On the kinetics of erythroid cell differentiation in fetal mice. II. DNA and hemoglobin measurements of individual erythroblasts during gestation

Microspectrophotometric absorption measurements were used to determine the hemoglobin content of erythroid cells derived from the yolk sac during gestation of fetal C3H mice, from day 9 to day 15. Using the DNA content as a marker for the mitotic state between 2C and 4C phase, five successive cell generations and their mean hemoglobin contents were distinguished: 12 pg (pg, picogram = 10^?12 gm). 22.2 pg, 37 pg, 50 pg and 56 pg. In the final state, nucleated erythrocytes contained 98 ± 22 pg hemoglobin. Erythroid cells derived from the liver were measured on day 15 of fetal gestation. The hemoglobin content of proerythroblasts was below 0.3 pg. The two cell generations in the basophilic state had 0.6 pg and 1.7 pg respectively. Polychromatic erythroblasts yielded a hemoglobin content of 5.1 pg in the first cell generation and 7.5 pg in the second one. Orthochromatic erythroblasts contained 8 pg, reticulocytes 12 pg and mature erythrocytes 28 ± 7 pg hemoglobin. Calculations based on these data suggest that the rate of total hemoglobin synthesis is similar in both yolk sac and liver erythropoiesis. The difference between the final hemoglobin content in nucleated erythrocytes of yolk sac origin and that in hepatic erythrocytes can be explained by the different cell generation times.     

Mouse hemoglobin during gestation. Absence of fetal hemoglobin

A "fetal hemoglobin' has been reported to exist during mouse gestation, Investigations using CMC chromatography, starch gel electrophoresis or isoelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated a "fetal hemoglobin'. In the current study isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no "fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as "fetal' in our laboratory was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.     

Changes in globin messenger RNA content during erythroid cell differentiation.

Previous studies have shown that mouse fetal erythroid precursor cells isolated by an immunological technique synthesize little or no globin and contain little, if any, globin mRNA, as assayed in a cell-free system (translatable mRNA). After culture for 10 hours in the presence of erythropoietin, there is a marked increase in globin synthesis and in translatable globin mRNA. The present studies were designed to measure directly the content of globin mRNA sequences during erythroid cell differentiation, by molecular hybridization with 3H-labeled DNA complementary to globin mRNA. The results indicate that few, if any, globin mRNA sequences are present in the total RNA of erythroid precursor cells. There is little or no pool of untranslated globin mRNA in these cells. After 10 hours of culture with erythropoietin, there is an increase in globin mRNA content, as ;easured by a change in the Cot1/2 values obtained by cDNA: mRNA hybridization with (Co) representing the concentration of RNA. Between 0 and 22 hours of culture, there is a 250-fold rise, and between 22 and 44 hours, a further 2-fold increase in globin mRNA content. During the 44 hours in culture, the number of cells in culture increases 2- to 3-fold. The number of globin mRNA molecules rises in erythroid precursor cells to an average value of 1800 molecules/cell during 22 hours of culture. In cultures without added erythropoietin, the absolute number of cells decreases, however, cells presumably induced to differentiate by exposure to erythropoietin in vivo continue to differentiate in vitro, accumulating globin mRNA and initiating globin synthesis.     

DNA synthesis and content and the accumulation of total protein and hemoglobin during the differentiation of primary erythroid cells in chickens

Using cytophotometric and autoradiographic methods, it was shown that on days 2-3 of embryogenesis primary erythroid cells (PEC) divided actively. The distribution of erythroblasts (EB) according to their DNA content is not, however, typical of a proliferating population: it contains an unusually large number of 4c cells resulting from the cell cycle arrest at the G2 phase. It is established that reticulocytes (RC) do not divide and are arrested at G1 or G2 phases, since they do not incorporate 3H-thymidine after their formation is complete and their DNA contents are strictly confined to either 2c or 4c. All types of PEC include a large number of cells containing H2c DNA which is due either to the cell cycle arrest at the S phase, or to the formation of accessory nuclei. All PECs have much higher contents of hemoglobin and total protein than do adult hen erythrocytes (EC). Hemoglobin and total protein contents of H2c and accessory nuclei containing cells are much higher than those in 2c-cells. We have calculated that adult birds and embryos contain the same amount of hemoglobin per gram of weight, but the quantity of red blood cells in the former is ten times higher. A conclusion is drawn that proliferation and cytodifferentiation regulation mechanisms are directed, in primary erythropoiesis, to intense hemoglobinization of the cells, and, in adult erythropoiesis, to increasing their number. In both the cases homeostatic regulation of erythropoiesis works.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylation of human fetal hemoglobin occurs throughout erythroid cell maturation

The biosynthesis of human acetylated fetal hemoglobin (Hb F1) has been examined by incubating the following cell types with [3H]leucine: (a) burst-forming unit erythroid cells cultured from umbilical cord mononuclear cells, (b) infant bone marrow, (c) umbilical cord blood, and (d) peripheral blood cells from adults with elevated fetal hemoglobin. Newly synthesized Hb F1 was 18-20% that of Hb F0 in burst-forming unit erythroid cells which were immature, mature, or in an intermediate state of development. In infant marrow and in infant and adult peripheral blood the extant Hb F1 comprised 10.8 +/- 1.8% of the total Hb F. In marrow cells the specific radioactivity (cpm/mg) of Hb F1 was 1.4-2.0-times greater than that of Hb F0. In peripheral blood cells these ratios were slightly greater. [3H]Leucine-labeled infant bone marrow, umbilical cord blood, and adult peripheral blood cells were subjected to density gradient ultracentrifugation. The ratios of specific radioactivity for Hb F1/Hb F0 increased from 1.0-1.8 in the lightest cell zone to 5.2-9.0 in the more dense cells. Thus the biosynthesis of Hb F1 is enhanced in cells which are more mature than those responsible for the bulk of hemoglobin synthesis, and the acetylation of Hb F provides a marker of erythroid cell maturation.     

Vanadate elevates fetal hemoglobin in human erythroid precursors by inhibiting cell maturation

Increased fetal hemoglobin (HbF) in erythroid precursors of patients with beta-hemoglobinopathies (sickle cell anemia and beta-thalassemia), in which adult hemoglobin synthesis is defective, ameliorates the clinical symptoms of the underlying diseases. The production of erythroid precursors depends on the action of erythropoietin (EPO), which prevents their apoptosis and stimulates their proliferation. EPO binds to its surface receptor, induces its homodimerization, and initiates a cascade of phosphorylation and dephosphorylation of a series of proteins by kinases and phosphatases, respectively. Vanadate inhibits various phosphatases, including those that are involved in the EPO pathway, thereby intensifying the signal. In this study, we investigated the effect of vanadate on the proliferation and maturation of human erythroid precursors in culture. When vanadate was added to cells derived from normal donors, cell maturation was delayed, as indicated by cell morphology, cell growth kinetics, the rate of appearance of hemoglobin-containing cells, and the expression of surface antigens (CD117, CD71, and glycophorin A). Analysis by high-performance liquid chromatography and flow cytometry of the hemoglobin profile of vanadate-treated normal cells revealed a higher proportion of HbF than was found in untreated cells. When vanadate was added to cells derived from patients with beta-thalassemia, a significant increase in HbF was observed. The results suggest that intensification of the EPO signal by vanadate results in maturation arrest and increased HbF production. Thus, inhibitors that are more specific and less toxic than vanadate may present a novel option for elevating HbF in patients with beta-hemoglobinopathies, as well as for intensifying the EPO response in other forms of anemia.     

Fetal erythropoiesis in steel mutant mice : I. A morphological study of erythroid cell development in fetal liver

A method of definitive identification of mutant (S1/S1^d) and wild-type (+/+) mouse embryos in segregating litters is described, based on the total number of circulating erythrocytes in a unit volume of embryonic blood and the relative proportion of nonnucleated vs. nucleated red blood cells. Evidence is presented that from days 13–17 of gestation, S1/S1^d embryos have many fewer fetal liver derived nonnucleated erythrocytes whereas the number of yolk sac-derived nucleated red blood cells is similar between S1/S1^d and +/+. Erythroid precursor cells at various stages of maturation in mutant fetal livers are studied by light and electron microscopy, and their fine structure is found to be identical to those present in normal embryos. The number of hemoglobin-containing mature erythroblasts in mutant fetal livers is far fewer than that of the normal, whereas the number of immature erythroid precursors present in a unit area of fetal liver is not significantly different between S1/S1^d and +/+. It is suggested that the mutant S1 gene product(s) interferes with or fails to support the differentiation of immature erythroid precursors into hemoglobin synthesizing cells.     

Alterations in lysine transfer RNA during erythroid differentiation of the Friend cell.

The proportion of lysine tRNA represented by the isoacceptor species lysine tRNA4 has previously been shown to be largest in cells with the greatest ability to proliferate. Using reverse phase chromatography (RPC-5), we have analyzed the changes in the relative quantities of lysine tRNA species which occur in different cellular states of the Friend cell, a transformed murine cell infected with Friend erythroleukemia virus complex. This cell undergoes erythroid differentiation when exposed to various chemicals. Lysine tRNA4 comprises 32% of the total lysine tRNA in rapidly dividing, uninduced Friend cells, but only 16% of the total lysine tRNA in uninducase. Friend cells undergoing erythroid differentiation divide more slowly than uninduced cells, and finally cease proliferation, but lysine tRNA4 becomes the major lysine tRNA species (greater than 50%). This does not appear to reflect erythroid properties of the cell, since the lysine tRNA of the mouse reticulocyte contains very little lysine tRNA4. The non-dividing erythroid Friend cell, therefore, represents an exception to the finding that non-dividing cells usually have little or no lysine tRNA4 present.     

Microspectrophotometric studies of Romanowsky stained blood cells. IV. Maturation of myeloid and erythroid cell lines in bone marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the "true" classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.     

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G₁/G₀ arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours. Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G₁/G₀ arrest. Lastly, in XX germ cells we observed a down‐regulation of genes involved in both G₁‐ and G₂‐phases of the cell cycle consistent with their entry into meiosis. Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.     

Alterations in tRNA isoaccepting species during erythroid differentiation of the Friend leukemia cell.

The chromatographic profiles of isoaccepting tRNAs were analyzed at five time points during the 96 hr, dimethylsulfoxide induced, erythroid-like differentiation of Friend leukemia cells. Sixty-four isoaccepting species of tRNA for 16 amino acids were resolved by RPC-5 chromatography. The relative amounts of tRNAphe, tRNAile, and tRNAval species were maintained by the cells during differentiation; whereas the relative amounts of some of the isoacceptor tRNAs for the other 13 amino acids changed significantly. Fluctuations in amounts of isoacceptors occurred between 36 and 72 hr after addition of dimethysulfoxide, corresponding to globin mRNA appearance and hemoglobin synthesis, respectively. In most cases, thepredominant tRNA isoacceptors of uninduced cells were retained throughout differentiation. Notable exceptions were tRNA species for threonine, proline, and methionine. Some of the isoacceptors occurring in relatively smaller amounts were not expressed at all times. These changes possibly reflect the cell's functional adaptation of tRNA in differentiation for hemoglobin synthesis.     

Characterization of amino acid transport during erythroid cell differentiation

We have studied the changes in amino acid transport in fetal erythroid cells isolated from rat fetal liver at different gestation days. Our results show that System A transport as measured by the Na+-dependent uptake of 2-(methylamino)isobutyric acid (MeAIB) was conspicuous at day 13 but virtually disappeared between days 16 and 18. In contrast, the activity of System ASC measured by the Na+-dependent uptake of MeAIB-insensitive threonine uptake increased after day 14 and was optimal between days 16 and 18. This transport system regressed in activity with further maturation, but remained conspicuously saturable in the matured red blood cell. Interestingly, the newly discovered Na+-independent System asc (Vadgama, J. V., and Christensen, H.N. (1985) J. Biol. Chem. 260, 2912-2921), selective for the uptake of test substrates threonine, serine, and alanine, was present in these erythroid cells. Its activity increased during gestation days 16-18. System L transport was present simultaneously with the Na+-independent System asc. As we had previously demonstrated for the pigeon red blood cell, these two transport systems are kinetically independent as confirmed with inhibition studies and the special selectivity of System L to trans stimulation. Tryptophan uptake could be attributed predominantly to System L, as also observed for the nucleated pigeon red blood cells and certain other cells. Arginine showed its familiar Na+-independent mode of uptake as a cation throughout the interval of study. An exceptional Na+-dependent component of arginine uptake emerged after day 14, peaked at day 18, and then disappeared on further maturation of the erythroid cell.