Formation of transient polykaryons by fusion of erythrocytes of different developmental programs

We report here two methods of fusing erythroid cells from bullfrogs (Rana catesbeiana), using polyethylene glycol or calcium phosphate, which yield masses of polykaryons in which the cytoplasms and nuclei of tadpole and adult frog erythroid cells are intermixed. The masses of fused cells carry out protein synthesis in culture, including the assembly of normal hemoglobin (Hb) tetramers. In these polykaryons there is reactivation of the expression of specific Hbs that have previously been "turned off" in vivo as the result of either a developmental Hb switch or normal cellular differentiation and RBC maturation. For example, the products of fusion of tadpole erythroblasts with adult frog mature RBCs synthesize adult Hb, whereas neither cell population alone does so. Recent experiments have taken advantage of a Hb-expression polymorphism that we discovered in this species, such that some tadpoles have greatly reduced expression of one of the larval Hbs (Hb Td-4). Fusion of erythroblasts from such tadpoles with RBC from frogs that had expressed Hb Td-4 when they were tadpoles produces polykaryons that synthesize Hb Td-4, indicating there is a trans factor that stimulates Td-4 expression. Heterospecific erythroid cell polykaryons can be constructed in an analogous manner, facilitating the study of trans-acting factors that regulate specific globin gene expression during development.     

Use of somatic cell fusion to reprogram globin genes.

The developmental phenomenon of hemoglobin switching occurs in all classes of vertebrates and is due to differential regulation of divergent globin genes which are arranged in chromosomally clustered families. By fusing erythroid cells of different developmental programs, it has been shown that erythroid nuclei of either early or late developmental stage can be reprogrammed, i.e. the gene switch can be reversed in adult erythroid nuclei and/or prematurely-induced in fetal/embryonic erythroid nuclei. Experiments with heterokaryons demonstrate that the reprogramming is due to trans-acting factors that are developmental-stage-specific. These results suggest the feasibility of using fusisome-carried sets of nuclear factors to reprogram somatic cells.     

Analysis of Xenopus laevis globins during development and erythroid cell maturation and the construction of recombinant plasmids containing sequences derived from adult globin mRNA

Developmental changes in the globin polypeptide composition of Xenopus laevis erythrocytes were examined by acid-urea polyacrylamide gel electrophoresis and a major switch from tadpole to adult globins was detected after metamorphic climax. The coding capacity of mRNA derived from mature adult erythroid cells was examined by cell-free translation and the products were shown to coelectrophorese with the globins of the adult erythroid cells. We describe the molecular cloning of sequences present in this mRNA and the characterisation of clones derived from one of the major globins and one clone derived from a minor adult globin mRNA.     

Expression of adult and tadpole specific globin genes from Xenopus laevis in transgenic mice.

Transgenic mice were generated which carried the adult alpha and beta-globin genes and the major tadpole specific beta-globin gene of Xenopus laevis. The adult specific alpha and beta genes were found to express in erythroid tissues in adult mice, while the major tadpole specific beta gene (beta T1) was expressed in blood from 12.5 day embryos. The pattern of expression of the beta T1 gene during mouse development was consistent with its being regulated as an embryonic globin gene in the mouse. This observation suggests that some of the factors mediating globin switching have been conserved during the evolution of modern amphibia and mammals and raises interesting questions concerning the evolution of vertebrate globin gene switching.     

Rapid reprogramming of globin gene expression in transient heterokaryons

Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell. Therefore, erythroid cells contain transacting regulatory factors that are capable of activating the expression of globin genes in a stage- and tissue-specific manner. These observations also indicate that globin genes are not irreversibly repressed in differentiated cells and that their expression can be rapidly reprogrammed in the presence of the appropriate regulatory factors.     

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.     

Human globin knock-in mice complete fetal-to-adult hemoglobin switching in postnatal development

Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

Distribution of developmentally regulated hemoglobins in embryonic erythroid populations.

Both cellular and molecular mechanisms regulate the expression of globin genes during development and differentiation.When a change occurs in the type of hemoglobin synthesized, it may be the result of a substitution of erythroid stem cell lineages or may arise through a modulation of globin gene expression after cells become committed to erythroid differentiation. We have investigated the relationship between the early to late embryonic hemoglobin switch and the primary to definitive erythrocyte change in chick embryos. Using double-label fluorescent antibody technique, we find the simultaneous presence of early and late hemoglobins in single erythrocytes of the definitive cell type. Synthesis of early embryonic hemoglobin is not restricted to the primary cell lineage. This evidence is most compatible with the hypothesis that erythroid cells become committed to the synthesis of specific globins after they have become committed to hemoglobin synthesis in general.     

Two Transitions of Haemoglobin Expression in Xenopus: from Embryonic to Larval and from Larval to Adult

Abstract. Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins ( X. borealis ) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Human erythroid progenitors from adult bone marrow and cord blood in optimized liquid culture systems respectively maintained adult and neonatal characteristics of globin gene expression

In vitro suspension culture procedures for erythroid progenitor cells make it possible for us to obtain large cultures of erythrocyte populations for the investigation of globin gene switching. In this study we aimed to establish optimized culture systems for neonatal and adult erythroblasts and to explore the globin expression patterns in these culture systems. To culture CD34+ cells purified from human umbilical cord blood (CB) and adult bone marrow (BM), we respectively replaced the fetal bovine serum (FBS) with human cord serum and human adult serum. These CD34+ cells were then induced to erythroid differentiation. All the globin mRNA (including alpha-, zeta-, beta-, gamma-and epsilon-globin), the hemoglobin (Hb)-producing erythroid cells and the cellular distribution of fetal hemoglobin (Hb F) were identified during the culture process. The results showed that the globin expression pattern during erythroid differentiation in our culture systems closely recapitulated neonatal and adult patterns of globin expression in vivo, suggesting that our specially optimized culture systems not only overcame the higher Hb F levels in the BM-derived CD34+ culture in FBS-containing medium but also eliminated the disadvantages of low cell proliferation rate and low globin mRNA levels in serum-free medium.     

Two transitions of haemoglobin expression in Xenopus: from embryonic to larval and from larval to adult

Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins (X. borealis) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.     

Effect of AGM and fetal liver-derived stromal cell lines on globin expression in adult baboon (P. anubis) bone marrow-derived erythroid progenitors

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction.     

Characterization of embryonic globin genes of the zebrafish

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.