Embryonic Hemoglobins Research Articles

New Approaches to the Regulation of Erythropoiesis and Hematoglobin Synthesis in β-Hemoglobinopathies: Studies with the Immunomodulatory Drug CC-4047.

Sickle cell disease (SCA) and β-thalassemia constitute worldwide public health problems and new therapies are needed. Inhibition of hemoglobin S (HbS) polymerization is a major target for therapeutic approaches in SCA. New experimental therapies including hydroxyurea (HU) have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve upon current treatment. Clinical trial results have demonstrated that lenalidomide (Revlimid®), recently approved by the FDA, reduces or even eliminates the need for red blood cell transfusions in some anemic myelodysplastic patients. We have examined whether CC-4047, another IMiDs® immunomodulatory drug currently under evaluation for the treatment of hematological cancers could regulate erythropoiesis and hemoglobin synthesis. For this purpose, we used an in vitro culture model to differentiate human erythroid progenitors from bone marrow or peripheral blood CD34+ cells. We demonstrate that CC-4047 is a potent inducer of HbF that synergizes with HU, the sole drug approved for SCA, during erythroid differentiation of CD34 progenitors isolated from healthy donors and patients with SCA. In addition CC-4047 modulates erythropoiesis, slowing erythroid maturation and increased proliferation of immature erythroid cells. Unlike other inducers of HbF such as HU, 5-aza-cytidine and butyrate, CC-4047 is not cytotoxic. The percentage of F-cells are increased by 2 to 3 fold with pan-cellular expression of HbF and an increase of Fetal/Adult hemoglobin ratio that are relevant for SCA. Gene expression profiling of erythroid differentiated cells shows that CC-4047 regulates specific erythroid transcription factors and genes that participate in hemoglobin synthesis, and genes involved in cell cycle and cellular differentiation. CC-4047 controls globin gene expression during erythroid differentiation by increasing the rate of γ-globin and ε-globin transcription and by inducing sustained expression of fetal and embryonic hemoglobin synthesis.Our results support the hypothesis that CC-4047, alone or in combination with certain current approved therapies comprising HU, can restore effective erythropoiesis and increase the ratio of fetal to adult hemoglobin. Moreover, CC-4047 has demonstrated anti-inflammatory and immunomodulatory effect in vivo that could help to limit the inflammatory state in sickle cell patients. In conclusion, administration of CC-4047 may represent an innovative new therapy for β-hemoglobinopathies.

Regulation of primitive hematopoiesis in zebrafish embryos by the death receptor gene

We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach. MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDR(mo), zChd(mo), and zRS(mo), respectively. Those co-injected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DR(mo) and zChd+RS(mo). zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChd(mo) embryos. At 48 hours postfertilization, zDR(mo) embryos showed increased Hb compared with WT or zRS(mo) embryos (2.36 x 10(-2) +/- 1.13 x 10(-3) vs 1.85 x 10(-2) +/- 5.60 x 10(-4) vs 1.79 x 10(-2) +/- 1.31 x 10(-3) U, p < 0.05). zChd+DR(mo) embryos also showed increased Hb compared with zChd(mo) or zChd+RS(mo) embryos (4.60 x 10(-2) +/- 2.79 x 10(-3) vs 3.17 x 10(-2) +/- 1.07 x 10(-3) vs 3.05 x 10(-2) +/- 1.25 x 10(-3) U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DR(mo) compared with zChd+RS(mo) embryos and caspase-3 activity in zDR(mo) vs zRS(mo) (0.525 +/- 0.094 vs 0.953 +/- 0.113 U, p < 0.05), and zChd+DR(mo) vs zChd+RS(mo) embryos (0.247 +/- 0.121 vs 1.180 +/- 0.082, p < 0.05). zChd+DR(mo) embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene. Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development.

Erythropoiesis and red cell function in vertebrate embryos

All vertebrate embryos produce a specific erythroid cell population--primitive erythrocytes--early in development. These cells are characterized by expression of the specific embryonic haemoglobins. Many aspects of primitive erythropoiesis and the physiological function of primitive red cells are still enigmatic. Nevertheless, recent years have seen intensive efforts to characterize in greater detail the molecular events underlying the initiation of erythropoiesis in vertebrate embryos. Several key genes have been identified that are necessary for primitive and the subsequent definitive erythropoiesis, which differs in several aspect from primitive erythropoiesis. This review gives in its first part a short overview dealing with comparative aspects of primitive and early definitive erythropoiesis in higher and lower vertebrates and in the second part we discuss the physiological function of primitive red cells based mainly on results from mammalian and avian embryos.

The changing cellular environments of hematopoiesis in human development in utero

The hematopoietic system is indispensable from the earliest stages of development and adapts to the rapidly changing anatomy of the embryo and fetus; this takes place in such different anatomic locations as the yolk sac blood island, hepatic parenchyme, aorta-gonads-mesonephros paravascular mesenchyme, and bone marrow primary logette. We herein summarize our investigation of these serial blood-forming events in the human embryo and fetus. The access to early stages of human development, availability of a large panoply of molecular markers for human blood cell lineages, and recent development of robust assays for the earliest human hematopoietic stem cells have allowed us to gain relatively clear insight into the developmental sequence that underlies the ontogeny of human blood cells. Conversely, the control exerted by these diverse cellular environments on the emergence of human hematopoietic cells remains elusive, as is the case in animal models. We nonetheless present preliminary attempts to decipher the structure and molecular characteristics of the distinct cellular "niches" in which blood cells are produced during human gestation.

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.

Erythroid-induced commitment of K562 cells results in clusters of differentially expressed genes enriched for specific transcription regulatory elements

Understanding regulation of fetal and embryonic hemoglobin expression is critical, since their expression decreases clinical severity in sickle cell disease and beta-thalassemia. K562 cells, a human erythroleukemia cell line, can differentiate along erythroid or megakaryocytic lineages and serve as a model for regulation of fetal/embryonic globin expression. We used microarray expression profiling to characterize transcriptomes from K562 cells treated for various times with hemin, an inducer of erythroid commitment. Approximately 5,000 genes were expressed irrespective of treatment. Comparative expression analysis (CEA) identified 899 genes as differentially expressed; analysis by the self-organizing map (SOM) algorithm clustered 425 genes into 8 distinct expression patterns, 322 of which were shared by both analyses. Differential expression of a subset of genes was validated by real-time RT-PCR. Analysis of 5'-flanking regions from differentially expressed genes by PAINT v3.0 software showed enrichment in specific transcription regulatory elements (TREs), some localizing to different expression clusters. This finding suggests coordinate regulation of cluster members by specific TREs. Finally, our findings provide new insights into rate-limiting steps in the appearance of heme-containing hemoglobin tetramers in these cells.

Erythroblasts derived in vitro from embryonic stem cells in the presence of erythropoietin do not express the TER-119 antigen

Objective In this study, we analyzed murine primitive erythropoiesis by coculturing Flk-1 + ES-derived cells with OP9 to find efficient culture conditions for erythroid cell induction. We utilized a nonserum culture system and EPO (erythropoietin) and found that this cytokine had unique properties. Materials and methods ES cells (E14.1) were first differentiated to Flk-1 + cells and then cocultured with OP9 stromal cells. BIT9500 was used as a serum replacement. The erythroid morphology, hemoglobin types, and TER-119 expression levels were analyzed. Results Primitive erythroid cells with embryonic hemoglobin were generated very efficiently when the serum-containing culture was converted to the nonserum system. In this serum-free culture, TER-119 + erythroblasts appeared first on day 2 and maturation proceeded until day 7. When EPO was added to this coculture, the number of induced floating cells increased twofold to threefold. Unexpectedly, the erythroid-specific antigen TER-119 expression of these cells was drastically reduced. Since reduced TER-119 expression is usually interpreted as maturation arrest, we examined the phenotypic features of the EPO-treated cells. We found, however, no evidence of maturation arrest in the aspects of morphology and hemoglobin content. EPO did not suppress TER-119 expression of erythroblasts derived from fetal liver or adult bone marrow. Conclusions Our results showed that EPO had the unusual property of inducing TER-119 − erythroblasts in ES-derived primitive erythropoiesis. It is likely that this effect is unique to primitive erythropoiesis.

Fetal and embryonic hemoglobins in erythroblasts from fetal blood and fetal cells enriched from maternal blood in pregnancies complicated by maternal diabetes mellitus

Objectives: To determine whether there is a delay or reversal in switch mechanisms from embryonic (ε and ζ) to fetal (γ) hemoglobins accompanying the erythroblastosis in fetuses of diabetic mothers, and whether the increased erythroblast count in the fetal blood is associated with an increase in fetomaternal cell trafficking.Materials and methods: Fetal and maternal blood samples were obtained from 11 fetuses and five pregnant women in pregnancies complicated by maternal diabetes mellitus. Blood samples were also taken as controls from 35 fetuses and 33 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with anti-CD71 antibody. Fluorescent antibodies were used to immunostain for gamma (γ), epsilon (ε) and zeta (ζ) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization for X and Y chromosomes was also carried out, to confirm the presence and proportion of enriched fetal cells from the maternal blood.Results: In both fetal and maternal blood the median percentages of erythroblasts positive for γ-globin, ε-globin and ζ-globin chains were significantly higher in fetuses of diabetic mothers compared to controls (fetus, γ-globin, 76 vs. 64%, p<0.0001; ε-globin, 4 vs. 0%, p<0.0001; ζ-globin, 4 vs. 0%; p<0.0001; mother, γ-globin, 14 vs. 1%, p<0.0005; ε-globin, 0.25 vs. 0%, p<0.0003; ζ-globin, 0.2 vs. 0%, p<0.0003). The median percentage of cells with Y signals in maternal blood was also higher in diabetic pregnancies compared to normal controls (7.5 vs. 1%, p<0.002).Conclusions: The findings suggest that the fetal erythroblastosis in diabetic pregnancies is accompanied by a delay in the switch from embryonic to fetal hemoglobin chains. In addition, it is associated with an increase in fetomaternal cell trafficking.

Measuring Assembly and Binding in Human Embryonic Hemoglobins

Publisher Summary Although the adult human hemoglobin is the most thoroughly studied protein and arguably one of the best understood, in terms of its function, the methods and models developed to investigate the detailed actions of this protein have not been widely applied to other hemoglobin systems. This chapter seeks to explore the methods and results obtained from the application of a range of techniques used to measure the assembly and binding processes that occur in the human embryonic hemoglobins. The human embryonic hemoglobins consist of a group of three hemoglobins synthesized in yolk sac-derived megaloblasts from approximately week 3 to week 16 of gestation. This chapter serves to illustrate the fact that, in order to assign the biological activity of a novel hemoglobin system, it is necessary to apply a wide range of methodologies. Only then, a proper understanding of this complex protein that shows both ligand and redox sensitivity within a multisubunit allosteric context can be obtained. Specifically, the methodologies described in this chapter have been applied successfully to the study of the three human embryonic hemoglobins obtained from recombinant yeast expression systems. Each of these hemoglobin expression systems consists of Saccharomyces cerevisiae cells containing plasmids based on the naturally occurring yeast 2μ plasmid. The plasmids have been engineered to contain each of the appropriate globin genes, linked in tandem to artificial galactose promoters. Nevertheless, the approaches described here are, in principle, applicable to any hemoglobin system.

Developmental regulation of the allosteric effector pattern of avian embryonic hemoglobin

Developmental regulation of the allosteric effector pattern of avian embryonic hemoglobin

Sensitivity and Specificity of the Identification of Fetal Cells in Maternal Blood by Combined Staining with Antibodies against Beta-, Gamma- and Epsilon-Globin Chains

Objectives: Antibodies against fetal and embryonic hemoglobins may identify fetal cells in maternal blood. Both γ- and Ε-globins are used as fetal cell markers. γ-Globin is not fetus specific. So far Ε-globin has been claimed to be fetus specific. In this communication, we compare the specificity of anti-Ε- and anti-γ-globin staining when combined with staining for β-globin. Methods: We applied single and double color immunofluorescent staining techniques in combination with XY chromosome hybridization. The blood sample was taken after chorion villus biopsy at 11 weeks of gestation from a woman carrying a male fetus. Results: By γ-globin staining alone, 21 fetal and 2 maternal nucleated red blood cells (NRBCs) were identified. Only 1 of the 2 maternally derived NRBCs expressed β-globin. By Ε-globin staining, 92 additional fetal NRBCs were identified. Conclusions: Ε-Globin antibody and combined Ε- and γ-globin antibody staining of a blood sample from a pregnant woman at 11 gestational weeks showed higher sensitivity but lower specificity for the fetal origin of erythroblasts with combined compared with separate staining. The final decision of the origin of cells was made by gender determination by FISH. Out of 2 γ-positive maternal cells 1 was β-globin antibody positive, 1 was β-globin negative, indicating that 100% specificity for fetal origin could not be obtained by combining all 3 hemoglobin types. Although only 1 blood sample was tested and only 2 γ-positive maternal NRBCs were identified, the result indicates that β-hemoglobin does not discriminate completely between γ-positive NRBCs of fetal and maternal origin.

Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human β-globin locus in erythropoietic cells

Reactivation of fetal hemoglobin genes has been proposed as a potential therapeutic procedure in patients with beta-thalassemia, sickle cell disease, or other beta-hemoglobinopathies. In vitro model systems based on small plasmid globin gene constructs have previously been used in human and mouse erythroleukemic cell lines to study the molecular mechanisms regulating the expression of the fetal human globin genes and their reactivation by a variety of pharmacologic agents. These studies have led to great insights in globin gene regulation and the identification of a number of potential inducers of fetal hemoglobin. In this study we describe the development of enhanced green fluorescence protein (EGFP) reporter systems based on bacterial artificial chromosomes (EBACs) to monitor the activity of the epsilon-, (G)gamma-, (A)gamma-, delta-, and beta-globin genes in the beta-globin locus. Additionally, we demonstrate that transfection of erythroleukemia cells with our EBACs is greatly enhanced by expression of EBNA1, which also facilitates episomal maintenance of our constructs in human cells. Our studies in human cells have shown physiologically relevant differences in the expression of each of the globin genes and also demonstrate that hemin is a potent inducer of EGFP expression from EGFP-modified epsilon-, (G)gamma-, and (A)gamma-globin constructs. In contrast, the EGFP-modified delta- and beta-globin constructs consistently produced much lower levels of EGFP expression on hemin induction, mirroring the in vivo ontogeny. The EGFP-modified beta-globin eukaryotic BAC (EBAC) vector system can thus be used in erythroleukemia cells to evaluate induction of the epsilon- and gamma-globin genes from the intact human beta-globin locus.

A human embryonic hemoglobin inhibits Hb S polymerization in vitro and restores a normal phenotype to mouse models of sickle cell disease

The principle that developmentally silenced globin genes can be reactivated in adults with defects in beta-globin gene expression has been well established both in vitro and in vivo. In practice, levels of developmental stage-discordant fetal gamma globin that can be achieved by using currently approved therapies are generally insufficient to fully resolve typical clincopathological features of sickle cell disease. The therapeutic potential of another developmentally silenced globin--embryonic epsilon globin--has been difficult to evaluate in the absence of a convenient expression system or an appropriate experimental model. The current work analyzes the antisickling properties of an epsilon -globin-containing heterotetramer (Hb Gower-2) both in vitro as well as in vivo in a well-established mouse model of sickle cell anemia. These animals, expressing 100% human Hb S, display a chronic hemolytic anemia with compensatory marrow and extramedullary erythropoiesis, abundant circulating sickled erythrocytes, and chronic tissue damage evidenced by parallel histopathological and functional deficits. By comparison, related mice that coexpress Hb S as well as Hb Gower-2 exhibit normal physiological, morphological, histological, and functional attributes. Subsequent in vitro analyses substantiate results from whole-animal studies, indicating that the polymerization of deoxygenated Hb S can be significantly slowed by relatively small quantities of Hb Gower-2. Together, the in vivo and in vitro analyses suggest that reactivation of epsilon-globin gene expression would be therapeutically beneficial to adults with sickle phenotypes, and provide a rationale for detailed investigations into the molecular basis for its developmental silencing.

Electron transfer between cytochrome b5 and some oxidised haemoglobins: the role of ionic strength

We have compared experimental measurements and Brownian dynamic calculations for the reduction of oxidised adult human haemoglobin by reduced bovine cytochrome b5 over a range of ionic strengths. Our calculations suggest that the presence of molecular electrostatic fields have a significant role to play in the formation of the electron transfer complexes. These results predict that electron transfer occurs within an ensemble of similarly weakly docked complexes, the formation of which is strongly ionic strength dependent. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes as a function of ionic strength. The outcome of this theoretical study is compared with experimental rate measurements. A comparison of the results obtained from adult and embryonic haemoglobins, at a fixed ionic strength, indicates a significant difference in the characteristics of complex formation. These data emphasise the role played by electrostatic interactions in this important physiological reaction.

Detection of fetal NRBCs in maternal blood of pregnant carriers of beta-thalassemia using anti-gamma and anti-epsilon monoclonal antibodies.

Fetal nucleated red blood cells (NRBCs) entering maternal circulation during pregnancy constitute a potential source of material for safe and reliable noninvasive prenatal diagnosis. The increased prevalence of beta-thalassemia mutations in countries like Greece may create a problem, making it difficult to distinguish between NRBCs of fetal or maternal origin. Use of Ab against embryonic hemoglobin epsilon may increase specificity for fetal NRBC detection. In the present study, Ab against embryonic hemoglobin epsilon was used in the first and second trimesters of pregnancy in order to determine if specificity for fetal NRBC detection could be increased.

A modeling study of the interaction and electron transfer between cytochrome b5 and some oxidized haemoglobins.

Using Brownian motion simulations we have studied the formation of docked complexes of reduced cytochrome b5 and oxidized haemoglobin. Our results indicate that the presence of molecular electrostatic fields has a significant role to play in the formation of these complexes. In contrast to previous modeling studies on this system, we clearly identify electron transfer within an ensemble of similarly docked complexes rather than the formation of a single complex. Docking involves a number of acidic residues surrounding the exposed haem edge of cytochrome b5 and a set of basic residues surrounding the exposed haem edge of the globins. Although amino acids from the partner globin proteins are involved to a small extent in the binding of some of the complexes, the reactivity of any particular globin is essentially independent of the nature of its partner globin chain within the haemoglobin molecule. Comparison of results from adult and embryonic haemoglobins indicates a significant difference in complex formation. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes. These data provide a theoretical insight into the important process of re-reduction of oxidized haemoglobins as well as explaining the general inability to produce crystalline forms of many docked electron transfer complexes.

The ferric form of the three human embryonic hemoglobins and their reactions with azide ions

The ferric forms of the three human embryonic hemoglobins exhibit pH titration’s with p K a values in the range 8.25–8.6, which correlate with the reduced proteins affinity for oxygen. Azide ions bind to each ferric protein in a process consisting of two separate binding steps. The equilibrium constants for this process are in the 10–30 μM range and can be assigned to the individual chain within the proteins as reactivity appears independent of the nature of the partner chain. The kinetics of the azide binding reactions occur on the second time scale at mM ligand concentrations and also indicate the presence of two distinct processes that have been assigned to each of the two chains within the proteins. Non of these processes indicate any evidence of heme–heme interactions within the ferric form of the proteins. These findings are discussed in comparison with previously reported properties of adult ferric hemoglobin.

Genetic analysis of fetal nucleated red blood cells from CVS washings

Chorionic villus sampling (CVS) is an established invasive prenatal diagnostic method for the detection of fetal chromosome aberrations. In 1-2% the karyotype result of CVS is inconclusive and follow-up confirmation will be required. To avoid another invasive procedure we examined fetal nucleated red blood cells (NRBCs) from CVS washings for genetic analysis. We analysed the washings of 20 chorionic villi samples of male fetuses. Fetal NRBCs were immunostained by an antibody against embryonic haemoglobin (HbE). FISH was performed with probes specific for the X and Y chromosome and the nucleus was counterstained with DAPI. Cells positive for the antibody, as well as for DAPI, were collected and stored by a semi-automated microscope. An operator reviewed those cells for their FISH signals. In 19 out of 20 CVS washings we found nucleated cells positive for HbE together with XY FISH signals. In none of the washings HbE positive cells with two X signals were found. Our results indicate that anti-HbE is a very specific antibody for identifying fetal NRBCs. NRBCs from CVS washings can be used as an additional fetal tissue for first trimester prenatal diagnosis.

Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP Signaling of Embryonic Red Blood Cells: Role of cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin

We have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, β-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and β-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.

Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP Signaling of Embryonic Red Blood Cells: Role of cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin

AbstractWe have previously shown that the cAMP signaling pathway controls major aspects of embryonic red blood cell (RBC) function in avian embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and Dragon et al, Am J Physiol 271:R982, 1996) that are important for adaptation of the RBC gas transport properties to the progressive hypercapnia and hypoxia of later stages of avian embryonic development. Data about the ontogeny of receptor-mediated cAMP signaling are lacking. We have analyzed the response of primitive and definitive chick embryo RBC harvested from day 3 to 18 of development towards forskolin, β-adrenergic, and A2 receptor agonists. The results show a strong response of immature definitive and primitive RBC to adenosine A2 and β-adrenergic receptor agonists, which is drastically reduced in the last stage of development, coincident with the appearance of mature, transcriptionally inactive RBC. Modulation of cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence on cAMP accumulation in definitive RBC. Under physiological conditions, PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC). Inhibition of sGC with the specific inhibitor ODQ decreases receptor-mediated stimulation of cAMP production; this effect is reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric oxide (NO), but we found no evidence for production of NO by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO in an oxygen-linked manner that may activate guanylyl cyclase.