Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry.

Human erythroblasts which are prereticulocyte maturation stages of red blood cells were studied by light microscopic cytochemistry and electron microscopy to provide more information on the ultrastructure of the micronucleoli which are terminal stages of nucleolar changes found during maturation of these cells. As indicated by light microscopy of smeared cells, micronucleoli were virtually the only types of nucleoli present in the last stages of maturing erythroblasts, i.e., polychromatic and orthochromatic (late polychromatic) erythroblasts. Accordingly, they were not portions of the periphery of other nucleoli. Inasmuch as most of the micronucleoli exhibited characteristic segregation of nucleolar fibrillar and granular components they presumably are producing little if any preribosomal RNA, since such segregation generally reflects inhibition of nucleolar RNA synthesis.     

UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis

UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and reactive oxygen species (ROS) modulation. High levels of UCP2 mRNA were recently found in erythroid cells where UCP2 is hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. We examined UCP2 protein expression and role in mice erythropoiesis in vivo. UCP2 was mainly expressed at early stages of erythroid maturation when cells are not fully committed in heme synthesis. Iron incorporation into heme was unaltered in reticulocytes from UCP2-deficient mice. Although heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of progenitor cells from bone marrow and fetal liver both in vitro and in vivo revealed that UCP2 deficiency results in a significant decrease in cell proliferation at the erythropoietin-dependent phase of erythropoiesis. This was accompanied by reduction in the phosphorylated form of ERK, a ROS-dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid cells revealed altered distribution of ROS, resulting in decreased cytosolic and increased mitochondrial ROS. Restoration of the cytosol oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.     

Asynchrony of erythroblast maturation induced by riboflavin deficiency

Ultrastructural studies indicate that galactoflavin-induced riboflavin deficiency induces asynchrony of rat erythroblast maturation. During the latter stages of maturation erythroblasts retain significantly larger numbers of ribosomes as compared to control cells. Nucleoli are not evident in erythroblasts whose nuclei indicate cells in the latter stages of development. Membrane whorls develop within the mitochondria of plasma cells, eosinophils and neutrophils during the fifth week of riboflavin deficiency. No further evidence of degeneration was noted among additional cell organelles.     

Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.     

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes

Haemolytic anaemia is one of the characteristics of life‐threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34⁺ haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a‐mediated cellular injury of developing erythrocytes. CD34⁺ haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a‐binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx‐mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx‐producing pathogens.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.     

Functional but abnormal adult erythropoiesis in the absence of the stem cell leukemia gene

Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.     

Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2

Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian erythroblasts.     

Cyclic AMP phosphodiesterase activity during differentiation of rabbit erythroid bone marrow cells.

Changes in the activity of cyclic AMP phosphodiesterase during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic cells, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts, which also have the highest content of cyclic AMP. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity was observed. In these cells the amount of cyclic AMP was about 80% lower than that in proerythroblasts. In polychromatic cells a further drop in phosphodiesterase activity occurred. After the final cell division the enzyme activity was very low and the levels of cyclic AMP in the early and late orthochromatic cells remained constant. Kinetic studies demonstrated a heterogeneity of erythroid cell cyclic AMP phosphodiesterase: high affinity, low-Km (5.5 X 10(-6) M) and low affinity, high-Km (0.1 X 10(-3) M) enzymes were found. The phosphodiesterase activity was dependent on the presence of Mg2+ and was activated by Ca2+ at low Mg2+ concentrations (1 mM). The changes in cyclic AMP phosphodiesterase activity during differentiation and maturation of erythroid cells suggest the possible importance of this enzyme in the physiological control of cyclic AMP concentrations in developing erythroblasts. The loss of cyclic AMP phosphodiesterase activity after cessation of cell division supports the concept of the significance of the final cell division in erythroblast differentiation.     

The Notch2–Jagged1 interaction mediates stem cell factor signaling in erythropoiesis

Stem cell factor (SCF), the ligand for the c-kit receptor, is essential for the production of red blood cells during development and stress erythropoiesis. SCF promotes erythroblast proliferation and survival, while delaying erythroid differentiation through mechanisms that are largely unknown. In cultures of primary human differentiating erythroblasts, we found that SCF induces an increase in the expression of Notch2, a member of the Notch family implicated in the control of cell growth and differentiation. Functional inhibition of either Notch or its ligand Jagged1 inhibited the effects of SCF on erythroid cell expansion. SCF also induced the expression of Hes-1 and GATA-2, which may contribute to transduce Notch2 signals in response to SCF. Transduction of primary erythroid precursors with a dominant-negative Notch2 mutant inhibited both basal and SCF-mediated erythroblast expansion, and counteracted the effects of SCF on erythroblast differentiation. These findings provide a clue to understand the effects of increased proliferation and delayed differentiation elicited by SCF on the erythroid compartment and indicate Notch2 as a new player in the regulation of red cell differentiation.     

On the yolk sac of the cat

Summary The phase of primitive erythropoiesis in the feline yolk sac lasts from the 14th to the 20th day after mating. The globular nucleated primitive erythroblasts are formed extravascularly to some extent, but they can be clearly distinguished from the endoderm. They do not undergo a denucleation and are still present in the circulating blood on the 45th day. Aging primitive erythroblasts are characterized by a loss of polysomes, by the appearance of long intracytoplasmic electron-lucent channels, and by a nuclear pyknosis which can turn into a karyolysis. Definitive erythropoiesis begins around the 17th day but, even by the 19th day, it is not particularly prominent. It ends around the 45th day. It is almost exclusively intravascular. The distinction of immature primitive erythroblasts from erythroblasts of the definitive series is difficult, because it is based upon only slight differences in the heterochromatinization, in the nuclear-cytoplasmic ratio, and in the organelle content of the cells. In the definitive series, the nuclear divisions follows the law of the rhythmical halving of the nuclear volume. The cells exhibit more clearly identifiable maturation stages here, and the checkerboard nucleus is more distinct. The vascular endothelium is largely attenuated and moderately fenestrated; it lacks a distinct basement membrane. Organelle-rich adventitial cells are found in close apposition.     

AN ULTRASTRUCTURAL STUDY OF EARLY MORPHOGENETIC EVENTS DURING THE ESTABLISHMENT OF FETAL HEPATIC ERYTHROPOIESIS

Morphogenetic events are described which characterize early stages of the interaction between mesenchyme and expanding epithelial cell cords derived from the hepatic endodermal diverticulum in the C57BL/6J mouse. This interaction culminates in the differentiation of hepatic epithelial and hematopoietic tissues. No basement membrane separates the presumptive hepatic epithelial cells from the adjacent mesenchyme, while intercellular attachments, both adherent junctions and desmosomes, are established transiently between heterologous cell types across this epithelio-mesenchymal interface. Yolk sac-derived erythroblasts found in the primitive liver are distinguished morphologically from endogenous hepatic erythroid cells; they are confined to the vascular compartment and are not, apparently, precursors for hepatic erythropoiesis. The earliest recognizable endogenous hepatic hematopoietic cells appear, extravascularly, among those mesenchymal cells in intimate contact with the endodermal epithelium between the 10¼ and 10½ gestational day. Definitive erythropoiesis commences between the 10½ and 11th fetal days. The ultrastructure of these primitive hepatic erythroid cells (proerythroblasts) and their transition to more mature forms (basophilic and polychromatophilic erythroblasts) are described.     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.     

A non-apoptotic role for Fas/FasL in erythropoiesis

Issues remain to be elucidated in the developmental regulation of erythropoiesis. In particular the role of Fas, a member of the tumor necrosis factor family of receptors despite much work remains unclear. During erythropoiesis, Fas is expressed at low levels on erythroblasts. For most cell types, Fas to FasL interaction causes apoptotic cell death via caspase activation. Here, we show that in humans, early erythroid progenitors are refractory to apoptosis triggered through Fas. Further during early human erythropoiesis, Fas triggered caspase activation provides a positive stimulus for erythroid maturation, and does not alter cellular proliferation or trigger apoptosis.     

Endothelial cells create a hematopoietic inductive microenvironment preferential to erythropoiesis in the mouse spleen

The spleen is an erythropoietic organ in mouse. To reconstruct a microenvironment essential for erythropoiesis in vitro, the stroma (MSS31) cell line had been established from a newborn mouse spleens. MSS31 cells exhibited properties of endothelial cells: (a) the cells showed the activity to uptake acetylated low-density lipoprotein (Ac-LDL) and (b) the cells can form a capillarylike structure by a phenotypic modulation in collagen matrices. MSS31 cells selectively supported the proliferation and differentiation of the erythroid progenitor cells by direct cell-to-cell contact in a semisolid medium in the presence of erythropoietin. These layers also supported erythrocyte maturation and enucleation of erythroblasts. This suggests that spleen endothelial cells are a new type of stromal cell with erythropoietic stimulation activity and may have a critical function in the hemopoietic inductive microenvironment of the mouse spleen.     

Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerable less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythrosynthesis in whole cells and in isolated nuclei.