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.     

Ontogenetic changes of circulating erythroid cells and haemoglobin components in Esox lucius

Using light microscopy the morphology, the mitotic index and levels of erythroid cell types were detected from 48 h pike Esox lucius embryos before hatching to adult specimens. At the same developmental stages, the haemoglobins and globin chains expressed were electrophoretically characterized. The erythroid cells of the primitive generation were the most abundant from 48 h before hatching until 15–20 days after hatching, then their number decreased and only rare cells remained in the 3 month‐old juvenile specimens. These cells divided and differentiated in the blood and were substituted by the definitive erythrocyte series. As in other vertebrates, the immature cells of the two generations differed in morphological properties and in the synthetized haemoglobin. The circulating erythroid cells of the definitive population cell lineage were, at all differentiation stages, smaller than those of the primitive generation. The definitive erythrocytes appeared in blood smears of 7 days post‐hatching larvae, they increased rapidly and at 20 days they represented the predominant red blood cell population in the circulation of young pike. Electrophoretic analysis of haemolysates obtained from different developmental stages indicated the presence of distinct embryonic, larval and adult haemoglobins. The embryonic haemoglobins differed from those of the older larva and juvenile specimens and were detectable within the first week of post‐hatching development when only primitive erythrocytes were present in the blood.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

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.     

Erythrocyte AMP-deaminase: an investigation of the increase in activity during chick maturation.

1. AMP-deaminase activity in erythrocytes increases gradually during chick (Gallus domesticus) maturation, reaching the adult level of enzymatic activity at about 16 weeks after hatching. 2. Adenosine deaminase activity increases approximately two-fold during this period. 3. Substrate specificity and immunoinhibition studies indicate that erythrocytes from adult chickens and newly-hatched chicks contain the same AMP-deaminase isozyme. 4. Comparison of temporal changes in RBC AMP-deaminase with those previously described for this enzyme in muscle and brain suggests that the level of this enzyme is regulated differently in these tissues.     

Hybrid isozymes of pyruvate kinase appear during avian cardiac development

Pyruvate kinase isozyme patterns in the ventricle of developing chicks shift gradually from one dominated by type K at ten days of embryonic development to the adult pattern, which is dominated by type M. Hybrid isozymes are apparent throughout development and are most prominent from two days before hatching until at least 14 days after hatching. These hybrid isozymes indicate simultaneous synthesis of the two subunit types in the same cells.The complex isozyme patterns of the chick heart probably limit the usefulness of simple kinetic analyses on tissue extracts for determing isozymic compositions during development.     

The Wavy Erythropoiesis of Developing Chick Embryos. Isolation of Each Wave by a Differential Lysis and Identification of the Constituent Erythroid Types

Erythroid carbonic anhydrase (CA) activity of chick embryos from the third day of incubation to the egg hatching has been determined. Five minor activity peaks with maxima at 3, 6, 9, 15 and 17 days of development and a major one with maximum at 19 days have been found. The correlation between the peak distribution and the timing of release into the blood stream of waves of newly produced erythroid cells has been demonstrated on the basis of the following observations: 1) a linear correlation exists between red cell maturation and increase of CA activity; 2) chick red cells undergo lysis in the "Ørskov" medium when their CA activity exceeds a threshold value (23±3 Units/10⁹ red cells); and 3) the lysis kinetics of red cells in the Ørskov medium is proportional to their CA content. We have thus been able to distinguish the immature erythroid forms from the mature ones on the basis of their behaviour in the Ørskov medium. In the blood of developing chick embryos, we have found waves of newly produced red cells at about 2, 4, 7, 10, 16 and 18 days of development.
The same experimental criteria allowed us to detect the waves of red cell production in the erythropoietic organs. One wave has been detected in the blood islands at about 2 days; four waves in the yolk sac at about 5, 6, 11 and 15 days; two waves in the spleen at about 18 and 20 days; two waves in the bone marrow at about 19 days of incubation and 1 day after hatching.
Primitive erythroid cells are produced in the first two waves: that of blood islands at 2 days and that of yolk sac at 5 days. Definitive red cells are produced in the other waves with the exception of the second wave of spleen and of the second wave of bone marrow, which are constituted by red cells of adult type.     

Hemoglobin function in the developing goat and sheep

Comparative studies of the blood of newly born goats and sheep have indicated a number of mechanisms which are responsible for a decreasing affinity for molecular oxygen in these developing animals during the first 40 to 60 days after birth.
The concentration of 2,3-DPG in the red cells of young goats increases four to sixfold during the first 3 to 4 days of life, and this increase is associated with a marked decrease in the cellular pH; 2,3-DPG does not bind to hemoglobins of goats and the decreased affinity for oxygen of goat blood at this period is apparently due to the lowered pH produced by the large increase of intracellular anions. Similar changes occur in young lambs.
After 15 to 20 days the changes of the dissociation curve are related more to structural differences between adult and fetal hemoglobins; cellular pH moves closer to the values of adult red cells. In goats of this age the predominate hemoglobins are those with β chains and these have dissociation curves shifted further to the right than other adult hemoglobins.
In young lambs Hb-C is found only in association with Hb-A but the amount present seldom exceeds 5 to 10%. The oxygen affinities of sheep Hb-A and Hb-C are identical but higher than that of sheep Hb-B.     

Intraerythrocytic organic phosphates of fetal and adult seaperch (Embiotoca lateralis): Their role in maternal-fetal oxygen transport

Summary The viviparous seaperch,Embiotoca lateralis, has unique fetal and adult hemoglobins. Stripped fetal hemoglobin has a higher oxygen affinity than stripped adult hemoglobin at pH 6.5–7.1. The oxygen affinities of both adult and fetal hemoglobins are lowered allosterically by ATP at pH 7.1. Both fetal and adult seaperch erythrocytes include approximately 82% ATP and 18% GTP of the total nucleotide triphosphates (NTP) with a trace of AMP. No 2,3-diphosphoglycerate or inositol polyphosphate was detected. Mid- and late-gestation erythrocytes contain less NTP/mole hemoglobin tetramer than do adult cells. The effective NTP concentration in adult cells is higher than that of the fetal erythrocytes even when the intracellular concentration of Mg²⁺, which complexes with NTP, is accounted for. The difference in adult and fetal intraerythrocytic NTP concentration should enhance transfer of oxygen from maternal to fetal blood. Thus, the teleostEmbiotoca lateralis may employ a dual mechanism in maternal-fetal oxygen transfer. A difference in fetal and maternal hemoglobin structure and oxygen affinities is enhanced by a difference in their respective intraerythrocytic organic phosphate concentrations.     

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.     

Some observations on the primitive and definitive erythrocytes of the developing chick

Summary The cytological changes in the primitive and definitive erythrocytes of the incubating chick have been followed. Observations have been made on the nucleoli, vital granules, mitochondria,Golgi apparatus, reticulum ofSinigaglia and the reticulation patterns of the basophilic substance. The cells of the primitive and definitive lines are ordinarily readily distinguished from one another. Data are included on the rate of disappearance of the primitive cells from the circulation. They may persist as long as two weeks after hatching. Giant primitive erythrocytes are common during the first week of incubation. The cells have one, two three or four nuclei. The nuclearplasma relationship is maintained somewhere near a constant. These atypical cells are due to aberrations in mitosis. Data on the percentage of mitosis in both types of erythrocytes are also included. The initial activity of the spleen and bone-marrow is reflected in the blood stream. There is a distinct rise in the proportion of young definitive erythrocytes. An attempt is made to correlate the findings ofHall (1934) on the changing affinity of the hemoglobin for oxygen with the changing blood picture. The primitive line does not persist long enough to account for the phenomenon. It is suggested, however, that the hemoglobin of the erythrocytes produced by the yolk sac may differ from that of the cells produced by the spleen and bone-marrow. With Plates I–III.     

Fetal hemoglobin distributions among adult baboon and macaque species

Heritable variation in fetal hemoglobin (Hb F) in erythrocytes of the adult human has been shown to occur at more than one genetic locus. Heritable variation has also been reported in adult baboons. Nonhuman primates thus may serve as useful models for understanding how Hb F is regulated in the human. In the study reported here we identified Hb F in hemolysates from 27 of 32 rhesus macaques, from 32 of 32 baboons, and from none of 35 cynomoglus macaques. Hb F as a percentage of total hemoglobin occurred as a normally distributed variable among rhesus macaques but among baboons the distribution was both skewed and kurtotic. Such difference could be either a consequence of nonrandom sampling of the gene pool in one of the species, or a consequence of species evolution. A technique of single cell hemoglobin electrophoresis was applied to erythrocytes from three adult pig-tailed macaques. This demonstrated that erythrocytes which contain Hb F (F-cells) also customarily contain Hb A and that the proportions of these two hemoglobins varies substantially among the F-cells, as we previously noted for human F-cells. We conclude that the macaques could serve as useful models for understanding Hb F regulation in the human.     

Morphogenesis of intestinal villi. I. Scanning electron microscopy of the duodenal epithelium of the developing chick embryo

Development of villi in the duodenum of the chick was studied in stages ranging from 11 days of incubation to one week after hatching. Formation of definitive villi is preceded by development of a set of previllous ridges that run lengthwise along the duodenum. The first set of 16 previllous ridges (Set I) is complete by about 13 days of incubation; all ridges in the set are fairly uniform and proceed through their subsequent development in synchrony. Previllous ridges in Set I fold into a highly regular zigzag pattern between 14 and 16 days of incubation. Definitive villi develop from Set I ridges beginning at about 17 days when populations of distinct cells appear on the crests of the ridges between angles in the zigzag folds. Cells in these populations lack the rounded appearance of cells seen in earlier stages; their apical surfaces are densely covered with microvilli. A second set of villi (Set II) develops at about 16 days of incubation when about 16 rows of tongue-like flaps erupt between the previllous ridges of Set I. At hatching, Set II villi are still smaller than villi of Set I; this distinction disappears by about the fourth day after hatching. The significance of the morphological changes in epithelial cells is discussed in terms of several hypotheses bearing on the mechanisms of villus formation.     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

Hemoglobin from the antarctic fish Notothenia coriiceps neglecta. Amino acid sequence of the beta chain

1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.     

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.     

The primary structure of the hemoglobins of a southern hemisphere lamprey (Mordacia mordax, Cyclostomata)

Mordacia mordax is a southern hemisphere lamprey belonging to Mordaciidae, a primitive family of Cyclostomata. Adult erythrocytes contain three monomeric hemoglobins which can be easily separated by cellulose acetate electrophoresis and isolated by ion-exchange chromatography. The N-terminal regions, and the tryptic peptides from each chain were submitted to automated Edman degradation; the alignment of the fragments was obtained by homology with the other Petromyzonoidea hemoglobins hitherto sequenced. Our results confirm the phylogenic distance between lampreys and hag-fish hemoglobins. As was observed for Petromyzon marinus species, two hemoglobins of Mordacia mordax are very close, as they differ only at 7 positions.     

Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.