) are not present on these cells at E9.5. Consistent with the lack of lymphoid markers, we also do not observe short-term development of B-cells (CD19+B220+ expressing Rag2 RNA) in cultures of the E9.5 sorted EMP, while bone marrow Lin-/ckit+/ScaI- cells do form B-cells under the same conditions. Clonal analysis of sorted EMP cells revealed single cells with both erythroid and granulocyte potential, similar to the common myeloid progenitors in adult bone marrow. Though these EMP are enriched at E9.5 in the yolk sac, they are also found at low levels in the fetal blood, embryo proper and placenta, consistent with their entrance into the circulation. By E10.5, EMP were most highly enriched in the newly formed fetal liver. Additionally by E12.5, a time when the first few HSCs are detected in the fetal liver, we find active erythropoiesis and granulopoiesis in the liver and the first definitive red blood cells and neutrophils in the bloodstream. Therefore, we believe the yolk sac definitive progenitors' fate is to populate the fetal liver and thus provide the first definitive erythrocytes and granulocytes for the embryo. The differentiation of embryonic stem cells (ES) and induced pluripotent stem cells (iPS) cells into mature cells types offers the hope of cell-based therapies. Analysis of differentiating murine ES cells reveals overlapping waves of primitive and definitive hematopoietic colony forming potential. We demonstrate the appearance of an EMP-like (ckit+/CD41+/FcGR+) population coincident with the emergence of definitive hematopoietic progenitors during murine ES cell differentiation as embryoid bodies. We have confirmed with colony forming assays that definitive hematopoietic potential is associated with this immunophenotypic group. Our studies support the concept that blood cell emergence during ES cell differentiation closely mimics pre-HSC hematopoiesis in the yolk sac. Disclosures:No relevant conflicts of interest to declare.

Regulation of Gata1 Expression by HS+3.5

AbstractAbstract 3864Precise regulation of Gata1 expression is required in order to control the balance between lymphoid/granulomonocytic (GM) and megakaryocytic-erythroid (MegE) specification, as well as to ensure correct differentiation of the MegE lineages. Transcriptional control is conferred in part by cis regulatory elements. An upstream enhancer, HS-3.5, and the erythroid first exon IE of Gata1 are necessary and sufficient to direct transgene expression in primitive but not definitive erythroid cells. Transgene expression in definitive red blood cells is restored by inclusion of an intronic DNaseI hypersensitive site, HS+3.5. Here we report the characterization of the HS+3.5 null embryonic stem cells and the HS+3.5 knockout mouse.In vitro differentiation of HS+3.5 null ES cells resulted in reduced myeloid and megakaryocytic colony formation compared to wild type. The ΔHS+3.5 ES cells retained normal primitive erythroid colony formation. ΔHS+3.5 definitive erythroid colony progenitors displayed a decreased sensitivity to Interleukin 3 (IL3) signalling compared to wild type.ΔHS+3.5 mice were viable and had normal blood counts and films. GM and erythroid progenitors also developed normally. However, there was a mild expansion of the E14.5 foetal liver Megakaryocytic Progenitor (MkP) compartment and an increase in Gata2 expression in both the bone marrow and foetal liver MkPs. Turning to Gata1, a decrease in Gata1 expression was observed in the following compartments: the bone marrow long term haematopoietic stem cell (LT-HSC) and the foetal liver common myeloid progenitor (CMP). The relationship between the effect of the HS+3.5 deletion on Gata1 expression and the haematopoietic phenotype will be discussed. Disclosures:No relevant conflicts of interest to declare.

The -566 Aγ-Globin HPFH Mutation Disrupts Temporal Repression of Fetal Hemoglobin Synthesis During Fetal Liver Definitive Erythropoiesis.

Abstract 2019Poster Board I-1041Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T>G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Development of a Preclinical Model for An Erythropoietin Deficient Anemia

The production of red blood cells is tightly controlled by the growth factor erythropoietin (EPO). Mice lacking a functional EPO gene die in-utero due to the complete absence of definitive red blood cells. Thus, studying the role of EPO in adult animals has been limited to the use of artificial anemia models such as phenylhydrazine (PHZ) treatment or phlebotomy. In addition, injections of rHuEPO have been shown to be a valuable tool to study erythropoiesis. However, these approaches have limited use as preclinical models since they do not recapitulate an anemia due to EPO deficiency as it is seen in patients with chronic kidney disease (CKD). These patients develop a chronic anemia due to the lack of a sufficient EPO gene expression response. In order to obtain a preclinical model for an EPO deficient anemia we developed a mouse model in which the EPO gene is flanked by two loxP sites (flox allele). In addition, these animals carry the Cre-ERT2 transgene which allows for a tamoxifen controlled activation of the Cre recombinase. Thus, these mice enable the inactivation of the EPO gene in adult animals by tamoxifen administration.After induction of Cre activity, mice heterozygous for the EPO allele (EPOko/flox) develop a moderate to severe anemia within 3 weeks. Although anemic, these mice display only low levels of EPO in their serum. However, within 3 months after induction of the knock-out the anemia resolves. This indicates that the Cre mediated excision of the EPO gene is insufficient and some EPO expressing cells remain. This mimics the clinical situation of patients with CKD.In order to study the response of EPO deficient mice to a severe anemia we treated animals with phenylhydrazine and observed the recovery of these animals. Interestingly, EPO deficient animals, although slightly delayed, fully recover from the induced anemia. Although the measured sEPO levels are significantly lower compared to control animals an increase in splenic erythropoiesis was observed in these animals. This indicates that the hypoxic response triggered by a sudden severe anemia is activating pathways other than EPO that promote erythropoiesis. Taken together, these results show that this novel mouse model will be useful tool as a preclinical model for the anemia associated with CKD. In addition, this model will allow the study of stress erythropoiesis under EPO limiting conditions. The Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International

BetaA, the major beta globin in definitive red blood cells, is present from the onset of primitive erythropoiesis in chicken

Reflecting physiological changes in oxygen acquisition and regulatory changes in globin transcription, the makeup of globin chains in erythrocytes varies in development and disease. The relationship between the globin chain composition and erythropoietic lineages/niches is not well-understood. Using a combination of proteomic-, genomic-, and intron-based in situ hybridization analyses, we show that the transcripts and protein product of the major adult beta globin, betaA, are present as early as the major embryonic beta globins during chicken primitive erythropoiesis. A rapid rise in betaA percentage is seen from embryonic day (E) 5, reaching adult profile by E7. Our data suggest that betaA locus is active from the onset of primitive erythropoiesis and that beta globin switching during development may reflect a change in relative transcript abundance rather than a strict on/off switch in gene activation.

Investigations of the effects of the antimalarial drug dihydroartemisinin (DHA) using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)

Artemisinin derivatives are effective and safe drugs for treating malaria, but they are not recommended during the first trimester of pregnancy because of resorptions and abnormalities observed in animal reproduction studies. Previous studies in rats showed that artemisinin embryotoxicity derives from the depletion of primitive red blood cells (RBCs) over a narrow critical time window (gestation Days 9–14). In order to further investigate the susceptibility of primitive RBCs to artemisinins and to establish whether this susceptibility is species-specific or inherent to the compound, we studied dihydroartemisinin (DHA), both a drug in its own right and the main metabolite of current artemisinin derivatives in use, in the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). This model readily allows investigation and monitoring of primitive and definitive RBCs. Effects on frog larvae exposed to DHA for 48 h during early embryonic development, starting from 24 h post fertilization, were similar to those on rat embryos in terms of reduction in the number of primitive RBCs (clonally produced within the ventral blood island). In contrast, RBCs of older larvae (stage 47, produced at the definitive sites of hematopoiesis) were affected minimally and subsequently recovered. Compared to rat embryos, the frog larvae had no areas of necrosis but they shared similar heart defects. The mitochondrion appeared to be the main subcellular target, similar to observations in Plasmodium. These results implicate artemisinin-induced embryotoxicity through perturbation of metabolically active RBCs; whereas this mode of action does not appear to be species-specific, the stages of susceptibility varied between different species. The window of susceptibility and duration of exposure must be considered to evaluate the clinical relevance of these findings.

Indian Hedgehog Is Essential for Definitive Haematopoiesis in the Fetal Liver.

Indian hedgehog (Ihh) is a member of the hedgehog family of secreted proteins which play diverse roles in development. Members of this family have established roles in craniofacial development, long bone formation and spermatogenesis. Mammals have three hedgehog proteins which all act through the Patched receptor to activate smoothened and downstream zinc finger transcription factors of the Gli family. Ihh can induce differentiation of primitive red blood cells during primitive streak formation, and definitive red blood cells from CD34+ cord blood stem cells. Both Ihh and the Patched receptor are expressed in the fetal liver. In this report we show Ihh knockout mice display a partially penetrant defect in fetal liver haematopoiesis characterised by severe anaemia and apoptosis of the fetal liver erythroid compartment. Primitive haematopoiesis is normal, so mutant embryos survive until ∼E14. Components of the hedgehog signalling pathway are expressed in stromal, stem cell and progenitor cell components of the haematopoietic system. Lastly, fetal liver progenitor cells (CFU-e, BFU-e, and myeloid CFUs) are normal in Ihh null mice, suggesting a critical requirement for Ihh in the fetal liver stem cell niche.

Erythroid pyrimidine 5′-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia

AbstractA characteristic process of terminal erythroid differentiation is the degradation of ribosomal RNA into mononucleotides. The pyrimidine mononucleotides can be dephosphorylated by pyrimidine 5′-nucleotidase (P5N-I). In humans, a lack of this enzyme causes hemolytic anemia with ribosomal structures and trinucleotides retained in the red blood cells (RBCs). Although the protein/nucleotide sequence of P5N-I is known in mammals, the onset and regulation of P5N-I during erythroid maturation is unknown. However, in circulating chicken embryonic RBCs, the enzyme is induced together with carbonic anhydrase (CAII) and 2,3-bisphosphoglycerate (2,3-BPG) by norepinephrine (NE) and adenosine, which are released by the embryo under hypoxic conditions. Here, we present the chicken P5N-I sequence and the gene expression of P5N-I during RBC maturation; the profile of gene expression follows the enzyme activity with a rise between days 13 and 16 of embryonic development. Thep5n-I expression is induced (1) in definitive but not primitive RBCs by stimulation of β-adrenergic/adenosine receptors, and (2) in definitive RBCs by hypoxic incubation of the chicken embryo. Since embryonic RBCs increase their hemoglobin-oxygen affinity by degradation of nucleotides such as uridine triphosphate (UTP) and cytidine triphosphate (CTP), the induction ofp5n-I expression can be seen as an adaptive response to hypoxia. (Blood. 2003;102:4198-4205)

Erythroid carbonic anhydrase and hsp70 expression in chick embryonic development: role of cAMP and hypoxia.

In the second half of avian embryonic development cAMP affects major aspects of red blood cell (RBC) function. At day 13/14, progressive developmental hypoxia causes the release of norepinephrine and erythroid beta-adrenergic receptor stimulation initiates the coordinate induction of adaptive key events of erythroid differentiation like carbonic anhydrase (CAII) and 2,3-biphosphoglycerate synthesis. Although cAMP-dependent regulation of CAII protein synthesis has been described in detail, no data exist about the transcriptional regulation in embryonic RBC. Here we report that after day 12 of embryonic development, the caII mRNA is accumulating. Hypoxic incubation at day 10 as well as in vitro incubation of isolated RBC with cAMP-elevating agonists strongly induces erythroid caII expression. The induction of caII occurs fast and does not require new protein synthesis. By screening several late erythroid genes, we could identify hsp70 as another cAMP-induced gene in definitive RBC. Because caII (but not hsp70) is also induced by cAMP in primitive RBC, the signal may regulate key events of late primitive and definitive erythropoiesis.

Fetal expression of a human Aγ globin transgene rescues globin chain imbalance but not hemolysis in EKLF null mouse embryos

Mice lacking the erythroid Kruppel-like factor (EKLF) die in utero at embryonic day 15 (E15) from severe anemia. EKLF(-/-) embryos display a marked deficit in beta-globin gene expression. To test whether beta-globin deficiency was solely responsible for the anemia and intrauterine death, we corrected the globin chain imbalance in EKLF(-/-) embryos by breeding with a strain of mice that express high levels of human gamma-globin. Despite efficient production of hybrid malpha(2)-hgamma(2) hemoglobin in the fetal livers of EKLF(-/-) animals, hemolysis was not corrected and survival was not prolonged. We concluded that deficiency of nonglobin EKLF target genes is a major contributor to the definitive red blood cell abnormalities and prenatal death in EKLF(-/-) embryos. These results suggest that strategies designed to antagonize EKLF function in adults with hemoglobinopathy, in an attempt to reactivate gamma-globin gene expression, may adversely affect other essential aspects of red blood cell physiology. (Blood. 2000;95:1827-1833)

Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP Signaling of Embryonic Red Blood Cells: Role of cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin

We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, β-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and β-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP Signaling of Embryonic Red Blood Cells: Role of cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin

AbstractWe have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, β-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and β-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

Adenosine causes cAMP-dependent activation of chick embryo red cell carbonic anhydrase and 2,3-DPG synthesis.

In late chick embryos, coordinate activation of red cell carbonic anhydrase II (CAII) and 2,3-diphosphoglycerate (2,3-DPG) synthesis is initiated by hypoxia. The effects are mediated by unidentified hormonal effectors resident in chick plasma. In the present investigation, we have analyzed the effect of adenosine receptor stimulation on embryonic red cell CAII and 2,3-DPG synthesis. We find that primitive and definitive embryonic red blood cells from chick have an A2a adenosine receptor. Stimulation of the receptor with metabolically stable adenosine analogues causes a large increase of red cell adenosine 3',5'-cyclic monophosphate (cAMP) and subsequent activation of red cell CAII and 2,3-DPG production in definitive red blood cells and of CAII synthesis in primitive red blood cells. Direct stimulation of adenylyl cyclase with forskolin has the same effect. Analysis of red cell protein pattern after labeling with [35S]methionine shows that stimulation of red cell cAMP levels activates synthesis of several other proteins aside from CAII. Presence of actinomycin D inhibits cAMP-dependent changes of protein synthesis, indicating that cAMP-dependent transcriptional activation is required. In contrast to the stable adenosine receptor analogues, adenosine itself was a very weak agonist, unless its metabolism was significantly inhibited. Thus, besides adenosine, other effectors of the adenylyl cyclase system are likely to be involved in the O2 pressure-dependent regulation of red cell metabolism in late development of avian embryos.