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.     

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 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.     

Line-restricted hemoglobin synthesis in chick embryonic erythrocytes

The presence of embryonic hemoglobin in early definitive erythrocytes was checked by indirect immunofluorescence assay, using specific antibodies raised against embryonic Hb P. As positive control we used anti-Hb A which reacted with the alpha A chain shared by the minor embryonic Hb E and the adult Hb A. The assay was performed using blood smears from embryos between 6 and 15 days of incubation and yolk sac sections from embryos between 4 and 6 days. Hb P was never detected in the definitive line in circulating erythrocytes or in maturing erythroblasts still sequestered in the blood islands of the yolk sac. The expression of the 'specific' embryonic genes is thus restricted to the primitive line (as the 'specific' adult beta gene is restricted to the definitive line), and the hemoglobin switch is the result of the progressive substitution of the primitive line by the definitive one.     

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.     

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.     

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.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Hemoglobins, haptoglobins, and transferrins in indigenous populations of Kenya

Hemoglobin, haptoglobin, and transferrin phenotypes were determined by means of starch-gel electrophoresis for a sample of 226 Kenya hospital patients. Allele frequencies were: Hb_β^S=0.081; Hp¹=0.057; Tf^D=0.038. Hemoglobin S was the only aberrant hemoglobin found in this sample. Transferrins C and D were the only transferrins found. Hemoglobin and transferrin phenotypes were also determined for a sample of 201 newborn Kenya infants. One of these infants had hemoglobins F, S, and C, eight had hemoglobins A, F, and S, and the remainder had hemoglobins A and F. Transferrins C, B, and D were found in this sample. Allele frequencies were: Tf^B=0.008; Tf^D=0.019.     

Separation of primitive and definitive erythroid cells of the chick embryo

The primitive and definitive erythroid cells of the chick embryo are separated preparatively by means of velocity sedimentation at unit gravity in BSA gradients. Analyses of the hemoglobins contained by the fractionated cells show a segregation of different hemoglobins between the primitive and definitive cells. Studies of the incorporation of [³H]leucine show that the fractionated cells are normal with respect to their protein synthetic activities and that their relative rates of incorporation are markedly different.     

Embryonic hemoglobins of different animal species

Summary The existence of embryonic hemoglobins is demonstrated in sheep-, calf and pig embryos. The occurrence and disappearance of these hemoglobins is quantitatively determined by cellulose acetate gel electrophoresis; hemoglobins as well as the globin chains, dissociated in 8 molar urea were quantitated. Sedimentation and diffusion experiments in the analytical ultracentrifuge revealed a S₂₀ of 4.3 and a D₂₀ of 6.6 for the examined hemoglobins. Therefore it is concluded that all hemoglobins occurring at different stages of embryonic and fetal development consist of 4 polypeptide chains with a total molecular weight of 66,000. The subsequent formation of the different polypeptide chains during ontogenesis is shown: At first only -chains are formed as demonstrated by the existence of Hb Gower I, consisting of four identical -chains. Subsequently the -chain appears, which leads to Hb Gower 2 (₂₂). The third polypeptide chain formed during the ontogenesis the -chain results finally in the appearance of HbF.In addition the existence of a HbF pig is demonstrated by the fingerprint technique.     

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.     

Glycosylated minor components of human fetal hemoglobin. Chromatographic separation, identification, and functional characterization

Human adult red cell lysate contains glycosylated minor hemoglobins AIa1, AIa2, AIb, and AIc. Similar minor hemoglobins, designated FIa1, FIa2, Fib, and FIc, have been separated by a Biorex 70 column chromatographic procedure from red cell lysates of newborn children and from an adult homozygote for hereditary persistence of fetal Hb. The minor Hb components were characterized by analyzing for carbohydrate and phosphate contents, by oxygen equilibrium analysis, and by comparing the chromatographic elution profiles of naturally occurring and in vitro synthesized minor components. The results indicate that Hb FIa1, Hb FIa2, and Hb FIc have been formed by the modification of gamma chains of Hb F by reacting with fructose-1,6-P2, glucose-6-P, and glucose, respectively. Hb FIb is a glycoprotein; the mechanism of its formation is unclear. Hb FIa1 and Hb FIa2 had significantly lower oxygen affinities and n values than the other minor components and the major Hb F0. Moreover, 2,3-diphosphoglycerate did not influence the oxygenation of the minor or the major fetal Hb components. Incubations of Hb F with [14C]hexoses and subsequent chromatographic separation of hemoglobins and their globin chains confirm the previous findings that the binding of carbohydrate to Hb involves both specific and nonspecific reactions.     

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.     

Identification of hemoglobin types contained in single chicken erythrocytes by fluorescent antibody technique

It has been suggested that the switch in hemoglobin (Hb) types (from embryonic to adult) during chicken embryonic development is associated with the substitution of one erythroid cell line (“primitive”) for another (“definitive”). For the detection of two Hb types inside single erythroid cells, rabbit antibodies specific for embryonic and adult Hbs were prepared. Rabbit antibody specific for embryonic Hb cross-reacted only with embryonic major Hb components, while antibody specific for adult Hb did solely with adult minor Hb component. The antibodies were conjugated with fluorescein isothiocyanate. The conjugated antibodies were used for the fluorescent staining of blood smears of developing chicken embryos at different ages. Direct fluorescent antibody technique demonstrated that the major components of embryonic Hb and the minor component of adult Hb were not present within the same erythrocyte during chicken ontogenesis. It strongly suggested that embryonic-type Hb and adult-type Hb do not coexist within the same cell.     

Contribution of ventral and dorsal mesoderm to primitive and definitive erythropoiesis in the salamander Hynobius retardatus

Previously, we found that the conversion of hemoglobins (Hbs) from the larval to the adult type occurred within a single erythroid cell population in a salamander, Hynobius retardatus ("Hb switching" model), whereas the transition involves replacement of red-blood-cell (RBC) populations ("RBC replacement" model) in many amphibians (M. Yamaguchi, H. Takahashi, and M. Wakahara, 2000, Dev. Gene Evol. 210, 180-189). To further characterize the Hb transition, developmental changes in the erythropoietic sites have been intensively analyzed using larval- and adult-specific globin antibodies and globin and GATA-3 RNA probes. Cells of the ventral blood island (VBI) and the dorsolateral plate (DLP) in embryos differentiate in situ to erythroid cells that contain larval globin mRNA, suggesting that both the VBI and the DLP contribute to "primitive" erythropoiesis. In contrast, the expression pattern of the GATA-3 gene suggests that cells of the DLP may contribute to "definitive" hematopoiesis. In order to determine whether it is possible to define a definitive erythropoiesis in H. retardatus or not, further experiments were done: (1) when metamorphosing larvae were treated with phenylhydrazine to induce anemia and then bled at the postmetamorphic stage after recovery from the anemia, a precocious Hb transition was observed in these animals; (2) an RBC population expressing only adult Hb was confirmed by subtracting the number of RBCs expressing larval Hb from the total number of RBCs during metamorphosis. All these results support the existence of a definitive erythroid cell population that contributes only adult RBCs in this species.     

The maternal and zygotic roles of the gene Polycomb in embryonic determination in Drosophila melanogaster

The mutation Polycomb (Pc) is known to cause a variety of intersegmental transformations in homozygous and heterozygous individuals of Drosophila melanogaster; Pc⁺ is thought to act as a negative regulator of genes of the bithorax complex. The function of this gene in the maternal germ line has been assessed by examining the variation in expression of these homoeotic phenotypes in individuals derived from a maternal germ line with a single or no dose of the Pc⁺ allele. Mosaic individuals with a homozygous or heterozygous Pc germ line were produced by transplantation of pole cells, the embryonic precursors of the germ line. By employing an X-linked dominant female-sterile mutation, the identification of mosaic females and the study of progeny derived from the exogenous germ line were greatly simplified; the advantages of this system for the transplantation of pole cells for such analyses are described. In general, all thoracic and abdominal segments of homozygous Pc embryos differentiate characteristics of the eighth, most posterior, abdominal segment. The extent and uniformity of this transformation as well as other manifestations of the homozygous Pc genotype are described and shown to be correlated with the maternal germ line genotype; homozygous Pc embryos derived from a homozygous Pc maternal germ line show greater expression of these phenotypes than do genetically identical embryos derived from a heterozygous Pc maternal germ line. The expression of some homoeotic phenotypes typical of heterozygous Pc adults shows only a slight correlation with the maternal genotype, while no homoeotic transformations are clearly evident in heterozygous larvae of either origin. Thus, the maternal effect of Pc is rescuable. The results suggest that the Pc⁺ gene is active in the maternal germ line but that the absence of the maternally derived Pc⁺ product can be largely compensated by the introduction of a wild-type allele upon fertilization; this rescue indicates that the maternal activity of Pc⁺ plays no major role in the normal process of embryonic segmental determination. The normal fertility of males and females with a homozygous Pc germ line and of their progeny suggests that Pc⁺ plays no role in the determination or development of the germ line in either the maternal or zygotic genome.     

Metabolism of 25-hydroxy-vitamin D3 by primary cultures of chick kidney cells

The primary culture of kidney cells from vitamin D deficient chicks is described. After four days in culture the cells reach confluency and retain their ability to metabolize 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃. Addition of one unit of bovine parathyroid hormone to the culture medium for 48 hours prior to assay had no effect on the cells' ability to produce 1,25-dihydroxy vitamin D₃, whereas after 24 hours in the presence of 5×10^?8$\text{M}$ 1,25-dihydroxyvitamin D₃ the cells produced not this metabolite, but 24,25-dihydroxyvitamin D₃. This cell culture system will allow the investigation of the regulation of renal 25-hydroxyvitamin D₃ metabolism under controlled $\text{in vitro}$ conditions.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Inhibition of DNA synthesis in embryonic mouse retina as a result of gene interaction.

It has been shown by autoradiography using ³H-thymidine that 11-day mouse embryos doubly homozygous for the autosomal recessive genes fidget (gene symbol fi) and ocular retardation (or), have three to five times fewer labelled nuclei in their retina anlages as do normal (genotype +/+ +/+) embryos singly homozygous for fidget (+/+ $\text{fi}\text{fi}$) or ocular retardation (+/+ $\text{or}\text{or}$). In 11-day embryos of +/+ +/+, +/+ $\text{fi}\text{fi}$ and +/+ $\text{or}\text{or}$ genotypes the labelled nuclei are localized mainly in the inner zone of the retina anlage. However, in double homozygotes the indices of labelled nuclei were not significantly different in the inner and outer zones of the retina anlage. The retina anlage of 12-day double homozygote, $\text{fi}\text{fi}\text{or}\text{or}$, has practically no nuclei synthesizing DNA. Consequently, the mutant genes fi and or which prolong the G₁ period of the cell cycle in single homozygotes, act synergetically to stop DNA synthesis in the retina anlage cells of 12-day $\text{fi}\text{fi}\text{or}\text{or}$ embryos.