Primitive Erythropoiesis Research Articles

Bipotential Primitive-Definitive Hematopoietic Progenitors in the Vertebrate Embryo

Two regions of the vertebrate embryo, the blood islands and the dorsal lateral plate (DLP), participate in early hematopoietic development. In Xenopus, primitive erythrocytes are derived solely from the ventral blood islands (VBI), while definitive hematopoietic cells such as lymphocytes are derived from both VBI and DLP. We have utilized a transplantation technique to demonstrate in vivo that all hematopoietic cells (embryonic, fetal, or adult) originate from ventral mesoderm. Reciprocal grafts between VBI and DLP demonstrated that both regions are bipotential with respect to primitive and definitive hematopoiesis. Commitment of the VBI to primitive erythropoiesis and restriction of the DLP to definitive hematopoiesis occurs during neurula stages. Thus, hematopoietic development involves the induction of the blood program on the ventral axis of the embryo followed by environmentally regulated specification to the primitive or definitive lineages.

GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis

Transcription factor GATA-1 is required for the terminal differentiation of both the primitive and definitive erythroid cell lineages, and yet the regulatory mechanisms of GATA-1 itself are not well understood. To clarify how the GATA-1 gene is transcriptionally controlled in vivo, presumptive regulatory regions of the gene were tested by fusion to a reporter gene and then examined in transgenic mice. We found that a transcriptional control element located between -3.9 and -2.6 kb 5' to the erythroid first exon serves as an activating element and that this sequence alone is sufficient to recapitulate the expression of GATA-1 (but uniquely in primitive erythroid cells). Addition of sequences from the GATA-1 first intron to this upstream element provides a necessary and sufficient condition for complete recapitulation of GATA-1 expression in both primitive and definitive erythroid cells. The first intron element does not possess intrinsic transcriptional activation potential when linked to the GATA-1 gene promoter but rather requires the upstream activating element for its activity. These experiments show that GATA-1 gene expression is regulated by discrete transcriptional control elements during definitive and primitive erythropoiesis: The 5' element displays properties anticipated for a primitive erythroid cell-specific activating element, and the novel element within the GATA-1 first intron specifically augments this activity in definitive erythroid cells.

Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta.

Core binding factor beta (CBF beta) is considered to be a transcriptional coactivator that dimerizes with transcription factors core binding factor alpha 1 (CBFA1), -2, and -3, and enhances DNA binding capacity of these transcription factors. CBF beta and CBFA2, which is also called acute myeloid leukemia 1 gene, are frequently involved in chromosomal translocations in human leukemia. To elucidate the function of CBF beta, mice carrying a mutation in the Cbfb locus were generated. Homozygous mutant embryos died between embryonic days 11.5-13.5 due to hemorrhage in the central nervous system. Mutant embryos had primitive erythropoiesis in yolk sac but lacked definitive hematopoiesis in fetal liver. In the yolk sac of mutant embryos, no erythroid or myeloid progenitors of definitive hematopoietic origin were detected, and the expression of flk-2/flt-3, the marker gene for early precursor cells of definitive hematopoiesis, was absent. These data suggest that Cbfb is essential for definitive hematopoiesis in liver, especially for the commitment to early hematopoietic precursor cells.

Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor

Erythropoietin (EPO) is the principal growth factor regulating the production of circulating erythrocytes. We introduced null mutations into both Epo and the EPO receptor ( EpoR) gene. Both heterozygotes appeared normal. Homozygous animals exhibited reduced primitive erythropoiesis and died around embryonic day 13, owing to failure of definitive fetal liver erythropoiesis. Both types of mutations exhibited identical phenotypes, indicating that EPO and the EPOR are crucial for definitive erythropoiesis in vivo and that no other ligands or receptors can replace them. Committed erythroid BFU-E and CFU-E progenitors were present in both homozygous fetal livers. Thus, neither EPO nor the EPOR is required for erythroid lineage commitment or for the proliferation and differentiation of BFU-E to CFU-E progenitors. EPO and the EPOR are crucial in vivo for the proliferation and survival of CFU-E progenitors and their irreversible terminal differentiation.

Initiation of Hematopoiesis and Vasculogenesis in Murine Yolk Sac Explants

The blood islands of the visceral yolk sac (VYS) are the initial sites of hematopoiesis in mammals. We have developed a yolk sac explant culture system to study the process of blood cell and endothelial cell development from extraembryonic mesoderm cells. No benzidine-positive cells or beta H1-globin mRNA expression was detected at the primitive streak or neural plate stage of development (E7.5). However, when isolated E7.5 dissected tissues were cultured for 36 to 72 hours in serum-free medium, hundreds of hemoglobin-producing cells and embryonic globin gene expression were identified in both intact yolk sac and VYS mesoderm explants. Explanted E7.5 extraembryonic mesoderm tissues thus recapitulate in vivo primitive erythropoiesis and do not require the presence of a vascular network or the VYS endoderm. Yolk sac blood islands also contain endothelial cells that arise by vasculogenesis and express flk-1. We detected flk-1 mRNA as early as the primitive streak stage of mouse embryogenesis. Culture of embryo proper and intact VYS explants, which contain both mesoderm and endoderm cells, produced capillary networks and expressed flk-1. In contrast, vascular networks were not seen when VYS mesoderm was cultured alone, although flk-1 expression was similar to that of intact VYS explants. The addition of vascular endothelial growth factor to VYS mesoderm explants did not induce vascular network formation. These results suggest that the VYS endoderm or its extracellular matrix is necessary for the coalescence of developing endothelial cells into capillary networks.

Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.

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

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

Regulated Expression of Globin Chains and the Erythroid Transcription Factor GATA-1 during Erythropoiesis in the Developing Mouse

Erythropoiesis in vertebrates is characterized by sequential changes in erythropoietic site, erythroblast morphology, and hemoglobin synthesis. We have examined the expression of globin chains and the major erythroid transcription factor GATA-1 (previously known as GF-1/NF-E1/Eryf 1) from days 7.5 to 17.5 of mouse development. mRNAs for embryonic (epsilon y2, beta H1, and zeta) and adult (alpha and beta) globin chains were quantitated by RNase protection assays. Switching of globins within the alpha-globin cluster (alpha and zeta) was not strictly coordinated with that within the beta-globin cluster (epsilon y2, beta H1, and beta). Regulation of globin switches during development was primarily transcriptional. Of particular note, we found two developmental switches (beta H1 to epsilon y2 and epsilon y2 to beta) in the mouse, more analogous than previously thought to shifts found in human development. The erythroid transcription factor GATA-1, believed to be a principal regulator of genes expressed in erythroid cells, first appeared in the embryo in yolk sac at the time of blood island formation and remained at a low level during embryonic erythropoiesis (8 to 11 days) relative to that found later in fetal liver (12 to 15 days). The rise in GATA-1 mRNA in fetal liver paralleled and preceded the rapid accumulation of adult beta-globin RNA. RNase protection assays and a GATA-1-specific peptide antiserum were used to establish that a single GATA-1 polypeptide is expressed throughout mouse development. Overall, these findings suggest that the levels of this erythroid transcription factor during development may contribute to the differential gene activation characteristic of definitive versus primitive erythropoiesis.

Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse.

Erythropoiesis in vertebrates is characterized by sequential changes in erythropoietic site, erythroblast morphology, and hemoglobin synthesis. We have examined the expression of globin chains and the major erythroid transcription factor GATA-1 (previously known as GF-1/NF-E1/Eryf 1) from days 7.5 to 17.5 of mouse development. mRNAs for embryonic (epsilon y2, beta H1, and zeta) and adult (alpha and beta) globin chains were quantitated by RNase protection assays. Switching of globins within the alpha-globin cluster (alpha and zeta) was not strictly coordinated with that within the beta-globin cluster (epsilon y2, beta H1, and beta). Regulation of globin switches during development was primarily transcriptional. Of particular note, we found two developmental switches (beta H1 to epsilon y2 and epsilon y2 to beta) in the mouse, more analogous than previously thought to shifts found in human development. The erythroid transcription factor GATA-1, believed to be a principal regulator of genes expressed in erythroid cells, first appeared in the embryo in yolk sac at the time of blood island formation and remained at a low level during embryonic erythropoiesis (8 to 11 days) relative to that found later in fetal liver (12 to 15 days). The rise in GATA-1 mRNA in fetal liver paralleled and preceded the rapid accumulation of adult beta-globin RNA. RNase protection assays and a GATA-1-specific peptide antiserum were used to establish that a single GATA-1 polypeptide is expressed throughout mouse development. Overall, these findings suggest that the levels of this erythroid transcription factor during development may contribute to the differential gene activation characteristic of definitive versus primitive erythropoiesis.

A karyometrical study of circulating erythroblasts of yolk sac origin in the mouse embryo.

Circulating erythroblasts of embryonic mice were karyometrically examined by light microscopy. In the erythroid cells in embryonic blood vessels, mitoses were encountered from 9 to 12 days of gestation. At 9 days, the circulating blood cells consisted of proerythroblasts and less mature cells. The nuclear diameter ranged from 4.8 to 9.8 micron, the majority ranging between 6 and 8 micron. At 11 days, hemopoietic cells with a nuclear diameter larger than 6 micron disappeared from embryonic circulation, and more than 90% had a nuclear diameter of less than 5 micron. Between 12 and 16 days of gestation, the smallest orthochromatic erythroblasts measuring 3.4 micron nuclear diameter showed the highest peaks. The progress of primitive erythropoiesis in embryonic circulation is discussed in comparison with that of definitive erythropoiesis.

Morphological aspects of prenatal haematopoietic development in the cat.

AbstractThe appearance of haematopoietic cells and the development of haematopoiesis in certain embryonic organs of the cat were studied using light microscopic (cytological smears and paraffin embedded tissues) and transmission electron microscopic methods. Primitive erythropoiesis occurred predominantly in the yolk sac whereas definitive erythropoiesis occurred in the yolk sac, liver, bone marrow and, to a lesser extent, in the spleen. In the yolk sac, erythropoiesis was predominantly intravascular whereas in the liver and bone marrow it was usually extravascular. Granulopoiesis occurred mainly in the liver and bone marrow. In the liver it was predominantly extravascular and occurred around vessels, bile ducts and in perisinusoidal spaces. Megakaryocytopoiesis occurred in the yolk sac, liver, bone marrow and spleen. The megakaryocytic line of cells were similar to those occurring in adult cats except that in the yolk sac unusual small, mononuclear to binuclear thrombocytogenic megakaryocytes were present also.The relative contributions of the embryonic organs of the cat to haematopoiesis were similar to those described for man and certain other mammals but the results for the sites of development and the appearance of early forms in the erythrocytic, granulocytic and megakaryocytic lines varied at times from those reported for man and other mammals.

Adult hemoglobins are synthesized in yolk sac microenvironment obtained from murine cultured blastocysts

The capability of yolk sac (YS) hematopoietic stem cells to produce in vitro either primitive or definite erythrocytes under the influence of stimulating agents was analyzed. Two systems were used: either 9-day-old yolk sacs were explanted and cultured or 3-day-old blastocysts were cultured under conditions in which they give rise only to YS tissue. In both systems, YS tissue produced only primitive erythrocytes. When the stimulating agents (spleen cell-conditioned medium or erythropoletin) were added to the medium, YS cultures gave rise to definitive erythrocytes. These data confirm the intrinsic ability of the YS stem cells to undergo primitive erythropoiesis; furthermore, they provide evidence that YS hemopoietic progenitor cells do not require any direct interaction with cells of the embryo to undergo definitive erythropoiesis but only stimulation by agents, such as pokeweed mitogen spleen conditioned medium or erythropoietin; the YS microenvironment does not counteract this capacity.

Genome Activity and Gene Expression in Avian Erythroid Cells

Publisher Summary This chapter discusses the genome activity and gene expression in avian erythroid cells. Avian embryogenesis involves at least three generations of erythroid cells, which are (1) the cells of primitive erythropoiesis occurring in the yolk sac, (2) the cells of definitive erythropoiesis in the yolk sac, and (3) the cells of definitive erythropoiesis in the bone marrow. Erythroid cells of different generations differ in morphology, dimensions, the size of the nucleus, and the type of hemoglobin. The functions of the globin genes coding for embryonic and adult globins are switched over at appropriate stages of development by starting a new cell generation rather than intracellularly through modulation of gene activity (repression of the genes for embryonic hemoglobins and activation of the genes for definitive hemoglobins). Analysis of heterogenous nuclear ribonucleic acid (hnRNA) metabolism in differentiating erythroid cells reveals a remarkable feature, the existence of stable hnRNA derivatives in their nuclei. The chapter also describes the possible mechanisms regulating expression of the genes in differentiating avian erythroid cells.

Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos.

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerable less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythrosynthesis in whole cells and in isolated nuclei.

On the yolk sac of the cat. II. Erythropoietic phases, ultrastructure of aging primitive erythroblasts, and blood vessels.

The phase of primitive erythropoiesis in the feline yolk sac lasts from the 14th to the 20th day after malting. The globular nucleated primitive erythroblasts are formed extravascularly to some extent, but they can be clearly distinguished from the endoderm. They do not undergo a denucleation and are still present in the circulating blood on the 45th day. Aging primitive erythroblasts are characterized by a loss of polysomes, by the appearance of long intracytoplasmic electron-lucent channels, and by a nuclear pyknosis which can turn into a karyolysis. Definitive erythropoiesis begins around the 17th day but, even by the 19th day, it is not particularly prominent. It ends around the 45th day. It is almost exclusively intravascular. The distinction of immature primitive erythroblasts from erythroblasts of the definitive series is difficult, because it is based upon only slight differences in the heterochromatinization, in the nuclear-cytoplasmic ratio, and in the organelle content of the cells. In the definitive series, the nuclear divisions follows the law of the rhythmical halving of the nuclear volume. The cells exhibit more clearly identifiable maturation stages here, and the 'checkerboard nucleus' is more distinct. The vascular endothelium is largely attenuated and moderately fenestrated; it lacks a distinct basement membrane. Organelle-rich adventitial cells are found in close apposition.

The erythroid cells and haemoglobins of the chick embryo

The changes in the types of erythroid cells produced during embryogenesis of the chick have been correlated with the changes in the types of haemoglobins found in the embryo. Primitive erythroid cells constitute the only red blood cells of 2- to 5-day embryos. The first recognizable immature definitive erythroid cells appear in the embryonic circulation at 5 to 6 days and progressively replace the primitive cells, such that by 14 to 16 days the primitive cells constitute less than 1 % of the circulating erythroid cells. Primitive erythropoiesis is strikingly different from definitive erythropoiesis. At any one time point between 2 and 16 days, all of the isolated primitive cells appear, by morphological criteria, to be at the same stage of maturation, and, although variation in cell size is observed, for an individual maturation stage, the small cells are not more mature than the medium-size cells, nor are the large cells less mature than the medium or small cells. Maturing primitive erythroid cells undergo the progressive changes in cell structure characteristic of erythroid maturation in mammalian erythropoietic systems, but do so as a uniform cell population. Haemoglobin, isolated from primitive erythroid cells of 2- to 5-day embryos, shows two components on polyacrylamide gel electrophoresis, haemoglobin E and haemoglobin P. The haemoglobin E/P ratio is constant in lysates from 2- to 5-day embryos. A t 6 to 7 days when the first haemoglobinized immature definitive erythroid cells appear in the embryonic circulation, two new haemoglobin components are observed in lysates of erythroid cells. These two new haemoglobin components are electrophoretically and immunologically identical to the two haemoglobin components of adult chickens, haemoglobins A and D. As the definitive erythroid cells replace the primitive erythrocytes in the embryonic circulation, the haemoglobins A and D increase in amount and replace haemoglobin P. Haemoglobin P cannot be detected immunologically in erythroid cell lysates from 16-day embryos which contain less than 1 % primitive cells. In erythroid cell lysates from late embryos, which contained few, if any, primitive erythrocytes, a minor haemoglobin, electrophoretically similar to haemoglobin E on pH 10.3 polyacrylamide gels, is consistently observed. This component differs from haemoglobin E on pH 8.9 polyacrylmide gels, on Sephadex G-100 columns, on polyacrylamide gels of different porosities, and shows a reaction of only partial identity with haemoglobin E by two-dimensional immunodiffusion. This haemoglobin component, haemoglobin H, is detectable electrophoretically in lysates from 12-day embryos and immunologically in lysates from 8-day embryos. Haemoglobin H has not been observed in adult chickens. The switch from the production of primitive to definitive erythroid cells during development of the chick embryo is associated with the initiation of synthesis of three new haemoglobins, the two adult haemoglobins and haemoglobin H. The haemoglobin D /A ratio of adult chicken haemoglobin, determined from the ratio of gel scan peak masses, is 0.30. When haemoglobins D and A first appear in erythroid cell lysates from 6- to 7-day embryos, the haemoglobin D /A ratio is about 0.9. T he D/A ratio of lysates falls to 0.5 by 16 to 18 days, a time when 99 % of the erythroid cells of the embryo are mature definitive erythrocytes. However, the haemoglobin D /A ratio of lysates from late embryos and young chicks of 0.5 to 20 days of age is consistently greater than that of adult chicken haemoglobin. Definitive erythrocytes of chick embryos and young chicks appear to differ from definitive cells of adult chickens in at least two ways: the presence of haemoglobin H and the higher haemoglobin D/A ratio.