Ontogeny of hemoglobins: Evidence for hemoglobin M

A new autosomal codominant hemoglobin mutation alters hemoglobin M of the primitive red cell line and hemoglobin D found in definitive cells. That Hb M and Hb D are altered by the same gene mutation supports the idea that Hb M shares a polypeptide chain with Hb D. It is concluded that in the switch from primitive hemoglobins to those of the definitive type, there are at least two α chains conserved; α^A of Hb E in Hb A and α^D of Hb M in Hb D.     

The hemoglobins of the developing chicken embryos. Fractionation and globin composition of the individual component of total erythrocytes and of a single erythrocyte type.

The hemoglobins of the chicken embryo at several stages of development have been isolated in pure form by column chromatography and their relative amounts and globin compositions determined. The analyses on separated primitive and definitive erythrocytes show that the first contain four hemoglobins different from the adult ones. The two major ones at four days, decrease gradually and are no longer detectable from 15 days on. The two minor ones increase up to 6-7 days, then decrease but are still present at hatching. The definitive embryonic erythrocytes contain two hemoglobins identical to the adult ones but their ratios change gradually during development and approach that of the adult hemoglobins at hatching.     

Evidences that hemoglobin switch in the chick embryo depends on erythroid cell line substitution

Chemical identifications of various hemoglobin types were performed on unfractionated erythroid cells derived from chicken embryos at 5 and 7 days of development and on purified primitive and definitive cells. Proteins were pulse-labelled in primitive erythroid cells at various times of culture to identify those actually synthesized. The data show that primitive cells contain and synthesize only embryonic hemoglobins at all stages of maturation and definitive cells contain adult and minor embryonic hemoglobins, but no major embryonic hemoglobins, not even in trace amounts. These results support a model for hemoglobin switch in the chicken embryo based on cell line substitution.     

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D_Davis (Hb D_D), an autosomal codominant in chickens, the α^D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D_D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M_D), on high-resolution isoelectric focusing gels. Comparison of $\text{P1}\text{P2}$ ratios in the four phenotypes indicated that homozygous Hb D_D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D_D) than did red cells from corresponding adult birds. Heterozygous Hb D_D embroys and adults had both Hb D and Hb D_D. Hb D_D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D_D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D_D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D_D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.     

Erythropoiesis in the developing chick embryo

The types of erythroid cells of chick embryos developing in ovo have been correlated with the hemoglobins of the embryos. Prior to 5 days, when primitive cells constitute the only erythroid cells, two hemoglobins can be resolved by polyacrylamide gel electrophoresis. The two adult hemoglobins and a minor hemoglobin found only in embryos and young chicks first appear simultaneously with initiation of definitive erythropoiesis.     

ERYTHROPOIETIC CELL CULTURES FROM CHICK EMBRYOS

Erythropoietic cell cultures from very early chick blastoderms survive for several days They show four to seven doublings of the erythroid cells and the appropriate morphological changes from proerythroblasts to mature erythrocytes Cell cycle times are the same as in ovo for the first day of culture, but slow down thereafter The hemoglobins of both the primitive and the definitive red cell series are produced. 5-Bromodeoxyuridine added to the cultures inhibits differentiation and hemoglobin synthesis, though not cell division, but quite soon the cells cease being sensitive The effect of the drug can be reversed by the addition of thymidine.     

Two different populations of primitive erythroid cells in the ehick embryo.

Antibodies prepared against the two hemoglobins of the adult chick cross react with the two minor hemoglobins and do not react with the two major hemoglobins isolated from lysates of primitive erythroid cells of the 4-day-old embryo. The different immunological reactivities of the two primitive hemoglobin pairs have permitted us to discriminate, in smears of primitive erythroid cells, two populations on the basis of their hemoglobin contents.     

Embryonic and adult chick haemoglobins: Their reaction with p-chloromercury benzoate

At least one of the primitive embryonic hemoglobins (Hbs) dissociates into monomers after treatment with p-chloromercury benzoate. The other primitive embryonic Hbs as well as the definitive embryonic and the adult Hbs are probably dissociated, by this reagent, into dimers. A globin from a primitive embryonic Hb component, which can be completely dissociated by this agent, may be isolated by gel filtration on Sephadex.     

Hemoglobin synthesis in isolated erythroid colonies from the chick embryo.

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived.Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [³H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P′, and P″) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed.     

Red cell ferritin and iron storage during chick embryonic development

Ferritin, the iron storage protein, is at least 10 times as abundant in the circulating primitive red cells of the chick embryo as in the circulating definitive red cells of adult roosters. The decline in the ferritin content of the circulating red cells in the embryo corresponded to the replacement of primitive red cells by definitive red cells, monitored by the disappearance of primitive and embryonic hemoglobins. Iron concentrations in the yolk, the major nutrient storage site, changed little during the period when ferritin was lost from the circulating red cells. The storage of iron in the ferritin of the primitive red cells and the preferential loss of the stored red cell iron that was observed in chickens also occur in mice and bullfrogs, which suggests a special role for red cell ferritin in developing animals.     

Dimethyl sulfoxide (DMSO) stimulates heme synthesis in quail embryonic yolk sac cells

Dissociated yolk sac cells from quail embryos at the definitive primitive streak stage were reaggregated, using a gyratory shaker with or without dimethyl sulfoxide (DMSO). After 24 h of incubation in the shaker, the aggregates were transferred onto a whole egg agar medium containing ⁵⁹Fe, and incubation was continued for an additional 48 h. It was clearly shown that DMSO-treated yolk sac aggregates showed a higher incorporation of radioactive iron into heme than the control culture without DMSO. The maximal stimulatory effect was observed at around 0.75% DMSO.     

Major Events in the Evolution of the Oxygen Carriers

SYNOPSIS. Major events in the history of the blood O² carriersin multicellular animals include: 1) The origin of the red bloodcell hemoglobins, which are both the most primitive and themost advanced O² carriers. At the molecular level, they arebelieved to have arisen only once; at the animal level, thereis no cogent reason to postulate more than two origins. 2) Theorigin of the hemerythrins, which occur at about the same phylogeneticlevel as the primitive red blood cell hemoglobins. They maynot have been selected in higher animals because of their temperaturesensitivity. The origins of 3) the molluscan hemocyanins and4) the arthropod hemocyanins. These two events occurred independently,though for similar reasons. Both kinds of hemocyanins offeredphysiological advantages over the primitive hemoglobins thatwere important in the context of the more advanced molluscanand arthropod cardiovascular systems. 5) The origins of extracellularheme proteins, which arose independently many tunes, and probablyfor as many different reasons. 6) The loss of urea sensitivity,and the acquisition of organic Po₄ sensitivity and additionalcooperativity and pH dependence by the red blood cell hemoglobins,which occurred well after the origin of the vertebrates.     

Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

During embryonic development, hematopoiesis occurs through primitive and definitive waves, giving rise to distinct blood lineages. Hematopoietic stem cells (HSCs) emerge from hemogenic endothelial (HE) cells, through endothelial‐to‐hematopoietic transition (EHT). In the adult, HSC quiescence, maintenance, and differentiation are closely linked to changes in metabolism. However, metabolic processes underlying the emergence of HSCs from HE cells remain unclear. Here, we show that the emergence of blood is regulated by multiple metabolic pathways that induce or modulate the differentiation toward specific hematopoietic lineages during human EHT. In both in vitro and in vivo settings, steering pyruvate use toward glycolysis or OXPHOS differentially skews the hematopoietic output of HE cells toward either an erythroid fate with primitive phenotype, or a definitive lymphoid fate, respectively. We demonstrate that glycolysis‐mediated differentiation of HE toward primitive erythroid hematopoiesis is dependent on the epigenetic regulator LSD1. In contrast, OXPHOS‐mediated differentiation of HE toward definitive hematopoiesis is dependent on cholesterol metabolism. Our findings reveal that during EHT, metabolism is a major regulator of primitive versus definitive hematopoietic differentiation.     

Differentiation of chick blood island erythroblasts into erythrocytes and stability in long-term culture

Clusters of 20-70 erythroblasts from blood islands of early chick blastoderm were cultured in serum-free chemically defined medium for a 3-month period. The erythroblast cluster produces erythroid cells and hemoglobins characteristic of the primitive and definitive erythroid cell lines. It seems there is a progenitor erythroid cell(s) in the erythroblast cluster which starts and/or continues maturing along various pathways of hemopoietic differentiation under simple culture conditions. The erythroid character of these cells is stable during the 3-month culture period.     

Studies on Sporopollenin Biosynthesis in Tulipa Anthers

Investigations were carried out to clarify sporopollenin biosynthesis. Tracer experiments were focussed on the incorporation of specifically labeled ¹⁴C-phenylalanine into sporopollenin. In addition, the incorporation of further ¹⁴C-labeled substances, such as glucose, acetate, malonic acid, mevalonate and tyrosine, was investigated. The sporopollenin fraction was isolated and purified by a gentle method including extractions by different solvents, incubations with hydrolyzing enzymes and fractionated saponifications. During the purification procedure the whereabouts of the initially applied radioactivity was followed. After each step the remaining as well as the released radioactivity was determined. Saponification of samples labeled after application of phenylalanine yielded p-coumaric acid and p-coumaric acid methyl ester as labeled products. In comparison with the other substances applied, the highest incorporation rates were obtained with phenylalanine, regardless of the position of labeling. After degradation of the sporopollenin sample labeled with ring-¹⁴C-phenylalanine, p-hydroxybenzoic acid was detected as the main labeled product. These results unequivocally show that an integral incorporation of the aromatic ring system occurred. Tracer experiments were carried out at different stages of development. Their results show that, although the incorporation rates of ¹⁴C-phenylalanine into sporopollenin differ, the substantial incorporation of this substance is not bound to defined stages of development.     

Membrane Channel Connexin 32 Maintains Lin−/c-kit+ Hematopoietic Progenitor Cell Compartment: Analysis of the Cell Cycle

Membrane channel connexin (Cx) forms gap junctions that are implicated in the homeostatic regulation of multicellular systems; thus, hematopoietic cells were assumed not to express Cxs. However, hematopoietic progenitors organize a multicellular system during the primitive stage; thus, the aim of the present study was to determine whether Cx32, a member of the Cx family, may function during the primitive steady-state hematopoiesis in the bone marrow (BM). First, the numbers of mononuclear cells in the peripheral blood and various hematopoietic progenitor compartments in the BM decreased in Cx32-knockout (KO) mice. Second, on the contrary, the number of primitive hematopoietic progenitor cells, specifically the Lin⁻/c-kit⁺/Scal⁺ fraction, the KSL progenitor cell compartment, also increased in Cx32-KO mice. Third, expression of Cx32 was detected in Lin⁻/c-kit⁺ hematopoietic progenitor cells of wild-type mice (0.27% in the BM), whereas it was not detected in unfractionated wild-type BM cells. Furthermore, cell-cycle analysis of the fractionated KSL compartment from Cx32-KO BM showed a higher ratio in the G₂/M fraction. Taken together, all these results imply that Cx32 is expressed solely in the primitive stem cell compartment, which maintains the stemness of the cells, i.e., being quiescent and noncycling; and once Cx32 is knocked out, these progenitor cells are expected to enter the cell cycle, followed by proliferation and differentiation for maintaining the number of peripheral blood cells.     

Bef medaka mutant reveals the essential role of c-myb in both primitive and definitive hematopoiesis

Vertebrate hematopoiesis is characterized by two evolutionally conserved phases of development, i.e., primitive hematopoiesis, which is a transient phenomenon in the early embryo, and definitive hematopoiesis, which takes place in the later stages. Beni fuji (bef) was originally isolated as a medaka mutant that has an apparently reduced number of erythrocytes in its peripheral blood. Positional cloning revealed that the bef mutant has a nonsense mutation in the c-myb gene. Previous studies have shown that c-myb is essential for definitive hematopoiesis, and c-myb is now widely used as a marker gene for the onset of definitive hematopoiesis. To analyze the phenotypes of the bef mutant, we performed whole-mount in situ hybridization with gene markers of hematopoietic cells. The bef embryos showed decreased expression of α-globin and l-plastin, and a complete loss of mpo1 and rag1 expression, suggesting that the bef embryos had defects not only in erythrocytes but also in other myeloid cells, which indicates that their definitive hematopoiesis was aberrant. Interestingly, we observed a diminution in the number of primitive erythrocytes and a delay in the emergence of primitive macrophages in the bef embryos. These results suggest that c-myb also functions in the primitive hematopoiesis, potentially demonstrating a link between primitive and definitive hematopoiesis.     

Variable Subunit Contact and Cooperativity of Hemoglobins

Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human, Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact. Received: 12 January 2001 / Accepted: 3 April 2001