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Comparative development hematology: Animal models to study human fetal erythropoiesis
THERIOGENOLOGY COMPARATIVE DEVELOPMENTAL HEMATOLOGY: ANIMAL MODELS TO STUDY HUMAN FETAL ERYTHROPOIESIS
(4
Hyram Kitchen Department of Environmental Practice College of Veterinary Medicine University of Tennessee Knoxville, Tennessee 37919
ABSTRACT
:
Developmental Hematology
A comparison of the ontogeny of hemoglobin is made in human, mouse and sheep. The differences and similarities are discussed to demonstrate the use of these animals as potential models to study regulation and transition of erythropoiesis during development. A general comparison of developmental erythropoiesis is made among various mananalswith a discussion of how various mammals facilitate oxygen transport across the placenta by differing mechanisms.
(a)
This work was supported in part by NIH Grant 7 ROl HD 10066-01 and the Agricultural Experiment Station, University of Tennessee.
The author wishes to acknowledge the suggestions and help of Drs. Robert L. Michel and Robert D. Lange, and the assistance of Mrs. Janice M. Bryan with the preparation of this manuscript.
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AliSW
CO~A~TI~ D~~P~~~~~~~Y: MODELS TO STUDY HUMAN FETAL ERYTRROPOIESIS
Introduction Many fascinating phenomena axe encountered in biology and serve to supply the investigator with an endless resource for the exploration of new knowledge and biological understanding, Of the many examples of such phenomena, one from the area of comparative hematology which is of extreme importance is the transition of embryonic and/or fetal hemoglobin to adult hemoglobin. This is of interest not only to the biologist, but to the medical scientist as well. The sequential emergence and disappearance of a variety of hemoglobin types associated with developxaent is not identical for all mammals. The purpose of this communication is to compare the regulation and control of erythropoiesis and hemoglobin types in various species. This comparison will'serve to point out how various molecular mechanisms can accomplish the same physiological objective. In this example, the facilitation of oxygen transport across the placenta to supply oxygen of the proper tension and saturation to the developing organism is a major objective. This report will be a review of work by others and a report of recent work performed in this laboratory. Functional erythrocytes are produced from early precursors. Each species has it's own distinctive type of hemoglobin. Although functionally identical, the hemoglobins of the various species differ with respect to their amino acid sequence (1). Not to be confused with the developmental cell series occurring during normal adult erythropoiesis are morphologically and biochemically distinctive red blood cells which can be related to the development of the organism. The temporal relationship of erythrocytes can be identified as embryonic cells, containing embryonic hemoglobin followed by transition to morphologically distinctive red blood cells. In other species, there are distinct embryonic cells with accompanying embryonic hemoglobin with a transition to fetal cells having a unique hemoglobin type and other red blood cell components followed by a further transition to the adult erythrocytes with the corresponding hemoglobin type.
Hemoglobin Synthesis and the Erythrocyte There is limited information regarding the control of hemoglobin synthesis. Even less is known regarding the relationship of the morphology of erythrocyte precursors to biochemical events. An excellent review of cellular differentiation, with the erythroid cell system of the mouse as a model, was published by Marks and Rifkind (2). The patterns of hemoglobins emerging at various stages of development differ in the mouse, sheep, and man; however, in each case the relationship of biochemical events and cell morphology is still to be made.
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There are numerous well defined intermediate stages of differentiation between the undifferentiated precursor (stem cell) and the mature erythrocyte. Whether all or some cells produce small quantities of all the possible hemoglobin types in a species is not known. It is possible that cells from the same sites of production have different capacities to synthesize hemoglobin types. The proportion of hemoglobin types in a cell population may be different. The distribution of hemoglobin type in individual erythrocytes may be related to the mean age of erythropoietin-sensitive cells (3). Whether a cell is committed to only one hemoglobin type is not known. There is an orderly succession of hemoglobin types within morphologically or biochemically different red blood cells which in general correspond to the embryonic, fetal and adult phases of life in most mammalian species (3, 4). See Figure 1. The ontogeny of human hemoglobin is well documented. The work done thus far indicates that the ontogeny of sheep hemoglobin is very similar to the human (5). The sheep and human embryonic, fetal and adult hemoglobins have structurally common alpha chains within each species. Variations that account for the hemoglobin type associated with the stages of development occur in the structurally distinct non-alpha chains (4). In contrast to the polypeptide chain type accounting for the various hemoglobins in the mouse,erythropoiesis occurs progressively in the mesoblastic yolk sac, hepatic and myeloid bone marrow tissue. Cellular and biochemical aspects of erythropoiesis have been studied in detail in the mouse, chicken and tadpole. Work and articles by Rifkind, et al . (2) and Ingram (6) give impetus to two major theories of divergence between primitive and definitive progenitor cells: 1) yolk sac primitive cells and definitive cell lines diverge from a common precursor cell, and 2) yolk sac primitive cells are seeds of the definitive cells. To define the use of animals as models to study the ontogeny and transition of hemoglobin, we must compare all three; man, sheep and mouse.
Comparison of Ontogeny of Hemoglobins in the Mouse, Sheep and Human A comparison of mouse, sheep and human developmental erythropoiesis will serve to identify what is currently known about the most often used models to study the ontogeny and transition of human hemoglobin. (See Figure 1) Smbryonic hemoglobin is associated with nucleated, ovalshaped cells produced in yolk sac for all species thus far studied (4). In man, embryonic hemoglobins Gower 1 and Gower 2 are characterized by structurally unique epsilon polypeptide chains; Gower 1 having the shorthand formula of EL,and Gower 2 cxp ~2 chains. In contrast to man and sheep, the mouse (the species in which the most detailed information is known) has no fetal hemoglobin (7, 8). Multiple polypeptide chains, both alpha and non-alpha, account for structural differences in the embryonic hemoglobin in the mouse (9). In the mouse, embryonic
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red blood cells differentiate as a relatively homogenous population. All embryonic hemoglobin synthesis occurs in cells which are synthesizing DNA and dividing. These cells are thought only to produce embryonic hemoglobin (7). The alpha chain is structurally identical to adult alpha chains within the respective species in most mammals. In the C57Bl-65 mouse, the embryonic hemoglobins are composed of x and y polypeptide chains for EI OLand y for EII and o and z for EIII (7). Adult hemoglobin has not been detected in the embryonic cells of these species. Erythropoiesis has been extensively characterized during embryonic life, after yolk sac erythropoiesis, and during the beginning of fetal life in the mouse. There is an alteration in the erythroid population in terms of the proportion of cells morphologically distinct during the gestation days 11 through 18 (2, 9). Erythropoiesis in the fetal liver proceeds with's rather heterogeneous population with regard to cell maturation contrasting to the homogenous yolk sac erythropoietic cells. This heterogeneous fetal cell population occurs in the absence of a fetal hemoglobin. In man and sheep, which do exhibit a fetal hemoglobin, the same type of erythropoiesis occurs. Djaldetti, et al.(lO) and Fantoni, et al.(U) have illustrated other interesting biochemical events occur-ring in the mouse during this period. Their experiments have demonstrated a definite time when continuous RNA formation is important to the perpetuation of the cell's ability to synthesize hemoglobin. However, red blood cells of the erythroid hepatic origin are no longer dependent on continuous RNA formation at a later time (the 13th day). One is tempted to consider the possibility of a different type of messenger being produced at this time, and to speculate on the possiblity of the more complex system, such as hemoglobin transition in man or sheep, being related to the type of messenger being produced. Whereas a morphologically identifiable population of cells can be related to the embryonic hemoglobin, it is more difficult to relate an erythrocyte population to fetal or adult hemoglobin alone. The problem of transition is more complex. Both fetal and adult hemoglobins are associated with non-nucleated, biconcave, disc-shaped cells which are produced successively during development in the liver and myeloid sites. Morphologically, fetal and adult erythrocytes are identical. Transition of hemoglobin may represent an alteration in hemoglobin synthesis in individual erythrocytes, particularly erythrocyte precursors. The level of regulation for hemoglobin biosynthesis can be genie or pretranscriptional. The degradation of a specific hemoglobin can be involved. The interaction of globin with heme and the interrelationship of their distinct or related control mechanism should be considered. types within individual cells, Transition of hemoglobin whether temporal or stimulated by environmental influences, most likely reflectdifferentjalgene activity and regulation at a level concomitant with the morphological differentiation of erythroid precursors. There being no fetal hemoglobin in the mouse, the same
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subsequent kind of transitional point of fetal to adult hemoglobin production in hepatic erythroid cells has not been studied. Although a shift from primitive to definitive erythroid cells, with an associated change in hemoglobin type from the embryonic to the fetal or from embryonic to adult is seen in the mouse, chicken, tadpole and man (2, 6, 11, 121, only the shift from embryonic hemoglobin is explained by change in erythrocyte population (13). However, the transition from a fetal to adult hemoglobin, which presumably occurs in the same cell type, appears to be more complex and represents a different level of differentiation. A theory by Baglioni (14) only considers fetal to adult transition, while Kabat (3) explains the molecular basis of an embryonic to fetal to adult transition in many mammals. The relationship of hemoglobin synthesis to the number of mitotic divisions has been offered as a model by Baglioni (14) to explain the switch of human fetal to human adult hemoglobin. Under certain pathological conditions, the number of mitotic divisions by a differentiating erythrocyte precursor may be fewer, thereby explaining the presence of fetal hemoglobin in these circumstances. The basis for many of these studies has been the examination of radioactive iron incorporation in hemoglobin produced within single cells. However, this technique cannot correlate hemoglobin type to differentiating cells Studies to date have not considered the possibility of a different (b) erythrocytic cell type associated with sheep hemoglobin C synthesis; that is, the existence of a morphological or structural difference in the erythrocytic cells containing hemoglobin C other than that due to hemoglobin C itself. The use of the sheep as a model to study the clustering of mutually exclusive hemoglobin genes in man and sheep was recently reviewed by Kabat (3). He also offered the looping-out excision theory as a possible explanation for hemoglobin differentiation. Nienhuis and Anderson (16) demonstrated that the control of the switch from A to C hemoglobin in sheep was not at the level of translation, but concluded the switch of sheep A to C was regulated by clone selection or at the transcriptional level. Sheep HbC can stimulate an -in vitro system using normal sheep marrow cells (17). This mechanism of activation of a normally "silent" hemoglobin gene offers a model for further studies. The importance of the relationship of hemoglobin synthesis and erythrocyte maturation is underlined by the necessity to determine quantitative and qualitative distribution of hemoglobin within single cells: 1) at different periods of gestation times; 2) at various stages of precursor maturation; and 3) during rapid erythropoiesis in growing animals, acute blood loss in adults and as stimulated by erythropoietin.
(b) Hemoglobin C is a unique hemoglobin found in sheep having AA or AB hemoglobin phenotype. HbC can be synthesized in adult animals which are anemic (15). This phenomena is reminicent of the transition of fetal to adult hemoglobin in the developing organism.
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Control of Hemoglobin Synthesis Control of the quantitative aspects of hemoglobin biosynthesis has various levels which need to be elucidated. Clearly, in order to understand the control of hemoglobin biosynthesis, several unique features of the relationship of hemoglobin to its erythrocyte must be considered. 1.
Red blood cells contain a hemoglobin concentration of approximately 35%. Maximum hemoglobin concentration per cell is from 34%-36%.
2.
Heme synthesis is coordinated to globin synthesis with the number of hemes equal to the number of globins.
3.
Equal numbersof alpha and non-alpha chains are synthesized per red blood cell.
4.
In the case of the human heterozygote having a normal and an abnormal hemoglobin (in which the abnormal subunit is a product of the same allele as the normal) these hemoglobins are not produced in a 1:l proportion. However, in contrast to this situation, domestic animals in which polymorphic hemoglobins are the products of the same pair of alleles, the proportion of hemoglobin component in the heterozygote is 1:l. There are other animals having multiple hemoglobin components in which the hemoglobin types are products of more than one pair of allelic genes, i.e., gene duplication or multiple cistrons. There is a quantitative difference in the proportion of hemoglobins in these animals. This phenomenon has been extensively studied in man and animals by Boyer -et a1.(18) and others (19, 20, 21).
5.
The sequential transition of epsilon, to gamma and to beta polypeptide chains accounts for embryonic, fetal and adult hemoglobin in most mammalian species. The switching from embryonic to either a fetal or adult type occurs in every species and the switching of fetal to adult hemoglobin occurs in many mammalian species. The switch of embryonic to adult is related to a morphologic change in the red blood cell population in every species thus far studied. This is not necessarily so for fetal to adult transition.
The mechanism which accounts for the transition or switching of hemoglobin types in mammals is an exciting area of investigation to study. Unique to the sheep and goat is the switching of an adult hemoglobin component "A" to adult hemoglobin C, under certain environmental circumstances (22, 23). This phenomenon unique to sheep and goats is given in detail later in this paper. In the mouse, the most abundant embryonic hemoglobin is type EI seen on the 10th day. By the 14th day, this is a relatively minor component. At this time, EII is a major component (24). All these studies demonstrate there are changes in the rates of synthesis of specific hemoglobins within the single red cell population. Regulation of globin synthesis at the cellular level is clearly indicated by the changing patterns of quantitative proportions of hemoglobin
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production seen in normal circumstances and in a variety of unique incidents. This disproportionate production of the betas and deltas explaining hemoglobin A2 in man is a normal situation, as is the small percent of fetal hemoglobin which represents persistence of gamma chain production in the adult. However, in man, there can be an increase in the proportion of fetal hemoglobin associated with certain severe anemias. There is a non-uniform distribution of fetal hemoglobin in the erythrocyte population in certain anemias: for example, the Thalassemias (25). This has been reflected as a specific population or clone of erythroid cells which have differential or quantitative differences in the polypeptide synthesis, hence hemoglobin types. These are all demonstrated by the elution technique of Betke and Kleihauer (26) In addition to seeing this type of change associated with unique pathological situations in man, we have the unique situation found in sheep (briefly discussed previously). Under conditions of severe blood loss or acute hemolysis induced by the administration of phenylhydrazine or erythropoietin, several workers have been able to show an increase of hemoglobin C (15, 27, 28). There is the cessation of production of one polypeptide chain (A) with the initiation of another (C), resulting in complete replacement of the normal adult hemoglobin A with the hemoglobin C (15). The same type of quantitative variation reported in the sheep has been seen in the duck by Bertles and Borgese (29). They have demonstrated a quantitative change in the hemoglobin produced in association with either acute hemolysis or bleeding. Alterations in hemoglobin synthesis for individual erythrocyte or erythrocyte precursors can occur at all levels of protein synthesis. An excellent review of this is given by Boyer (30). The unique ability of adult sheep to switch from A to C hemoglobin type under the influence of environment is reminiscent of the switch from fetal to adult hemoglobin during the late part of human fetal development. Sheep offer a model on which to perform experiments that would be impossible in man. Regulation of Erythropoiesis in the Fetus, Newborn and Adult Important in the control of erythropoiesis is the hormone erythropoietin. A complete review of the physiology and biochemical aspects of erythropoietin is not appropriate, although certain aspects should be emphasized. It is quite clear that erythropoietin is essential as a regulatory hormone in normal erythropoiesis and during severe stress and acute blood loss. Whether erythropoietin is vital for erythropoiesis in the embryo and fetus is open to question. For example, bilateral nephrectomy, which completely abolishes erythropoietin in the normal adult rat, has no deleterious effect upon erythropoiesis in the embryo or fetus (31). Stohlman has suggested several times that the regulation of erythropoiesis in the newborn differs from that in the adult. The production of erythropoietin in the newborn rat was examined by Carmena, Howard and Stohlman (32) during neonatal phases of life and compared to the adult animal. Erythropoietin could not be demonstrated
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in the plasma of neonatal rats. However, they reported that during neonatal development, rat fetuses placed in a hypoxic condition, demonstrated progressive increase in the capacity to produce erythropoietin as they developed. They suggested from these findings that during the hepatic erythropoiesis in fetal life, the red cell production is controlled differently than in the adult animal. It is possible that there is a gradual transition from a fetal to adult type of regulation, suggesting the possibility that there could be a unique erythropoietin of fetal origin for fetal regulation. The present biological assay for erythropoietin may not be sensitive to a fetal erythropoietin. In Stohlman's studies, it is important to note that they indicated that the transition of fetal to adult type of regulation was followed by a concomitant decrease in the number of syncytial cells present in fetal liver and myeloid erythropoiesis. These authors then proposed that syncytial cells serve as a fetal stem cell which is gradually replaced by more differentiated erythropoietin-sensitive cells. The syncytial cells described in the rat have also been observed in the human fetus. There is obviously some controversy regarding the ability and function of erythropoietin in the fetus. Zanjani, -et al. (33) for example, developed a successful surgical procedure to extensively study erythropoietin production in fetal lambs during the last third of the uterine development period. From these studies,theyconcluded that they could detect erythropoiesisstimulating activity or erythropoietin in the plasma obtained from the normal non-anemic fetus. Upon injection of phenylhydrazine into the fetus (resulting in acute anemia), highly significant amounts of erythropoietin or erythropoietic-stimulating activity material was apparrent in the circulation. This work offeredevidence that sheep in late pregnancy have the capacity to produce erythropoietin. Whether erythropoietin actually serves a role in the regulation of erythropoiesis was questionable. Because they did not measure hemoglobin types or relate hemoglobin to morphology, they were unable to determine if erythropoietin would have a direct effect on what type of hemoglobins were being produced. However, recent studies by Zanjani's laboratories have produced convincing evidence for the role of erythropoietin in fetal erythropoiesis (34). The administration of antibiody against erythropoietin to fetal sheep during the last third of the gestation period resulted in a supression of erythropoiesis in the fetus. Whether there is a fetal erythropoietin and adult erythropoietin is still up for question. Also, the effect of erythropoietin in early development is not known. The primary site of erythropoietic stimulating activity material is the kidney in the adult animal, however in the fetus the major source may be the spleen and liver (35). Equally important for the understanding of fetal erythropoiesis is the intriguing finding of erythropoiesis inhibiting factor in the fetus. (36). In studies of bone marrow in newborn calves, Gabuzda, et al. (37) observed that erythropoietin was capable of stimulating both adult and fetal hemoglobin synthesis. Gabuzda felt there was no direct effect of
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erythropoietinin differentiallydetermininggenetic transcriptionor translastiouof these globin types. And since the synthesisof the two hemoglobinsproceeded in a synchronousmanner, there was no evidence that erythropoietinstimulatedpreferentiallythe synthesisof fetal hemoglobin in the less mature erythrocyte. This eliminatedthe possibility that erythropoietinacts on fetal hemoglobinby inducing stem cells of a populationof immature erythroblasts. He felt this data was consistentwith -in vivo observationsin the human sickle cell anemia patient where the adult and fetal hemoglobinwere simultaneously suppressedfollowingtransfusion. In contrast to these findings,there is the data of Necheles, et al., (38) who concluded that erythropoietin in vitro stimulates adult but not fetal hemoglobin synthesis. These -investigators were working with adult human bone marrow tissue, which can be directly compared with bone marrow tissue of the newborn calf. On the other hand, the use of newborn calves is not comparable to studies performed on erythropoiesis of primarily hepatic origin. Studies cited here not only involve different stages of development and species, but also in some cases did not ascertain the hemoglobin types being produced. From limited studies, one can only conclude that each species reacts differently to the regulation by erythropoietin at different temporal times of development. In the study of the newborn sheep, this fetal work in human and calf must be contrasted to the direct mediation of the transition of A to C by erythropoietin, as reported by Boyer (15) and others (39). Present criteria indicate that yolk sac erythropoiesis, in which immature precursors do not persist, is not responsive to erythropoietin (40). Thirteen-fourteen day fetal erythroid cells of hepatic origin in the mouse are quite susceptible to the effect of erythropoietin (40, 41). The effects of erythropoietin on purified erythroid cell precursors Cantor, -et a1.(42) studied the erythropoietin in vitro were studied. _effect on isolated immature erythroid cell precursors. Their data now identifies the erythropoietic-sensitive cell as an early precursor which although descendant, is distinct from the multi-potent hemopoietic stem cell. It is morphologically indistinguishable from the class of cells identified as proerythroblasts. These cells exhibit a marked acceleration of RNA after exposure to erythropoietin. The cellular response to erythropoietin -in vitro includes proliferation of differentiating erythroblasts, initiation of hemoglobin synthesis and preservation of the proliferation for self-perpetuation (40, 41, 42). Basch (43) concludes that human fetal hemopoietic tissue is responsive to erythropoietin. He has concluded from his studies that stimulation by this hormone did not collectively stimulate the production of one or another of the human hemoglobins A or F. His work included cells of very young gestation age: 6 weeks. He felt that at least in the human, there was no evidence that erythropoietin was involved in the transition that occurs in sheep which have been injected with erythropoietin. From his studies, he then concluded that erythropoietin does not mediate the transition of F to A in man. Therefore, the transition of human hemoglobin from fetal to adult resembles that of cattle rather than the A to C transition in sheep which is mediated by erythropoietin. It is felt that on a cellular basis, erythropoietin
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induces replication of erythroid precursors and causes differentiation of these precursor cells to hemoglobin synthesizing cells. Its relationship to the transition of hemoglobins in animals other than the A to C transition in sheep remains quite nebulous.
Transition of Hemoglobin Type The control of the transition from fetal hemoglobin to adult or from adult to fetal is open to speculation. An excellent review of the "switch" from fetal to adult has been given by Bertles (44, 45). One can easily invoke a variety of mechanisms which include repression, derepression and control genes, etc. However, all explanations whether they apply to mammalian systems or not , must consider control at the cistron level and must explain the fact that the transition occurring in pathological events in man is often accompanied by pleiotropic regression (44, 45, 46). There is a transition or reversion to erythrocytes having fetal characteristics, enzymes and surface antigens. Although there is little doubt that transition occurs at a level of differentiation, one can consider some of the more gross aspects of control which have been associated with specific pathological conditions. In some of those not so associated, a simple approach might be to consider if there is a relationship between red cell production, oxygen demand and the hemoglobin type. Some examples would be: 1) simple anemia, cyanosis or hypoxia are pathological features which do not universally result in the transition of adult to fetal hemoglobin; 2) persistence of fetal hemoglobin has been associated with juvenile myelocytic leukemia; 3) untreated Addisonian pernicious anemia patients have had an increase in fetal hemoglobin; 4) the fact that hormones are related to regulation is suggested by the discovery of a definite increase in fetal hemoglobin in the blood of women in the second trimester of pregnancy; and 5) the genetically determined persistent high fetal hemoglobin is associated with no marked pathological features. These are a few examples which indicate there is no one universal mechanistic prediction that can be made. Acceleration of the transition from tadpole to frog hemoglobin occurred in studies by Moss and Ingram (47) by adding thyroid hormone to their environment. However, this transition is accompanied by a corresponding change in red cell population, similar to the embryonic to adult shift in the mouse rather than the fetal to adult in man and sheep.
Reversion to Fetal Erythropoiesis There is clear evidence in the human that the transition to fetal hemoglobin is accompanied by a switch to red blood cell populations which have different biochemical characteristics such as surface antigens and red blood enzymes. One recently reported phenomena which is unique to the fetal erythrocyte is the non-pathological transient fetal porphyria of the fetus of all mammals thus far studied (48). Characteristic of the phenomena are the occurrence of uroporphyrin I and III in the cartilage and bones and the km differences of the delta aminolevulinic acid
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THERIOGENOLOGY synthetase (49). Because of these fetal characteristics of the porphyrin pathway during a period in development, there is a difference in regulation and control of heme synthesis resulting in the accumulation of porphyrin compounds in the bone tissue for a period of time during gestation. This period of time differs for each species and is normally related to fetal development. The flourescence of the porphyrins in the fetal bones seen under ultraviolet light is not unlike the flourescence seen due to the accumulation of uroporphyin I in adult fox squirrel skeletal bones. The transient nature of the phenomena in developing fetuses can be related to the hepatic erythropoiesis. (See Figure 3) This evidence again underlines the distinct characteristics of fetal erythropoiesis. Conclusions In a recent review, the ontogeny of the hemoglobins of various animals has been made (4). From this and various other articles on fetal erythropoiesis (2, 3, 15, 44, 45) the following comparison of human developmental erythropoiesis can be made with that of other mammals. (See Table I). It is quite clear that there are similarities and differences upon comparison of the ontogeny of hemoglobin among species. The evidence for regulation of erythropoiesis by erythropoietin in late pregnancy is convincing. However, the role of erythropoietin in the transition of hemoglobin types has not been established with the exception of hemoglobin C in sheep. Further work needs to be done on regulation of erythropoiesis during early embryonic and fetal periods in mammals. Emphasis should be placed not only on hemoglobin types but also upon other markers related to fetal erythropoiesis. An increase in oxygen affinity by fetal red blood cells is common to all species thus far reported. However, the mechanism which accounts for the differences in oxygen affinity can be quite different in various species. Upon comparison the following generalizations can be made. Mechanisms By Which Fetal Blood Can Have A Higher Oxygen Affinity than Maternal Blood 1.
The fetal hemoglobin may have an intrinsically higher oxygen affinity independent of any intracellular co-factors. Examples include the sheep and the goat.
2.
Fetal hemoglobin may have impaired interaction with a red cell co-factor. For example, the oxygen affinity of “stripped” human fetal hemoglobin is very similar to that of adult hemoglobin - A. However, hemoglobin F binds less strongly to 2,3 DPG, and therefore, in the presence of 2.3 DPG within the red blood cell, its oxygen affinity is correspondingly greater.
3.
The content of 2,3 DPG may be significantly less in fetal red blood cells, and change rapidly after birth. Through a
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mechanism of modulation the 2,3 DPG content can alter the oxygen affinity. This is true for the horse and for the pig and dog. The first two mechanisms cited above require the presence of the fetal hemoglobin which differs structurally and functionally from the adult hemoglobin. But in the third, the hemoglobin may be identical in the adult and the fetus, and indeed, this is the case for the horse, dog and pig.
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TABLE
I
COMPARISON OF HUMAN EMBRYONIC AND FETAL ERYTHROPOIESIS WITH ERYTHROPOIESIS OCCURRING DURING DEVELOPMENT INOTHERMAMMALS
Human Fetal Erythropoiesis
Animal Fetal Erythropoiesis
Embryonic hemoglobin associated with an unique nucleated red blood cell
All mammals thus far studied have embryonic erythropoiesis with a corresponding embryonic hemoglobin type.
Unique fetal hemoglobin
Not all species demonstrate a unique fetal hemoglobin
Multiple cistrons for genetic control of fetal hemoglobin
Very limited evidence for multiple cistrons for genetic control of fetal hemoglobin (only in primates)
Persistence of fetal hemoglobin after birth and into the adult
Prolonged persistence of fetal hemoglobin after birth has not been shown in animals even at a very small level.
Relatively slow transition from fetal to adult
Relatively rapid transition from fetal to adult hemoglobin
Shortened life span of fetal cells
Little or no information is available regarding life span of fetal red blood cells in animals.
Red blood cell surface antigens unique to the fetal erythrocyte
Red blood surface antigens unique to fetal erythrocytes have not been reported
Changes in oxygen affinity due to modulation of 2,3 DPG
The hemoglobin within the erythrocytes of some animal species are affected by modulation of 2,3 DPG, others are not
Transient non-pathological porphyria during development
All animals show a transient porphyria during early gestation
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HUMAN
240
60
Red Blood
100
y.
4Birth DAYS
MOUSE
Embryonic
(/Adult
k,--
,
Cells
Containing a Distinct Hemoglobin DAYS
SHEEP
,Adul
I
100 9%
t
I
F&l
I I
Birth
Iy
fl
I Hb C
s.150 DAYS
Temporal Relation of Transition of Embryonic, Fetal and Adult Hemoglobins in Various Mammals Figure 1.
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FIGURE 2A. Subunit composition of mammalian hemoglobins. Four polypeptide chains, two non-identical pairs account for the subunit composition of mammalian hemoglobin. The pairs can be classified as alphas (a) and non-alphas (a 6,y, etc.)
i3 a
a
=
cr2f32
B
The shorthand formula can be illustrated as a2Bz for adult human hemoglobin and a2y2 for fetal or a2E2 for embryonic. Other minor components are given other Greek letter designations in human beings. In the embryonic mouse, non a chains are identified as either x, y, or 2.
FIGURE 2B. Ontogeny of hemoglobin: subunit comparison of the structure of embryonic, fetal and adult hemoglobin in various species. The amount of adult hemoglobin seen in each species is also given by estimate of the percent at birth.
Mouse
EMBRYONIC
Human
5
x2
y2
El
EQ
31
(32
y2
E
a2E2
511
a2
22
Es&
II
a2
y:
a2
Y2
A
FETAL
a2!32
a262
ADULT
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a2
E2
a2y2
a2B2
a262
100%
2040%
at Birth
at Birth
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O-10% at Birth
231
U n its/g
6ALAS
Liver
600
1200
35 40 DAYS
50 60
65
0
Figure 3.
Total Concentration of Porphyrins in Amniotic Fluid and Concentration of a-Amino Levulinic Acid Synthetase from Guinea Pig Liver as Related to Hepatic Erythropoiesis
30
50
100
Fluid
mg/lOOml Porphyrins in Amniotic
THERIOGENOLOGY REFERENCES
1.
Kitchen, H. Animal hemoglobin heterogeneity. York Academy of Sciences 241:12-24, 1975.
2.
Marks, Paul A. and Richard A. Rifkind. Protein synthesis: control in erythropoiesis. Science 175:955, 1972.
3.
Kabat, David. Gene selection in hemoglobin and in antibodysynthesizing cells. Science 175:134, 1972.
4.
Kitchen, H., and Brett, I. Embryonic and fetal hemoglobin in animals. Annals of the New York Academy of Sciences, 241:653671. 1974.
5.
Hanunerberg,G., Brett, I., and Kitchen, H. Ontogeny of hemoglobin in sheep. Ann. N. Y. Acad. Sci., Vol. 241:672, 1974.
6.
Ingram, Vernon M. 235L338m, 1972.
7.
Kovach, J.S., P. A. Marks, E. s. Russell and H. Epler. Erythroid cell development in fetal mice: Ultrastructural characteristics and hemoglobin synthesis. J. Mol. Biol. 25:131, 1967.
8.
Rifkind, Richard A., David Chui and Hazel Epler. An ultrastructural study of early morphogenetic events during the establishment of fetal hepatic erythropoiesis. J. Cell. Biol. 40:343, 1969.
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Fantoni, A. A., A. Bank and P. A. Marks. Globin composition and synthesis of hemoglobin in developing fetal mice erythoid cells. Science 157:1327, 1967.
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Djaldetti, Meir, David Chui, Paul A. Marks and Richard A. Rifkind. Erythoid cell development in fetal mice: Stabilization of the hemoglobin synthetic capacity. J. Mol. Biol. 50:345, 1970.
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Fantoni, A. A. de la Chapelle, Richard A. Rifkind and Paul A. Marks. Erythoid cell development in fetal mice: Synthetic capacity for different proteins. J. Mol. Biol. 33:79, 1968.
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Maniatis, George M. and Vernon M. Ingram. Erythropoiesis during amphibian metamorphosis. I. Site of maturation of erythrocytes in Rana catesbeiana. J. Cell Biol. 49:372, 1971.
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Matioli, G. T., N. Niewisch and H. Vogel. Distribution of hematologically competent stem cells in tissues of mice (fetus and newborn). Fed. Proc. 27:672, 1968.
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Baglioni, Corrado. Correlations between genetics and chemistry in human hemoglobins. In Molecular Biology, J. H. Taylor, Ed., p. 405, Academic Press, New York, 1964.
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15.
Boyer, Samuel H. Sheep hemoglobins, erythropoietin, and genetic regulation. In Hemopoietic Cellular Proliferation, Frederick Stohlman, Jr., Ed,, p. 141, Grune and Stratton, Inc,, New York, 1970.
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Nienhuis, Arthur W. and W. French Anderson. Hemoglobin switching in sheep and goats: Change in functional globin messenger RNA in reticulocytes and bone marrow cells. Proc. Nat, Acad. Sci., 69(8) :2184, 1972.
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Adamson, J.W., and G. Stamatoyannopoulos. The A+C switch in sheep hemoglobins: -In vitro studies with erythropoietin. Annals of New York Acad. of Sci., 241:556-565, 1974.
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Boyer, S. H., P. Hathaway, and M. C. Garrick. In symposium of quantitative biology: Human Genetics 29:333, 1964. cold Spring Harbor Laboratory of Ocean Biology, Cold Spring Harbor.
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Kitchen, H., E. 0. Kouba, and C. W. Easley. Animal models for the study of hemoglobinopathies. Animal Models for Biochemical Research III, p. 52, National Academy of Sciences, Washington, D.C. 1970.
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Kouba, Ethel Oliver and Hyram Kitchen. Hemoglobin in Macaca speciosa: Quantitative differences. Arch. Biochem. Biophys. 145 (1):283, 1971.
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Itano, H.A. The human hemoglobin: Their properties and genetic control. Adv. Protein. Chem. 12:216, 1957.
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Van Vliet, G. and T. H. J. Huisman. Changes in the hemoglobin types of sheep as a response to anemia. Biochem. J. 93:401, 1964.
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Huisman, T. H. J., J. P. Lewis, M.D. Blunt, H. R. Adams, A. Miller, Dozy Andree and Evalyn M. Boyd. Hemoglobin C in newborn sheep and goats: A possible explanation for its function and biosynthesis. Ped. Res. 3:189, 1969.
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Barker, Jane E. Development of the mouse hematopoietic system. Developmental Biology 18:14, 1968.
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Shepard, M. K., D. J. Weatherall and C. L. Conley. Semiquantitative estimation of the distribution of fetal hemoglobin in red cell populations. Bull. Johns Hopkins Hosp. 110:293, 1962.
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Betke, K., and E. Kleihauer. Fetaler and bleibender blutfarbstoff in erythrozyten und erythroblasten von menschlichen feten und neugeborenen. Blut 4:241, 1958.
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Kitchen, H., J. Eaton and W. J. Taylor. Rapid production of a hemoglobin by induced hemolysis in sheep: Hemoglobin C. Am. J. Vet. Res. 29(2):218, 1968.
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Bertles, 5. F., and T. A. Borgese. Disporportional synthesis of the adult's two hemoglobins during acute anemia. J. Clin. Invest,, 47:6?9, 1968.
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Boyer, S. H. An appraisal of genetic regulation of protein synthesis in higher organisms. In Modern Trend in Human Genetics, A. E. H. Embery, Ed., p. 1, Butterworth, London, 1969.
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Lucarelli, G., D. Howard, and E. Stohlman, Jr. Regulation of erythropoiesis XV neonatal erythropoiesis and effect of nephrectomy. J. Clin. Invest. 43:2195, 1964.
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Carmena, Albert O., Donald Howard and Frederick Stohlman, Jr. Regulation of erythropoiesis. XXII. Erythropoietin production in the newborn animal. Blood 32(3):376, 1968.
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Zanjani, Esmail, D., Edgar 0. Horger, III, Albert S. Gordon, Linda N. Cantor and Donald L. Hutchinson. Erythropoietin production in the fetal lamb. 3. Lab. Clin. Med. XXX 74(5):782, 1969,
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Zanjani, E. D., L. I. Mann, H. Barlington, A. S. Gordon, and L. R. Wasserman. Evidence for a Physiologic Role of Erythropoietin in Fetal Erythropoiesis. Blood, Vol. 44, No. 2:285-290, 1974.
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Kaplan, S. M., S. R. Weinberg, and A. S. Gordon. Erythropoietin production in the neonatal rat: Sites of erythrogenin production. Bio. Neonate 24:145-l.50,1914.
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Lindemann, R. Erythropoietin and erythropoiesis inhibitors in the neonatal period in erythropoiesis. Univ. of Tokyo Press, Ed. by K. Nakao, J. W. Fisher and T. Fumimaro. pp. 339-345, 1975.
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Gabusda, T. G., R. K. Silver, Chui Cheung Lily and H. G. Lewis. The formation of foetal and adult hamoglobin in cell cultures of neonatal calf marrow. Proc. Sot. Exp. Biol. Med. ll9:1207 (1965)
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Necheles, T. F., R. G. Sheehan and H. 3. Meyer. Effect of erythropoietin and oxygen tension on -in vitro synthesis of hemoglobin A and F by adult human bone marrow. Proc. Sot. Exp. Biol. Med. 119:1207, 1972.
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Moore, S. IL.,W. C. Godley, G. Van Vliet, J. P. Lewis, E. Boyd and T.H.J. Huisman. The production of hemoglobin C in sheep carrying the gene for hemoglobin A: Hematologic aspects. Blood 28:314, 1966.
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Cole, R. J. and John Paul. The effects of erythropoietin on haem synthesis in mouse yolk sac and cultural foetal liver cells. J. Embryol. Exp. Morph. 15:245, 1966,
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Paul, John and J. A, Hunter. Synthesis of macromolecules during induction of haemoglobin synthesis by erythropoietin. J. Mol. Biol, 42:31, 1969.
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Cantor, Linda N., Allan J. Morris, Paul A. Marks and Richard A. Rifkind, Purification of erythropoietin-responsive cells by immune hemolysis. Proc. Nat. Acad. Sci. 69(6):1337, 1972.
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Basch, Ross S. Hemoglobin synthesis in short-term cultures of human fetal hematopoietic tissues. Blood 39(4):530, 1972.
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Bertles, John F. The occurrence and significance of fetal hemoglobins in regulation of hematopoiesis, Albert S. Gordon, Ed., Appelton Century Crofts, New York, 1970.
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Bertles, J. F. 'Human fetal hemoglobin significance in disease. Ann. N. Y. Acad. Sci., Ed. H. Kitchen & S. Boyer. Vol 241:638, 1974.
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Charache, Samuel, John J. Schruefer and Wilma B. Bias. "Hereditary persistence of fetal" red cells. J. Clin. Invest. 47:17a, 1968.
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Moss, Bernard and Vernon M. Ingram. Hemoglobin synthesis during amphibian metamorphosis. II. Synthesis of adult hemoglobin following thyroxine administration. J. Mol. Biol. 32:493, 1968,
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Kitchen, H., Wood, D., and Brett, I. Hemoglobin transition and porphyria during development. Fed. Proc. 30(3): Part II, 1971.
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Bishop, D. F. Differentiation between isozymes of guinea pig 6-aminolevulinic synthetase by affinity chromatography. Fed. Proc.:l976.
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Kitchen, H., and H. F. Bunn. Ontogeny of equine haemoglobins. J. Reprod. Fert., Suppl. 23:595-598, 1975.
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(4
Hyram Kitchen Department of Environmental Practice College of Veterinary Medicine University of Tennessee Knoxville, Tennessee 37919
ABSTRACT
:
Developmental Hematology
A comparison of the ontogeny of hemoglobin is made in human, mouse and sheep. The differences and similarities are discussed to demonstrate the use of these animals as potential models to study regulation and transition of erythropoiesis during development. A general comparison of developmental erythropoiesis is made among various mananalswith a discussion of how various mammals facilitate oxygen transport across the placenta by differing mechanisms.
(a)
This work was supported in part by NIH Grant 7 ROl HD 10066-01 and the Agricultural Experiment Station, University of Tennessee.
The author wishes to acknowledge the suggestions and help of Drs. Robert L. Michel and Robert D. Lange, and the assistance of Mrs. Janice M. Bryan with the preparation of this manuscript.
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AliSW
CO~A~TI~ D~~P~~~~~~~Y: MODELS TO STUDY HUMAN FETAL ERYTRROPOIESIS
Introduction Many fascinating phenomena axe encountered in biology and serve to supply the investigator with an endless resource for the exploration of new knowledge and biological understanding, Of the many examples of such phenomena, one from the area of comparative hematology which is of extreme importance is the transition of embryonic and/or fetal hemoglobin to adult hemoglobin. This is of interest not only to the biologist, but to the medical scientist as well. The sequential emergence and disappearance of a variety of hemoglobin types associated with developxaent is not identical for all mammals. The purpose of this communication is to compare the regulation and control of erythropoiesis and hemoglobin types in various species. This comparison will'serve to point out how various molecular mechanisms can accomplish the same physiological objective. In this example, the facilitation of oxygen transport across the placenta to supply oxygen of the proper tension and saturation to the developing organism is a major objective. This report will be a review of work by others and a report of recent work performed in this laboratory. Functional erythrocytes are produced from early precursors. Each species has it's own distinctive type of hemoglobin. Although functionally identical, the hemoglobins of the various species differ with respect to their amino acid sequence (1). Not to be confused with the developmental cell series occurring during normal adult erythropoiesis are morphologically and biochemically distinctive red blood cells which can be related to the development of the organism. The temporal relationship of erythrocytes can be identified as embryonic cells, containing embryonic hemoglobin followed by transition to morphologically distinctive red blood cells. In other species, there are distinct embryonic cells with accompanying embryonic hemoglobin with a transition to fetal cells having a unique hemoglobin type and other red blood cell components followed by a further transition to the adult erythrocytes with the corresponding hemoglobin type.
Hemoglobin Synthesis and the Erythrocyte There is limited information regarding the control of hemoglobin synthesis. Even less is known regarding the relationship of the morphology of erythrocyte precursors to biochemical events. An excellent review of cellular differentiation, with the erythroid cell system of the mouse as a model, was published by Marks and Rifkind (2). The patterns of hemoglobins emerging at various stages of development differ in the mouse, sheep, and man; however, in each case the relationship of biochemical events and cell morphology is still to be made.
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There are numerous well defined intermediate stages of differentiation between the undifferentiated precursor (stem cell) and the mature erythrocyte. Whether all or some cells produce small quantities of all the possible hemoglobin types in a species is not known. It is possible that cells from the same sites of production have different capacities to synthesize hemoglobin types. The proportion of hemoglobin types in a cell population may be different. The distribution of hemoglobin type in individual erythrocytes may be related to the mean age of erythropoietin-sensitive cells (3). Whether a cell is committed to only one hemoglobin type is not known. There is an orderly succession of hemoglobin types within morphologically or biochemically different red blood cells which in general correspond to the embryonic, fetal and adult phases of life in most mammalian species (3, 4). See Figure 1. The ontogeny of human hemoglobin is well documented. The work done thus far indicates that the ontogeny of sheep hemoglobin is very similar to the human (5). The sheep and human embryonic, fetal and adult hemoglobins have structurally common alpha chains within each species. Variations that account for the hemoglobin type associated with the stages of development occur in the structurally distinct non-alpha chains (4). In contrast to the polypeptide chain type accounting for the various hemoglobins in the mouse,erythropoiesis occurs progressively in the mesoblastic yolk sac, hepatic and myeloid bone marrow tissue. Cellular and biochemical aspects of erythropoiesis have been studied in detail in the mouse, chicken and tadpole. Work and articles by Rifkind, et al . (2) and Ingram (6) give impetus to two major theories of divergence between primitive and definitive progenitor cells: 1) yolk sac primitive cells and definitive cell lines diverge from a common precursor cell, and 2) yolk sac primitive cells are seeds of the definitive cells. To define the use of animals as models to study the ontogeny and transition of hemoglobin, we must compare all three; man, sheep and mouse.
Comparison of Ontogeny of Hemoglobins in the Mouse, Sheep and Human A comparison of mouse, sheep and human developmental erythropoiesis will serve to identify what is currently known about the most often used models to study the ontogeny and transition of human hemoglobin. (See Figure 1) Smbryonic hemoglobin is associated with nucleated, ovalshaped cells produced in yolk sac for all species thus far studied (4). In man, embryonic hemoglobins Gower 1 and Gower 2 are characterized by structurally unique epsilon polypeptide chains; Gower 1 having the shorthand formula of EL,and Gower 2 cxp ~2 chains. In contrast to man and sheep, the mouse (the species in which the most detailed information is known) has no fetal hemoglobin (7, 8). Multiple polypeptide chains, both alpha and non-alpha, account for structural differences in the embryonic hemoglobin in the mouse (9). In the mouse, embryonic
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red blood cells differentiate as a relatively homogenous population. All embryonic hemoglobin synthesis occurs in cells which are synthesizing DNA and dividing. These cells are thought only to produce embryonic hemoglobin (7). The alpha chain is structurally identical to adult alpha chains within the respective species in most mammals. In the C57Bl-65 mouse, the embryonic hemoglobins are composed of x and y polypeptide chains for EI OLand y for EII and o and z for EIII (7). Adult hemoglobin has not been detected in the embryonic cells of these species. Erythropoiesis has been extensively characterized during embryonic life, after yolk sac erythropoiesis, and during the beginning of fetal life in the mouse. There is an alteration in the erythroid population in terms of the proportion of cells morphologically distinct during the gestation days 11 through 18 (2, 9). Erythropoiesis in the fetal liver proceeds with's rather heterogeneous population with regard to cell maturation contrasting to the homogenous yolk sac erythropoietic cells. This heterogeneous fetal cell population occurs in the absence of a fetal hemoglobin. In man and sheep, which do exhibit a fetal hemoglobin, the same type of erythropoiesis occurs. Djaldetti, et al.(lO) and Fantoni, et al.(U) have illustrated other interesting biochemical events occur-ring in the mouse during this period. Their experiments have demonstrated a definite time when continuous RNA formation is important to the perpetuation of the cell's ability to synthesize hemoglobin. However, red blood cells of the erythroid hepatic origin are no longer dependent on continuous RNA formation at a later time (the 13th day). One is tempted to consider the possibility of a different type of messenger being produced at this time, and to speculate on the possiblity of the more complex system, such as hemoglobin transition in man or sheep, being related to the type of messenger being produced. Whereas a morphologically identifiable population of cells can be related to the embryonic hemoglobin, it is more difficult to relate an erythrocyte population to fetal or adult hemoglobin alone. The problem of transition is more complex. Both fetal and adult hemoglobins are associated with non-nucleated, biconcave, disc-shaped cells which are produced successively during development in the liver and myeloid sites. Morphologically, fetal and adult erythrocytes are identical. Transition of hemoglobin may represent an alteration in hemoglobin synthesis in individual erythrocytes, particularly erythrocyte precursors. The level of regulation for hemoglobin biosynthesis can be genie or pretranscriptional. The degradation of a specific hemoglobin can be involved. The interaction of globin with heme and the interrelationship of their distinct or related control mechanism should be considered. types within individual cells, Transition of hemoglobin whether temporal or stimulated by environmental influences, most likely reflectdifferentjalgene activity and regulation at a level concomitant with the morphological differentiation of erythroid precursors. There being no fetal hemoglobin in the mouse, the same
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subsequent kind of transitional point of fetal to adult hemoglobin production in hepatic erythroid cells has not been studied. Although a shift from primitive to definitive erythroid cells, with an associated change in hemoglobin type from the embryonic to the fetal or from embryonic to adult is seen in the mouse, chicken, tadpole and man (2, 6, 11, 121, only the shift from embryonic hemoglobin is explained by change in erythrocyte population (13). However, the transition from a fetal to adult hemoglobin, which presumably occurs in the same cell type, appears to be more complex and represents a different level of differentiation. A theory by Baglioni (14) only considers fetal to adult transition, while Kabat (3) explains the molecular basis of an embryonic to fetal to adult transition in many mammals. The relationship of hemoglobin synthesis to the number of mitotic divisions has been offered as a model by Baglioni (14) to explain the switch of human fetal to human adult hemoglobin. Under certain pathological conditions, the number of mitotic divisions by a differentiating erythrocyte precursor may be fewer, thereby explaining the presence of fetal hemoglobin in these circumstances. The basis for many of these studies has been the examination of radioactive iron incorporation in hemoglobin produced within single cells. However, this technique cannot correlate hemoglobin type to differentiating cells Studies to date have not considered the possibility of a different (b) erythrocytic cell type associated with sheep hemoglobin C synthesis; that is, the existence of a morphological or structural difference in the erythrocytic cells containing hemoglobin C other than that due to hemoglobin C itself. The use of the sheep as a model to study the clustering of mutually exclusive hemoglobin genes in man and sheep was recently reviewed by Kabat (3). He also offered the looping-out excision theory as a possible explanation for hemoglobin differentiation. Nienhuis and Anderson (16) demonstrated that the control of the switch from A to C hemoglobin in sheep was not at the level of translation, but concluded the switch of sheep A to C was regulated by clone selection or at the transcriptional level. Sheep HbC can stimulate an -in vitro system using normal sheep marrow cells (17). This mechanism of activation of a normally "silent" hemoglobin gene offers a model for further studies. The importance of the relationship of hemoglobin synthesis and erythrocyte maturation is underlined by the necessity to determine quantitative and qualitative distribution of hemoglobin within single cells: 1) at different periods of gestation times; 2) at various stages of precursor maturation; and 3) during rapid erythropoiesis in growing animals, acute blood loss in adults and as stimulated by erythropoietin.
(b) Hemoglobin C is a unique hemoglobin found in sheep having AA or AB hemoglobin phenotype. HbC can be synthesized in adult animals which are anemic (15). This phenomena is reminicent of the transition of fetal to adult hemoglobin in the developing organism.
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Control of Hemoglobin Synthesis Control of the quantitative aspects of hemoglobin biosynthesis has various levels which need to be elucidated. Clearly, in order to understand the control of hemoglobin biosynthesis, several unique features of the relationship of hemoglobin to its erythrocyte must be considered. 1.
Red blood cells contain a hemoglobin concentration of approximately 35%. Maximum hemoglobin concentration per cell is from 34%-36%.
2.
Heme synthesis is coordinated to globin synthesis with the number of hemes equal to the number of globins.
3.
Equal numbersof alpha and non-alpha chains are synthesized per red blood cell.
4.
In the case of the human heterozygote having a normal and an abnormal hemoglobin (in which the abnormal subunit is a product of the same allele as the normal) these hemoglobins are not produced in a 1:l proportion. However, in contrast to this situation, domestic animals in which polymorphic hemoglobins are the products of the same pair of alleles, the proportion of hemoglobin component in the heterozygote is 1:l. There are other animals having multiple hemoglobin components in which the hemoglobin types are products of more than one pair of allelic genes, i.e., gene duplication or multiple cistrons. There is a quantitative difference in the proportion of hemoglobins in these animals. This phenomenon has been extensively studied in man and animals by Boyer -et a1.(18) and others (19, 20, 21).
5.
The sequential transition of epsilon, to gamma and to beta polypeptide chains accounts for embryonic, fetal and adult hemoglobin in most mammalian species. The switching from embryonic to either a fetal or adult type occurs in every species and the switching of fetal to adult hemoglobin occurs in many mammalian species. The switch of embryonic to adult is related to a morphologic change in the red blood cell population in every species thus far studied. This is not necessarily so for fetal to adult transition.
The mechanism which accounts for the transition or switching of hemoglobin types in mammals is an exciting area of investigation to study. Unique to the sheep and goat is the switching of an adult hemoglobin component "A" to adult hemoglobin C, under certain environmental circumstances (22, 23). This phenomenon unique to sheep and goats is given in detail later in this paper. In the mouse, the most abundant embryonic hemoglobin is type EI seen on the 10th day. By the 14th day, this is a relatively minor component. At this time, EII is a major component (24). All these studies demonstrate there are changes in the rates of synthesis of specific hemoglobins within the single red cell population. Regulation of globin synthesis at the cellular level is clearly indicated by the changing patterns of quantitative proportions of hemoglobin
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production seen in normal circumstances and in a variety of unique incidents. This disproportionate production of the betas and deltas explaining hemoglobin A2 in man is a normal situation, as is the small percent of fetal hemoglobin which represents persistence of gamma chain production in the adult. However, in man, there can be an increase in the proportion of fetal hemoglobin associated with certain severe anemias. There is a non-uniform distribution of fetal hemoglobin in the erythrocyte population in certain anemias: for example, the Thalassemias (25). This has been reflected as a specific population or clone of erythroid cells which have differential or quantitative differences in the polypeptide synthesis, hence hemoglobin types. These are all demonstrated by the elution technique of Betke and Kleihauer (26) In addition to seeing this type of change associated with unique pathological situations in man, we have the unique situation found in sheep (briefly discussed previously). Under conditions of severe blood loss or acute hemolysis induced by the administration of phenylhydrazine or erythropoietin, several workers have been able to show an increase of hemoglobin C (15, 27, 28). There is the cessation of production of one polypeptide chain (A) with the initiation of another (C), resulting in complete replacement of the normal adult hemoglobin A with the hemoglobin C (15). The same type of quantitative variation reported in the sheep has been seen in the duck by Bertles and Borgese (29). They have demonstrated a quantitative change in the hemoglobin produced in association with either acute hemolysis or bleeding. Alterations in hemoglobin synthesis for individual erythrocyte or erythrocyte precursors can occur at all levels of protein synthesis. An excellent review of this is given by Boyer (30). The unique ability of adult sheep to switch from A to C hemoglobin type under the influence of environment is reminiscent of the switch from fetal to adult hemoglobin during the late part of human fetal development. Sheep offer a model on which to perform experiments that would be impossible in man. Regulation of Erythropoiesis in the Fetus, Newborn and Adult Important in the control of erythropoiesis is the hormone erythropoietin. A complete review of the physiology and biochemical aspects of erythropoietin is not appropriate, although certain aspects should be emphasized. It is quite clear that erythropoietin is essential as a regulatory hormone in normal erythropoiesis and during severe stress and acute blood loss. Whether erythropoietin is vital for erythropoiesis in the embryo and fetus is open to question. For example, bilateral nephrectomy, which completely abolishes erythropoietin in the normal adult rat, has no deleterious effect upon erythropoiesis in the embryo or fetus (31). Stohlman has suggested several times that the regulation of erythropoiesis in the newborn differs from that in the adult. The production of erythropoietin in the newborn rat was examined by Carmena, Howard and Stohlman (32) during neonatal phases of life and compared to the adult animal. Erythropoietin could not be demonstrated
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in the plasma of neonatal rats. However, they reported that during neonatal development, rat fetuses placed in a hypoxic condition, demonstrated progressive increase in the capacity to produce erythropoietin as they developed. They suggested from these findings that during the hepatic erythropoiesis in fetal life, the red cell production is controlled differently than in the adult animal. It is possible that there is a gradual transition from a fetal to adult type of regulation, suggesting the possibility that there could be a unique erythropoietin of fetal origin for fetal regulation. The present biological assay for erythropoietin may not be sensitive to a fetal erythropoietin. In Stohlman's studies, it is important to note that they indicated that the transition of fetal to adult type of regulation was followed by a concomitant decrease in the number of syncytial cells present in fetal liver and myeloid erythropoiesis. These authors then proposed that syncytial cells serve as a fetal stem cell which is gradually replaced by more differentiated erythropoietin-sensitive cells. The syncytial cells described in the rat have also been observed in the human fetus. There is obviously some controversy regarding the ability and function of erythropoietin in the fetus. Zanjani, -et al. (33) for example, developed a successful surgical procedure to extensively study erythropoietin production in fetal lambs during the last third of the uterine development period. From these studies,theyconcluded that they could detect erythropoiesisstimulating activity or erythropoietin in the plasma obtained from the normal non-anemic fetus. Upon injection of phenylhydrazine into the fetus (resulting in acute anemia), highly significant amounts of erythropoietin or erythropoietic-stimulating activity material was apparrent in the circulation. This work offeredevidence that sheep in late pregnancy have the capacity to produce erythropoietin. Whether erythropoietin actually serves a role in the regulation of erythropoiesis was questionable. Because they did not measure hemoglobin types or relate hemoglobin to morphology, they were unable to determine if erythropoietin would have a direct effect on what type of hemoglobins were being produced. However, recent studies by Zanjani's laboratories have produced convincing evidence for the role of erythropoietin in fetal erythropoiesis (34). The administration of antibiody against erythropoietin to fetal sheep during the last third of the gestation period resulted in a supression of erythropoiesis in the fetus. Whether there is a fetal erythropoietin and adult erythropoietin is still up for question. Also, the effect of erythropoietin in early development is not known. The primary site of erythropoietic stimulating activity material is the kidney in the adult animal, however in the fetus the major source may be the spleen and liver (35). Equally important for the understanding of fetal erythropoiesis is the intriguing finding of erythropoiesis inhibiting factor in the fetus. (36). In studies of bone marrow in newborn calves, Gabuzda, et al. (37) observed that erythropoietin was capable of stimulating both adult and fetal hemoglobin synthesis. Gabuzda felt there was no direct effect of
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erythropoietinin differentiallydetermininggenetic transcriptionor translastiouof these globin types. And since the synthesisof the two hemoglobinsproceeded in a synchronousmanner, there was no evidence that erythropoietinstimulatedpreferentiallythe synthesisof fetal hemoglobin in the less mature erythrocyte. This eliminatedthe possibility that erythropoietinacts on fetal hemoglobinby inducing stem cells of a populationof immature erythroblasts. He felt this data was consistentwith -in vivo observationsin the human sickle cell anemia patient where the adult and fetal hemoglobinwere simultaneously suppressedfollowingtransfusion. In contrast to these findings,there is the data of Necheles, et al., (38) who concluded that erythropoietin in vitro stimulates adult but not fetal hemoglobin synthesis. These -investigators were working with adult human bone marrow tissue, which can be directly compared with bone marrow tissue of the newborn calf. On the other hand, the use of newborn calves is not comparable to studies performed on erythropoiesis of primarily hepatic origin. Studies cited here not only involve different stages of development and species, but also in some cases did not ascertain the hemoglobin types being produced. From limited studies, one can only conclude that each species reacts differently to the regulation by erythropoietin at different temporal times of development. In the study of the newborn sheep, this fetal work in human and calf must be contrasted to the direct mediation of the transition of A to C by erythropoietin, as reported by Boyer (15) and others (39). Present criteria indicate that yolk sac erythropoiesis, in which immature precursors do not persist, is not responsive to erythropoietin (40). Thirteen-fourteen day fetal erythroid cells of hepatic origin in the mouse are quite susceptible to the effect of erythropoietin (40, 41). The effects of erythropoietin on purified erythroid cell precursors Cantor, -et a1.(42) studied the erythropoietin in vitro were studied. _effect on isolated immature erythroid cell precursors. Their data now identifies the erythropoietic-sensitive cell as an early precursor which although descendant, is distinct from the multi-potent hemopoietic stem cell. It is morphologically indistinguishable from the class of cells identified as proerythroblasts. These cells exhibit a marked acceleration of RNA after exposure to erythropoietin. The cellular response to erythropoietin -in vitro includes proliferation of differentiating erythroblasts, initiation of hemoglobin synthesis and preservation of the proliferation for self-perpetuation (40, 41, 42). Basch (43) concludes that human fetal hemopoietic tissue is responsive to erythropoietin. He has concluded from his studies that stimulation by this hormone did not collectively stimulate the production of one or another of the human hemoglobins A or F. His work included cells of very young gestation age: 6 weeks. He felt that at least in the human, there was no evidence that erythropoietin was involved in the transition that occurs in sheep which have been injected with erythropoietin. From his studies, he then concluded that erythropoietin does not mediate the transition of F to A in man. Therefore, the transition of human hemoglobin from fetal to adult resembles that of cattle rather than the A to C transition in sheep which is mediated by erythropoietin. It is felt that on a cellular basis, erythropoietin
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induces replication of erythroid precursors and causes differentiation of these precursor cells to hemoglobin synthesizing cells. Its relationship to the transition of hemoglobins in animals other than the A to C transition in sheep remains quite nebulous.
Transition of Hemoglobin Type The control of the transition from fetal hemoglobin to adult or from adult to fetal is open to speculation. An excellent review of the "switch" from fetal to adult has been given by Bertles (44, 45). One can easily invoke a variety of mechanisms which include repression, derepression and control genes, etc. However, all explanations whether they apply to mammalian systems or not , must consider control at the cistron level and must explain the fact that the transition occurring in pathological events in man is often accompanied by pleiotropic regression (44, 45, 46). There is a transition or reversion to erythrocytes having fetal characteristics, enzymes and surface antigens. Although there is little doubt that transition occurs at a level of differentiation, one can consider some of the more gross aspects of control which have been associated with specific pathological conditions. In some of those not so associated, a simple approach might be to consider if there is a relationship between red cell production, oxygen demand and the hemoglobin type. Some examples would be: 1) simple anemia, cyanosis or hypoxia are pathological features which do not universally result in the transition of adult to fetal hemoglobin; 2) persistence of fetal hemoglobin has been associated with juvenile myelocytic leukemia; 3) untreated Addisonian pernicious anemia patients have had an increase in fetal hemoglobin; 4) the fact that hormones are related to regulation is suggested by the discovery of a definite increase in fetal hemoglobin in the blood of women in the second trimester of pregnancy; and 5) the genetically determined persistent high fetal hemoglobin is associated with no marked pathological features. These are a few examples which indicate there is no one universal mechanistic prediction that can be made. Acceleration of the transition from tadpole to frog hemoglobin occurred in studies by Moss and Ingram (47) by adding thyroid hormone to their environment. However, this transition is accompanied by a corresponding change in red cell population, similar to the embryonic to adult shift in the mouse rather than the fetal to adult in man and sheep.
Reversion to Fetal Erythropoiesis There is clear evidence in the human that the transition to fetal hemoglobin is accompanied by a switch to red blood cell populations which have different biochemical characteristics such as surface antigens and red blood enzymes. One recently reported phenomena which is unique to the fetal erythrocyte is the non-pathological transient fetal porphyria of the fetus of all mammals thus far studied (48). Characteristic of the phenomena are the occurrence of uroporphyrin I and III in the cartilage and bones and the km differences of the delta aminolevulinic acid
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THERIOGENOLOGY synthetase (49). Because of these fetal characteristics of the porphyrin pathway during a period in development, there is a difference in regulation and control of heme synthesis resulting in the accumulation of porphyrin compounds in the bone tissue for a period of time during gestation. This period of time differs for each species and is normally related to fetal development. The flourescence of the porphyrins in the fetal bones seen under ultraviolet light is not unlike the flourescence seen due to the accumulation of uroporphyin I in adult fox squirrel skeletal bones. The transient nature of the phenomena in developing fetuses can be related to the hepatic erythropoiesis. (See Figure 3) This evidence again underlines the distinct characteristics of fetal erythropoiesis. Conclusions In a recent review, the ontogeny of the hemoglobins of various animals has been made (4). From this and various other articles on fetal erythropoiesis (2, 3, 15, 44, 45) the following comparison of human developmental erythropoiesis can be made with that of other mammals. (See Table I). It is quite clear that there are similarities and differences upon comparison of the ontogeny of hemoglobin among species. The evidence for regulation of erythropoiesis by erythropoietin in late pregnancy is convincing. However, the role of erythropoietin in the transition of hemoglobin types has not been established with the exception of hemoglobin C in sheep. Further work needs to be done on regulation of erythropoiesis during early embryonic and fetal periods in mammals. Emphasis should be placed not only on hemoglobin types but also upon other markers related to fetal erythropoiesis. An increase in oxygen affinity by fetal red blood cells is common to all species thus far reported. However, the mechanism which accounts for the differences in oxygen affinity can be quite different in various species. Upon comparison the following generalizations can be made. Mechanisms By Which Fetal Blood Can Have A Higher Oxygen Affinity than Maternal Blood 1.
The fetal hemoglobin may have an intrinsically higher oxygen affinity independent of any intracellular co-factors. Examples include the sheep and the goat.
2.
Fetal hemoglobin may have impaired interaction with a red cell co-factor. For example, the oxygen affinity of “stripped” human fetal hemoglobin is very similar to that of adult hemoglobin - A. However, hemoglobin F binds less strongly to 2,3 DPG, and therefore, in the presence of 2.3 DPG within the red blood cell, its oxygen affinity is correspondingly greater.
3.
The content of 2,3 DPG may be significantly less in fetal red blood cells, and change rapidly after birth. Through a
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mechanism of modulation the 2,3 DPG content can alter the oxygen affinity. This is true for the horse and for the pig and dog. The first two mechanisms cited above require the presence of the fetal hemoglobin which differs structurally and functionally from the adult hemoglobin. But in the third, the hemoglobin may be identical in the adult and the fetus, and indeed, this is the case for the horse, dog and pig.
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TABLE
I
COMPARISON OF HUMAN EMBRYONIC AND FETAL ERYTHROPOIESIS WITH ERYTHROPOIESIS OCCURRING DURING DEVELOPMENT INOTHERMAMMALS
Human Fetal Erythropoiesis
Animal Fetal Erythropoiesis
Embryonic hemoglobin associated with an unique nucleated red blood cell
All mammals thus far studied have embryonic erythropoiesis with a corresponding embryonic hemoglobin type.
Unique fetal hemoglobin
Not all species demonstrate a unique fetal hemoglobin
Multiple cistrons for genetic control of fetal hemoglobin
Very limited evidence for multiple cistrons for genetic control of fetal hemoglobin (only in primates)
Persistence of fetal hemoglobin after birth and into the adult
Prolonged persistence of fetal hemoglobin after birth has not been shown in animals even at a very small level.
Relatively slow transition from fetal to adult
Relatively rapid transition from fetal to adult hemoglobin
Shortened life span of fetal cells
Little or no information is available regarding life span of fetal red blood cells in animals.
Red blood cell surface antigens unique to the fetal erythrocyte
Red blood surface antigens unique to fetal erythrocytes have not been reported
Changes in oxygen affinity due to modulation of 2,3 DPG
The hemoglobin within the erythrocytes of some animal species are affected by modulation of 2,3 DPG, others are not
Transient non-pathological porphyria during development
All animals show a transient porphyria during early gestation
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HUMAN
240
60
Red Blood
100
y.
4Birth DAYS
MOUSE
Embryonic
(/Adult
k,--
,
Cells
Containing a Distinct Hemoglobin DAYS
SHEEP
,Adul
I
100 9%
t
I
F&l
I I
Birth
Iy
fl
I Hb C
s.150 DAYS
Temporal Relation of Transition of Embryonic, Fetal and Adult Hemoglobins in Various Mammals Figure 1.
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FIGURE 2A. Subunit composition of mammalian hemoglobins. Four polypeptide chains, two non-identical pairs account for the subunit composition of mammalian hemoglobin. The pairs can be classified as alphas (a) and non-alphas (a 6,y, etc.)
i3 a
a
=
cr2f32
B
The shorthand formula can be illustrated as a2Bz for adult human hemoglobin and a2y2 for fetal or a2E2 for embryonic. Other minor components are given other Greek letter designations in human beings. In the embryonic mouse, non a chains are identified as either x, y, or 2.
FIGURE 2B. Ontogeny of hemoglobin: subunit comparison of the structure of embryonic, fetal and adult hemoglobin in various species. The amount of adult hemoglobin seen in each species is also given by estimate of the percent at birth.
Mouse
EMBRYONIC
Human
5
x2
y2
El
EQ
31
(32
y2
E
a2E2
511
a2
22
Es&
II
a2
y:
a2
Y2
A
FETAL
a2!32
a262
ADULT
AUGUST-SEPTEMBER
Sheep
a2
E2
a2y2
a2B2
a262
100%
2040%
at Birth
at Birth
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O-10% at Birth
231
U n its/g
6ALAS
Liver
600
1200
35 40 DAYS
50 60
65
0
Figure 3.
Total Concentration of Porphyrins in Amniotic Fluid and Concentration of a-Amino Levulinic Acid Synthetase from Guinea Pig Liver as Related to Hepatic Erythropoiesis
30
50
100
Fluid
mg/lOOml Porphyrins in Amniotic
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