Embryonic Erythroblasts Research Articles

Flumioxazin, a PPO inhibitor: A weight-of-evidence consideration of its mode of action as a developmental toxicant in the rat and its relevance to humans

Flumioxazin, is a herbicide that has inhibitory activity on protoporphyrinogen oxidase (PPO), a key enzyme in the biosynthetic pathway for heme. Flumioxazin induces anemia and developmental toxicity in rats, including ventricular septal defect and embryofetal death. Studies to elucidate the mode of action (MOA) of flumioxazin as a developmental toxicant and to evaluate its relevance to humans have been undertaken. The MOA in the rat has now been elucidated. The first key event is PPO inhibition, which results in reduced heme synthesis in embryonic erythroblasts. The critical window for this effect is gestational day 12 when almost all erythroblasts are at the polychromatophilic stage, synthesizing heme very actively. Embryonic anemia/hypoxemia is induced and the heart pumps more strongly as a compensatory action during organogenesis, leading to thinning of the ventricular walls and failure of the interventricular septum to build completely and close. Investigations showed that this MOA is specific to rats and has no relevancy to humans. Flumioxazin inhibited PPO in rat hepatocyte mitochondria more strongly than in human. A 3-dimensional molecular simulation revealed that species differences in binding affinity of flumioxazin to PPO, observed previously in vitro, were due to differences in binding free energy. In vitro studies using several types of rat and human cells (erythroblasts derived from erythroleukemia cell lines, cord blood, or pluripotent stem cells), showed that flumioxazin decreased heme synthesis in rat cells but not in human cells, demonstrating a clear, qualitative species difference. Considering all available information, including data from PBPK modelling in rat and human, as well as the fact that anemia is not a symptom in patients with variegate porphyria, a congenital hereditary PPO defect, shows that the sequence of events leading to adverse effects in the rat embryo and fetus are very unlikely to occur in humans.

Fluid Shear Stress Upregulates E-Tmod41 via miR-23b-3p and Contributes to F-Actin Cytoskeleton Remodeling during Erythropoiesis.

The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm2) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3’UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, PE0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and PE0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling.

Dihydroartemisinin (DHA) Treatment Causes an Arrest of Cell Division and Apoptosis in Rat Embryonic Erythroblasts in Whole Embryo Culture

Within 24 hr after oral administration of the antimalarial artesunate to rats on Day 10 or 11 postcoitum (pc), there is depletion of embryonic erythroblasts (EEbs), leading to embryo malformation and death. The proximate agent is dihydroartemisinin (DHA), the primary metabolite. We investigated the causes of EEb depletion by evaluating effects of DHA on EEbs in whole embryo culture (WEC). Rat embryos cultured starting on Day 9 pc were treated with 1 or 7 μM DHA for 24 hr starting after 19 hr of culture (∼Day 10 pc) and for 2 to 12 hr starting after 43 hr of culture (∼Day 11 pc). DHA effects indicating the depletion of EEbs were paling of the visceral yolk sac and reductions in visible blood cells, H&E-stained normal (Type II or III) EEbs, and dividing (BrdU-stained) EEbs. DHA-induced abnormal cell division was indicated by increases in symmetric and asymmetric binuclear cells. DHA-induced apoptosis was indicated by increases in TUNEL- and Caspase-3-positive cells and EEbs with fragmented nuclei. In addition, although the overall number of EEbs was decreasing, DHA caused increases in the numbers of circulating early-stage (Type I or earlier) EEbs that could not be accounted for by cell division, suggesting the release of new, less sensitive erythroblasts from the yolk sac. In summary, treatment of Day 10 or 11 pc rat embryos with DHA in WEC resulted in defective and arrested cell division in EEbs followed by apoptosis, suggesting a mechanism for their depletion after artesunate treatment in vivo.

Abstract 255: The Kidney and Heart are Hemogenic Organs During Embryonic Life.

During early embryonic life, the heart starts to beat before an effective circulation is established, and the kidney starts to form its vasculature before it connects to the general circulation. We and others have shown a close lineage relationship between endothelial cells (ECs) and hematopoietic cells. In fact, during embryonic development erythroblasts bud from the endothelium of developing vessels, a process we termed hemovasculogenesis. Those studies suggested the possibility that embryonic organs may have hemogenic potential. To test this hypothesis, we performed lineage studies and colony forming unit (CFC) assays to trace the fate of hematopoietic stem cells (HSCs), erythroid cells, and ECs in heart and kidney from embryonic mice. Using ER-GFPcre mice that express both GFP and cre under control of the erythropoietin receptor locus in the erythroid cells, we identified hematopoietic progenitors (Hb+Nanog+) within nascent vessels in the early embryonic kidney and heart. Using EC-SCL-Cre-ERT transgenic mice that specifically express tamoxifen inducible Cre in EC progenitors, we found both circulating and non-circulating cells from the EC lineage in the early embryonic heart and kidney. CFC assays using HSC-SCL-Cre-ERT; mTmG mice (which express GFP in the cells from the HSC lineage upon tamoxifen induction) showed that both the embryonic kidney and heart possess HSCs. Further, transplantation studies of pre-vascular embryonic kidneys from EC-SCL-Cre-ERT;R26R mice under the kidney capsule of WT adult mice showed blood cells derived from the embryonic kidney suggesting that the embryonic kidney also possesses HSCs that originate in situ. These studies indicate that the embryonic kidney and heart function as hematopoietic organs during early embryogenesis. In addition to solve an important scientific controversy in our understanding of lineage/fate relationships in the developing embryo, these findings are relevant for tissue repair/regeneration and may help explain why under pathological circumstances, hematopoiesis occurs in extramedullary organs.

Dihydroartemisinin inhibits the human erythroid cell differentiation by altering the cell cycle

Artemisinin derivatives such as dihydroartemisinin (DHA) induce significant depletion of early embryonic erythroblasts in animal models. We have reported previously that DHA specifically targets pro-erythroblasts and basophilic erythroblasts, when human CD34+ stem cells are differentiated toward the erythroid lineage, indicating that a window of susceptibility to artemisinins may exist also in human developmental erythropoiesis during pregnancy. To better investigate the toxicity of artemisinin derivatives, the structure–activity relationship was evaluated against the K562 leukaemia cell line, used as a model for differentiating early human erythroblasts. All artemisinins derivatives, except deoxyartemisinin, inhibited both spontaneous and induced erythroid differentiation, confirming that the peroxide bridge is responsible for the erythro-toxicity. On the contrary, cell growth was markedly reduced by DHA, artemisone and artesunate but not by artemisinin, 10-deoxoartemisinin or deoxy-artemisinin. The substituent at position C-10 is responsible only for the anti-proliferative effect, since 10-deoxoartemisinin did not reduce cell growth but arrested the differentiation of K562 cells. In particular, the results showed that DHA resulted the most potent and rapidly acting compound of the drug family, causing (i) the decreased expression of GpA surface receptors and the down regulation the γ-globin gene; (ii) the alteration of S phase of cell cycle and (iii) the induction of programmed cell death of early erythroblasts in a dose dependent manner within 24h. In conclusion, these findings confirm that the active metabolite DHA is responsible for the erythro-toxicity of most of artemisinins used in therapy. Thus, as long as no further clinical data are available, current WHO recommendations of avoiding malaria treatment with artemisinins during the first trimester of pregnancy remain valid.

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

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

Artesunate and artelinic acid: association of embryotoxicity, reticulocytopenia, and delayed stimulation of hematopoiesis in pregnant rats

The artemisinin antimalarials cause embryo death and malformations in animals by killing embryonic erythroblasts. Groups of pregnant rats (N = 4) were administered 35 and 48 µmol/kg artesunate and 17.2, 28.7, 48, 96, and 191 µmol/kg artelinic acid as a single oral dose on gestational day (GD) 12. Litters were examined on GD21. The ED(50) for embryo death with artelinic acid (23.4 µmol/kg) was just slightly lower than that for decreased reticulocyte count at 24 hr postdose (33.5 µmol/kg) and both had similarly steep dose responses (maximal effects of total litter loss and ∼60% decreases in reticulocyte count at 48 µmol/kg). Results with artesunate were similar. The correlation coefficient between embryo death and decreased reticulocyte count was 0.82 (p<0.01). The close relationship between embryotoxicity and reticulocytopenia is suggestive of a common mechanism-artemisinin-induced mitochondrial damage leading to cell death. At 9 days postdose, treatment with artesunate and artelinic acid also caused increases in counts of reticulocytes, lymphocytes, basophils, and monocytes (up to 3.7 ×, 1.7 ×, 4.7 ×, and 1.7 × control, respectively). This stimulation of hematopoiesis may have been mediated by the direct oxidative conversion of artesunate or artelinic acid to the artemisininyl hydroperoxide within the bone marrow cells or by an indirect increase in reactive oxygen species. The high correlation between embryotoxicity and reticulocytopenia further supports the assertion that therapeutic dosage regimens of artemisinins that cause decreases in reticulocyte count in pregnant women during the putative critical period (approximately postconception wk 3 to 9) are at risk of also causing adverse effects on the embryo.

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

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

Pregnancy exposure registries for assessing antimalarial drug safety in pregnancy in malaria-endemic countries.

Because pregnant women are routinely excluded from pre-licensure clinical trials for fear of harming the mother or the developing foetus [1], most drugs are marketed with limited information on their safety during pregnancy and therefore are not recommended for use by pregnant women. Yet drugs are widely used by pregnant women, and medication often cannot be avoided in chronic diseases such as epilepsy and HIV or other acute illness that harm the mother and the unborn child if left untreated.

Artesunate: developmental toxicity and toxicokinetics in monkeys

The developmental toxicity, toxicokinetics, and hematological effects of the antimalarial drug, artesunate, were previously studied in rats and rabbits and have now been studied in cynomolgus monkeys. Groups of up to 15 pregnant females were dosed on Gestation Days (GD) 20-50 or for 3-7-day intervals. At 30 mg/kg/day, 6 embryos died between GD30 and GD40. Histologic examination of 3 live embryos (GD26-GD36) revealed a marked reduction in embryonic erythroblasts and cardiomyopathy. At 12 mg/kg/day, 6 embryos died between GD30 and GD45. Four surviving fetuses examined on GD100 had no malformations, but long bone lengths were slightly decreased. At the developmental no-adverse-effect-level (4 mg/kg/day), maternal plasma AUC was 3.68 ng.h/mL for artesunate and 6.93 ng.h/ml for its active metabolite, dihydroartemisinin (DHA). No developmental toxicity occurred with administration of 12 mg/kg/day for 3 or 7 days, GD29-31 or GD27-33 (maternal plasma AUC of 9.84 ng.h/mL artesunate and 16.4 ng.h/mL DHA). Exposures at embryotoxic doses were substantially lower than human therapeutic exposures. However, differences in monkey and human Vss for artesunate (0.5 L/kg vs. 0.18 L/kg) confound relying solely on AUC for assessing human risk. Decreases in reticulocyte count occur at therapeutic doses in humans. Changes to reticulocyte counts at embryotoxic doses in monkeys (> or =12 mg/kg/day) were variable and generally minor. Artesunate was embryolethal at > or =12 mg/kg/day when dosed for at least 12 days at the beginning of organogenesis, but not when dosed for 3 or 7 days, indicating that developmental toxicity of artesunate is dependent upon duration of dosing in cynomologus monkeys.

Developmental toxicity of artesunate in the rat: comparison to other artemisinins, comparison of embryotoxicity and kinetics by oral and intravenous routes, and relationship to maternal reticulocyte count

The antimalarial, artesunate, is teratogenic and embryolethal in rats, with peak sensitivity on Days 10 and 11 postcoitum (pc). We compared the developmental toxicity of structurally related artemisinins, dihdyroartemisinin (DHA), artemether (ARTM), and arteether (ARTE) to that of artesunate after oral administration to rats on Day 10 pc. In separate studies, embryolethality was characterized after single intravenous (IV) administration of artesunate on Day 11 pc, and toxicokinetic parameters following oral and IV administration were compared. Lastly, to determine whether maternal hematologic effects occurred at doses that affect embryonic erythroblasts, artesunate was orally administered on Day 11 pc at a dose that caused 100% embryolethality. All artemisinins caused the same pattern of embryolethality and fetal cardiovascular and skeletal abnormalities as previously shown for artesunate. In the IV study, marked postimplantation loss occurred at 1.5 and 3 mg/kg artesunate, but not at 0.75 mg/kg. Among the toxicokinetic parameters evaluated, only the DHA AUC(0-t) was similar at embryolethal oral and IV doses of artesunate. An embryolethal dose of artesunate caused a 15% decrease in maternal reticulocyte counts and no other hematologic effects. Several structurally related artemisinins cause similar developmental toxicity, suggesting an artemisinin class effect. Equally embryotoxic oral and IV treatments of one artemisinin compound (artesunate) produced similar systemic exposure to the artesunate metabolite, DHA, suggesting that DHA may be the proximate developmental toxicant. Embryolethal doses of artesunate only caused minor changes in maternal reticulocyte counts indicating that adult hematology parameters are not as sensitive as embryonic erythroblasts.

An Erythoid-Specific Component of the Rhesus Stage Selector Protein, rNF-E4, Modulates γ-Globin Gene Expression Specifically.

Understanding the molecular mechanisms that regulate γ-globin gene expression is essential for development of new therapeutic strategies for individuals with sickle cell disease and β-thalassemia. We have previously identified a tissue- and developmentally- specific multiprotein transacting factor complex, the human stage selector protein (SSP), which facilitates the interaction of the g-globin gene promoters with the upstream locus control region enhancer in fetal erythoid cells. This complex interacts with the stage selector element (SSE) in the proximal g-globin promoter, a regulatory motif phylogenetically conserved in primate species with a distinct fetal stage of β-globin like gene expression. Given these observations, we hypothesized that a similar complex modulates γ-globin in the rhesus macaque, a non-human primate model that has been utilized to study β-globin like gene expression. We focused our efforts on NF-E4, given that a human isoform of this factor confers erythroid and fetal specificity to the SSP complex. Fetal liver erythroblasts were obtained from rhesus embryos and analyzed by reverse transcriptase(RT)-PCR analysis for NF-E4 expression. NF-E4 like transcripts were identified in day 60, 80 and 120 embryonic erythroblasts, but not other rhesus tissues, demonstrating an erythroid-specific pattern of expression. Utilizing 5′ RACE, we cloned a full length NF-E4 transcript, identifying an open reading frame encoding a 131 amino acid polypeptide. This 20kD polypeptide shares a high degree of homology with human NF-E4, especially in its carboxy-terminal domain. Like human NF-E4, GST pulldown chromatography confirmed the ability of the rhesus factor to interact directly with CP2 and ALY, the other core components of the SSP. To evaluate rNF-E4 function in vivo, we utilized retrovirally mediated gene transfer to enforce expression of this factor in K562 cells, a model of human fetal erythropoiesis. Initial co-immunoprecipitation studies confirmed the in vivo interaction of rNF-E4 with other components of the SSP. Interestingly, we observed a specific 3-fold induction of γ-globin gene expression in rNF-E4 expressing cells when compared to controls. Moreover, we demonstrated that, like enforced expression of human NF-E4, rNF-E4 induced a significant increase in ε-globin gene expression. Taken together, our results suggest a conservation of NF-E4 expression and function in species with a fetal stage of globin gene expression. Moreover, the identification of rNF-E4 provides a platform for the pre-clinical development of therapeutic agents that induce high levels of NF-E4 in adult erythroblasts.

Artesunate-induced depletion of embryonic erythroblasts precedes embryolethality and teratogenicity in vivo

Artesunate (ART), an artemisinin antimalarial, is embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum (pc), respectively (Clark et al.: Birth Defects Res B 71:380-394, 2004; White et al.: Birth Defects Res A 70:265, 2004). In this study, pregnant rats were administered a single oral dose of 17 mg/kg ART on Days 10-11 pc and conceptuses were evaluated through Day 14 pc. Paling of visceral yolk sacs was observed within 3-6 hr after treatment. Within 24 hr, marked paling and embryonic erythroblast depletion were observed macroscopically, which preceded malformations and embryo death, and persisted through Day 14 pc. Histologically, embryonic erythroblasts were reduced and cells showed signs of necrosis within 24 hr, were maximally depleted by 48 hr, and had partially rebounded within 3-4 days after treatment (Days 13 and 14 pc). Iron accumulation was evident in treated erythroblasts as early as 6 hr after treatment, suggesting impairment of heme synthesis. Heart abnormalities (swollen or collapsed chambers) were observed within 24 hr in approximately 25-60% of embryos and within 48 hr in 100% of embryos, correlating with histologic signs of cardiac myopathy (thinned and underdeveloped heart walls and enlarged chambers). Delays in limb and tail development occurred by Day 13 pc. Embryos were viable through Day 13 pc, but approximately 77% of embryos had died by Day 14 pc, presumably due to hypoxia and/or cardiac abnormalities. In summary, embryonic erythroblasts are the primary target of ART toxicity in the rat embryo after in vivo treatment, preceding embryolethality and malformations.

Gene expression in somatic cell hybrids derived from embryonic mice transgenic for human globin genes.

Somatic cell hybrids have been used previously to investigate the developmental regulation of globin gene expression. When a chromosome containing an active human fetal gamma gene is transferred to mouse erythroleukaemia (MEL) cells by cell fusion, there is persistent expression of this gene through multiple hybrid-cell divisions despite the 'adult' trans-acting factor environment of the MEL cell. We analysed hybrids formed between embryonic erythroblasts and MEL cells to determine whether the embryonic globin genes would be similarly regulated. Surprisingly, there was no detectable expression of either the endogenous (epsilon, beta h 1) or the transgenic (human epsilon) embryonic beta-like genes in a large number of hybrid pools and clones, even though these genes were expressed in the donor cells at the time of fusion. In contrast, the human gamma gene, which is also expressed as an embryonic gene in transgenic mice, continued to be expressed in these hybrids. This lack of embryonic gene expression in hybrids also applied to the alpha gene cluster; no mouse or human zeta mRNA was detectable in embryonic cell hybrids from mice transgenic for the human alphaglobin genes. Hybrids generated from embryonic non-erythroid cells resulted in expression that was largely restricted to the adult beta gene. It has previously been suggested that epigenetic modifications to the beta-globin gene cluster during normal development are responsible for the perpetuation of gamma and beta gene expression in this hybrid cell system. The results presented here suggest that no such stable modifications occur around the embryonic genes and therefore that the regulatory mechanisms determining embryonic globin gene transcription differ from those responsible for the expression of gamma and beta genes in this system.

Distribution of actin, myosin, and spectrin during enucleation in erythroid cells of hamster embryo.

Yolk-sac derived erythroblasts undergo semi-synchronous maturation and some of them enucleate in the peripheral blood of embryos. We have studied the assembly and distribution of actin, myosin, and spectrin during the enucleation of Syrian hamster embryonic erythroblasts. At day 11 of the gestation, that is just before the start of the enucleation, formation of a cytoskeletal structure consisted chiefly of particulate associations of F(filamentous)-actin was detected by the staining with rhodamine-labeled phalloidin. Stress-fiber-like structures were not observed in each differentiation stage after day 10. Distribution of myosin, actin, and spectrin was studied immunocytochemically to know the role of them in the enucleation of erythroid cells that starts at late day 11 or early day 12 in the gestation. The enucleation is preceded by the approach and the subsequent attachment of nucleus to the plasma membrane. At that time, actin and myosin are present in the cytoplasmic and cortical region of the cells. From the time when the extrusion of nucleus has started, condensation of actin and myosin was observed at the cell cortex area surrounding the extruding nucleus, and a contractile ring-like structure was infrequently observed. Spectrin was observed in the cortical region of the cells, and the change of the localization of spectrin was not observed throughout the terminal differentiation process (days 10-12) of the embryonic erythroid cells. The results show the possible involvement of a myosin-actin contractile system that appears around the extruding nucleus within the mechanism of erythroid enucleation.

The chicken c-erbA proto-oncogene is preferentially expressed in erythrocytic cells during late stages of differentiation.

We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.

The chicken c-erbA proto-oncogene is preferentially expressed in erythrocytic cells during late stages of differentiation.

We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.

Expression of Embryonic Haemoglobin in ts AEV-Transformed Embryonic Erythroid Cells During Temperature-Induced Differentiation

Cells prepared from 1-day-old chick blastoderms were infected with a temperature-sensitive mutant of avian erythroblastosis virus (tsAEV). Clonal strains of transformed erythroblasts were isolated from the infected blastoderm cells. By shift to the nonpermissive temperature, these cells could be induced to differentiate into erythrocyte-like cells which expressed embryonic haemoglobins. Embryonic haemoglobins could not be detected in tsAEV-transformed erythroblasts from adult bone marrow when induced to differentiate under the same conditions. In contrast to normal primitive erythrocytes, tsAEV-infected embryonic erythroblasts differentiated in vitro expressed also adult haemoglobin. These results suggest an influence of the haematopoietic environment on the switch from embryonic to adult erythrocytes.