Embryonic Globin Chains Research Articles

Evidence for embryonic haemoglobins from Sparus aurata under normal and hypoxic conditions.

Teleost haemoglobins vary in polymorphisms and primary structure, although display similar functional properties. Key amino acids for Root effect (a reduction in oxygen-carrying capacity and loss of cooperativity with declining pH) are conserved throughout fish evolution. For the first time, we cloned and characterised Sparus aurata L. embryonic globin chains (eα1, eα2, eβ). We also studied haemoglobins (eHbI, eHbII) behaviour in normal and low-oxygen conditions. Several amino acids in fry globins are different in chemical type (e.g. polar → non-polar and vice versa), compared to adult globins. His55α1, crucial for Root effect, is substituted by Ala in fry, presumably enhancing oxygen capture, transport and reducing the dependence of Root effect from pH. Phylogenetic trees demonstrate that eα1 globin diversified more recently than eα2; moreover, eα1, eα2 and eβ globins evolved earlier than adult α and β globins. In low-oxygen conditions, fry haemoglobins display the same behaviour of the adult haemoglobins (probably, embryonic and adult-type I Hbs display a higher oxygen affinity than type II Hbs, operating through a rapid cycle of heme-Fe auto-oxidation/reduction). Therefore, based on our results and on the comparison with adult haemoglobins, we hypothesise that embryonic haemoglobins have evolved to better adapt fry to variable habitats. We studied Sparus aurata for its economical relevance in Mediterranean aquaculture. The information we provide can help understand Sparus aurata behaviour in the wild and in rearing conditions. Further studies with functional assays will deepen the knowledge on the molecular mechanisms of fry haemoglobin physiology.

Sox6 is necessary for efficient erythropoiesis in adult mice under physiological and anemia-induced stress conditions.

BackgroundDefinitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions.Methodology/Principal Findings Sox6fl/flErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6fl/flCaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6fl/flErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling.Conclusions/SignificanceThis study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1.

Humanized Mouse Model of Cooley's Anemia

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

Embryonic Hemoglobins Are Expressed in Definitive Cells

Human embryonic zeta and epsilon globin chains are synthesized in yolk sac-derived primitive erythroid cells, and decrease rapidly during definitive erythropoiesis. Examination of zeta and epsilon globin expression at the cellular level using dual-color immunofluorescence staining with specific monoclonal antibodies showed that embryonic globin proteins are present in definitive erythroid cells. More than half of fetal erythrocytes were positive for zeta and approximately 5% for epsilon globin. Approximately one third of newborn red blood cells were zeta-positive and less than 1% epsilon-positive. Adult erythrocytes did not have embryonic globins. Erythroblasts that developed in liquid cultures also contained embryonic globin in amounts which declined with ontogenic age, and the proportion of positive cells in vitro was less than in the comparable erythrocytes that developed in vivo. Thus, embryonic globin chains are synthesized in definitive erythroid cells and decrease with ontogeny. Modulation of embryonic globin gene expression is not solely due to a switch from primitive to definitive erythropoiesis.

Translational control of globin chain ontogeny in hamster yolk sac erythroid cells.

Prior research has demonstrated that globin ontogeny of hamster proceeds nearly to completion during the several days that yolk sac erythroid cells (YSEC) circulate in the embryo; synthesis of embryonic globin chains gives way to synthesis of adult globin chains in these primitive cells. In the present study, we translated total cell RNA extracted from YSEC on days 9-13 of gestation in wheat germ cell-free extract, expecting to observe the same progressive rise that occurs in vivo in rates of translation of alpha- and beta-globin mRNA during ontogeny. The opposite occurred; translation rates of both globins decreased sharply. This disparity between synthesis of alpha- and beta-globins in vivo and in vitro suggested an element of control of translation attributable to the YSEC cytoplasm. We therefore assayed the effect of RNA-free clarified YSEC cytoplasm on cell-free translation of YSEC RNA. A repression of translation was detected of alpha- and beta-globin mRNA (not of embryonic globin mRNA), exercised strongly by cytoplasm from YSEC early in ontogeny (gestational day 9), and weakening as ontogeny progressed. The same effect was noted on alpha- and beta-globin mRNA of adult hamster and of rabbit. Heat treatment of cytoplasm abolished the greater part of the translation regulation, suggesting that the active agent is protein. Further characterization of this translational regulator included: (a) it binds to globin poly(A) mRNA but not to poly(A), (b) it was not detected in cell lysate of adult hamster brain, lung, or erythrocytes, and (c) it did not inhibit translation of adult hamster brain and liver RNA. We conclude that hamster globin ontogeny is substantially modulated by this translational regulation of alpha- and beta-globin expression.

Primitive erythropoiesis of mouse teratocarcinoma stem cells PCC3/A/1 in serum-free medium

Mouse teratocarcinoma stem cells PCC3/A/1 differentiated into various types of cells, such as red cells, when they were grown in serum-free medium containing transferrin and bovine serum albumin on a KCF cell feeder layer. These red cells were stained well with 2,7-diaminofluorene (DAF), and therefore were erythroid cells. They were nucleated and contained embryonic globin chains, immunologically identified with antiembryonic hemoglobin antisera after acid urea Triton X-100 polyacrylamide gel electrophoresis (UT-PAGE). The addition of erythropoietin to the culture medium enhanced the production of both embryonic and adult globin chains. The addition of interleukin-3 also enhanced the production of embryonic globin chains, but not the production of adult globin chains. These results indicated that primitive erythropoiesis of PCC3/A/1 teratocarcinoma cells did not require exogenous addition of any hematopoietic factor such as erythropoietin or interleukin-3. This culture system will be a new model system for investigating the factors regulating the primitive erythropoiesis in yolk sac blood islands.

Evidence for the synthesis of embryonic globin chains in adult erythroid progenitor cells

Embryonic globin chains were found to be synthesized in vitro by the BFU-E colonies derived from adult sickle cell anemia (SS) patients, their heterozygous relatives as well as a few normal controls. In the absence of sufficient material for conducting direct structural analyses of these peptides, they were confirmed by evaluating the co-migration of the epsilon and zeta-chains with the corresponding structurally characterized globin chains obtained from K562 cell lysates on a reversed phase high performance liquid chromatogram. The presence of zeta-chain was also confirmed using an immunologic procedure. Furthermore, significant 35S-methionine incorporation peak was observed corresponding to the zeta-chain synthesized by the BFU-E-derived colonies implying an active synthesis of this embryonic globin chain in BFU-E cells obtained from hemopoietically adult persons.

An embryonic pattern of expression of a human fetal globin gene in transgenic mice

During the evolution of the beta-globin family gene in vertebrates, different globin genes acquired different developmental patterns of expression. In mammals, specific 'embryonic' beta-like globins are synthesized in the earliest erythroid cells, which differentiate in the yolk sac of the embryo. In most mammals the embryonic globin chains are replaced by 'adult' beta-globins in fetal and adult erythrocytes, which arise in the liver and bone marrow, respectively. However, in simian primates (including humans), a distinct 'fetal' type of beta-like globin chain predominates in fetal erythroid cells. Based on the pattern of DNA sequence homologies between different mammalian species, these fetal globin genes, G gamma and A gamma, are thought to have descended from an ancestral gene, 'proto-gamma', which was embryonic in its pattern of expression. In the mouse, as well as in most other mammalian species, the descendants of the proto-gamma gene continue to function as embryonic genes. To investigate the evolutionary changes that led to the 'fetal recruitment' of the gamma-globin genes in primates, we have introduced the cloned human G gamma-globin gene into the mouse germ line. We report here that the human G gamma gene reverts to an embryonic pattern of expression in the developing mouse. This observation suggests that during evolution a shift occurred in the timing of expression of a trans-acting signal controlling the proto-gamma gene.

Influence of maternal diabetes in rats on hemoglobin synthesis and uridine uptake by fetal liver cells.

The effect of streptozocin (STZ)-induced maternal diabetes in rats on fetal erythropoiesis was studied in short-term cultures of fetal liver cells at the time of switch from embryonic to adult hemoglobins. Liver erythroid cell functions were monitored by measuring the incorporation of [3H]-uridine in trichloroacetic acid (TCA)-soluble and -insoluble cell fractions and of [3H]-leucine in hemoglobin chains. Fetal liver cells of diabetic rats showed a higher incorporation of [3H]-uridine compared with controls when the cells were obtained from 14-day-old fetuses. However, there were no significant differences in the uptake of uridine when cells were obtained from 16-day-old fetuses. In parallel cell cultures, incorporation of [3H]-leucine into adult and embryonic globin chains was studied by separation of the globin chains by high-performance liquid chromatography (HPLC). The overall globin chain synthesis was higher in the fetuses of diabetic mothers compared with controls on day 14 of gestation. Erythropoietin had similar effects on the stimulation of globin chains in the two groups of fetuses. However, in the fetuses of diabetic mothers, erythropoietin had a specific stimulatory effect on embryonic-type globins that was significantly higher in the fetuses of diabetic mothers compared with controls. Differences between fetuses of control and diabetic mothers completely disappeared at 16 days of gestation. It is concluded that maternal diabetes has an effect on the cells synthesizing embryonic hemoglobins on day 14 of gestation, but by the time the switch from embryonic to adult-type hemoglobins is complete, these differences are abolished.

Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x

Cytoplasmic poly(A) +mRNA from 12-day mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the α-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic α-like globin chain. The insert sequence is 316 nucleotides long, contains the codons corresponding to amino acid residues 43–141, and extends into the 3' untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known α globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the α- and β-globin gene complexes.

High performance liquid chromatography of rat globin chains of fetal liver during the switch from embryonic to adult hemoglobin.

A reversed-phase high performance liquid chromatography (HPLC) method for the direct separation of globin chains from cell lysates of peripheral blood and fetal liver from rat fetuses is described. Partial amino acid analysis of the globins eluted from the HPLC columns as well as comparison with the known elution positions of the adult globin chains in carboxymethyl cellulose chromatography indicated that alpha-chains are eluted first, followed by adult beta chains. The last chains to be eluted are considered as embryonic globins because of their absence in adult rats and their rapid disappearance from the liver after the 14th day of gestation. Liver erythroid cells isolated from rat fetuses on day 14 of gestation mainly synthesized alpha-chains and embryonic globin chains, whereas cells prepared from 16-day old fetuses synthesized almost exclusively alpha-chains and adult beta chains. When the fetal rat liver cells were cultured for 20h with erythropoietin there was a significant stimulation in the synthesis of alpha-globins and adult beta chains but not on the synthesis of embryonic globin chains. It is concluded that HPLC can be useful for the study of rat globin chain synthesis during fetal development, because it separates the globin chains in the three groups of globins, namely alpha, beta and embryonic chains which are important in the switch occurring in the liver.

Preferential induction of fetal versus embryonic globin chains in human leukemic cell lines

By use of a newly developed technique combining affinity chromatography of hemoglobin on haptoglobin-Sepharose and IEF of globin chains, we analyzed the globin synthetic pattern of human K562 cells in both the basal state and after addition of several potential inducers. Hemin only was found effective: its addition at 50 μM results in a quantitative increase of globin chain synthesis (from 0.3 to 1% up to 5%) and a qualitative “switch” with a striking increase of α and a decrease of ϵ and ξ chains (relative to the prevailing γ chains). This system, in which hemin induces changes that mimic to some extent the normal embryonic-fetal switch, might therefore provide a cellular model for investigating molecular mechanisms of globin gene regulation. In addition similar results were obtained with a different human myeloid leukemia cell line, the KG1, thus raising the possibility that the expression of embryonic globin genes in malignant cells might not be simply the consequence of abnormal gene expression but rather reflect a possibly physiological differentiation phenomenon.

Sequence differences between mouse embryonic y- and adult beta-globin messenger ribonucleic acids.

Poly(A)-containing RNA was isolated from yolk sac cells of BDF1 hybrid mice on the 12th day of gestation. In vitro translation of the poly(A)-containing RNA by a messenger RNA (mRNA) dependent reticulocyte lysate system revealed that this RNA contained a high concentration of mRNAs from two embryonic globin chain subunits, y-1 and y-2. Cross hybridization of complementary DNA (cDNA) to the embryonic globin mRNAs with adult reticulocyte globin mRNAs and with cloned cDNAs for adult alpha- and beta-globin chain mRNAs showed that the sequence of embryonic globin y-chain mRNA differed from that of adult beta-chain mRNA.

Embryonic-fetal erythroid characteristics of a human leukemic cell line.

We have studied a number of cell surface, enzyme, and protein markers in the human leukemic K562 cell line. We have confirmed previous observations that these cells accumulate human embryonic hemoglobins after exposure to hemin. In addition, our results demonstrated that these cells possess in the "ininduced" state i surface antigen, lactate dehydrogenase isoenzymes characteristic of embryonic or fetal erythroid cells, fetal and embryonic globin chains, and globin mRNAs. The levels if i antigen, embryonic globin chains, and embryonic globin mRNA increased substantially after exposure of the cells to hemin in suspension culture. In contrast, K562 cells lacked several surface, enzymatic, and functional properties typical of granulocytes, lymphocytes, monocytes, or adult erythroblasts, including HLA surface antigens, surface immunoglobulins, sheep erythrocyte rosetting, phagocytosis, terminal deoxynucleotidyl transferase, carbonic anhydrase, ABO and Rh blood groups, and adult hemoglobins. The K562 cell line therefore exhibits phenotypic properties of embryonic erythroid progenitor cells and a quantitative increase in the expression of some of these properties can be achieved by exposure of the cells to hemin.

Linkage of genes for adult alpha-globin and embryonic alpha-like globin chains.

In alpha-thalassemia, the genetic locus for the alpha chains of adult hemoglobin is not expressed. We have examined the hemoglobins of a number of individual mouse embryos heterozygous for a particular alpha-thalassemia (Hbath-J) and find no decrease in the proportion of hemoglobins containing the alpha chain as compared to the hemoglobin containing the alpha-like embryonic globin chain. This result suggests that the locus for this embryonic alpha-like chain is inactivated or deleted in these embryos as well. Because a single mutational event inactivated adult and embryonic loci, we conclude that they are probably closely linked to one another on the same chromosome. We also present evidence that an unusual hemoglobin in the blood of these embryos is composed only of an embryonic beta-like chain, and is thus analogous to the hemoglobin H (beta 4 tetramer) of adult alpha-thalassemics.

Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

Isolation and characterization of the messenger RNAs for mouse embryonic globin chains.

Messenger RNAs for mouse embryonic globins were purified from yolk sac erythroid cells by oligodeoxythymidilate-cellulose chromatography and sucrose density centrifugation. Full-sized complementary DNA copies of embryonic globin mRNAs were synthesized. Acrylamide gel electrophoresis of these RNAs indicate an average molecular weight of 220 000, including a polyadenylated sequence of about 35 residues, as determined by hybridization to [3H]polyuridylate. The wheat-germ translation products of mRNAs have the size and the ionic characteristics of the four embryonic globin chains alpha, x, y and z. Hybridization kinetics in vast RNA excess were performed and compared to standard r0t curves of adult globin messengers, demonstrating a total base sequence complexity of about 880 000 daltons, that is four different RNA sequences of 220 000 molecular weight. The titration of embryonic globin cDNAs with increasing amounts of their complementary RNA templates indicates that the embryonic globin messengers were isolated at a high degree of purity.