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.     

Immunohistochemical analysis of the distribution of histone H5 and hemoglobin during chicken development

We used immunohistochemical procedures to investigate embryonic erythropoiesis in serial sections of chicken embryos after 2-13 days of incubation. Antibodies specific for the erythrocyte-specific histone H5, for embryonic hemoglobin, and for adult hemoglobin were used as markers for general, primitive, and definitive erythropoiesis, respectively. Histone H5 was present in erythrocytes at all of the stages studied, i.e., in both the primitive and definitive cells. Cell of the definitive lineage were first detected, at about 5-6 days of incubation, in erythroid foci in the mesenchyme around the vitelline stalk. At 7-9 days of incubation, a massive mesenchymal conglomeration of erythropoietic cells developed, extending from the cervical to the abdominal region and ventrally to the vertebral body, with its largest extensions being around the arteries in the mediastinum. Immunostaining revealed that these erythroid cells belonged to the definitive erythropoietic lineage. These cells had disappeared completely after 12 days of incubation, i.e., before erythropoiesis is visible in the bone marrow. These observations are consistent with the notion that the yolk sac is essential for the formation of the definitive erythroid lineage.     

Distribution of developmentally regulated hemoglobins in embryonic erythroid populations.

Both cellular and molecular mechanisms regulate the expression of globin genes during development and differentiation.When a change occurs in the type of hemoglobin synthesized, it may be the result of a substitution of erythroid stem cell lineages or may arise through a modulation of globin gene expression after cells become committed to erythroid differentiation. We have investigated the relationship between the early to late embryonic hemoglobin switch and the primary to definitive erythrocyte change in chick embryos. Using double-label fluorescent antibody technique, we find the simultaneous presence of early and late hemoglobins in single erythrocytes of the definitive cell type. Synthesis of early embryonic hemoglobin is not restricted to the primary cell lineage. This evidence is most compatible with the hypothesis that erythroid cells become committed to the synthesis of specific globins after they have become committed to hemoglobin synthesis in general.     

Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.

In this study, we have mapped the onset of hematopoietic development in the mouse embryo using colony-forming progenitor assays and PCR-based gene expression analysis. With this approach, we demonstrate that commitment of embryonic cells to hematopoietic fates begins in proximal regions of the egg cylinder at the mid-primitive streak stage (E7.0) with the simultaneous appearance of primitive erythroid and macrophage progenitors. Development of these progenitors was associated with the expression of SCL/tal-1 and GATA-1, genes known to be involved in the development and maturation of the hematopoietic system. Kinetic analysis revealed the transient nature of the primitive erythroid lineage, as progenitors increased in number in the developing yolk sac until early somite-pair stages of development (E8.25) and then declined sharply to undetectable levels by 20 somite pairs (E9.0). Primitive erythroid progenitors were not detected in any other tissue at any stage of embryonic development. The early wave of primitive erythropoiesis was followed by the appearance of definitive erythroid progenitors (BFU-E) that were first detectable at 1-7 somite pairs (E8.25) exclusively within the yolk sac. The appearance of BFU-E was followed by the development of later stage definitive erythroid (CFU-E), mast cell and bipotential granulocyte/macrophage progenitors in the yolk sac. C-myb, a gene essential for definitive hematopoiesis, was expressed at low levels in the yolk sac just prior to and during the early development of these definitive erythroid progenitors. All hematopoietic activity was localized to the yolk sac until circulation was established (E8.5) at which time progenitors from all lineages were detected in the bloodstream and subsequently in the fetal liver following its development. This pattern of development suggests that definitive hematopoietic progenitors arise in the yolk sac, migrate through the bloodstream and seed the fetal liver to rapidly initiate the first phase of intraembryonic hematopoiesis. Together, these findings demonstrate that commitment to hematopoietic fates begins in early gastrulation, that the yolk sac is the only site of primitive erythropoiesis and that the yolk sac serves as the first source of definitive hematopoietic progenitors during embryonic development.     

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching.

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.     

Embryonic and fetal beta-globin gene repression by the orphan nuclear receptors, TR2 and TR4

The TR2 and TR4 orphan nuclear receptors comprise the DNA-binding core of direct repeat erythroid definitive, a protein complex that binds to direct repeat elements in the embryonic and fetal beta-type globin gene promoters. Silencing of both the embryonic and fetal beta-type globin genes is delayed in definitive erythroid cells of Tr2 and Tr4 null mutant mice, whereas in transgenic mice that express dominant-negative TR4 (dnTR4), human embryonic epsilon-globin is activated in primitive and definitive erythroid cells. In contrast, human fetal gamma-globin is activated by dnTR4 only in definitive, but not in primitive, erythroid cells, implicating TR2/TR4 as a stage-selective repressor. Forced expression of wild-type TR2 and TR4 leads to precocious repression of epsilon-globin, but in contrast to induction of gamma-globin in definitive erythroid cells. These temporally specific, gene-selective alterations in epsilon- and gamma-globin gene expression by gain and loss of TR2/TR4 function provide the first genetic evidence for a role for these nuclear receptors in sequential, gene-autonomous silencing of the epsilon- and gamma-globin genes during development, and suggest that their differential utilization controls stage-specific repression of the human epsilon- and gamma-globin genes.     

Evidences that hemoglobin switch in the chick embryo depends on erythroid cell line substitution

Chemical identifications of various hemoglobin types were performed on unfractionated erythroid cells derived from chicken embryos at 5 and 7 days of development and on purified primitive and definitive cells. Proteins were pulse-labelled in primitive erythroid cells at various times of culture to identify those actually synthesized. The data show that primitive cells contain and synthesize only embryonic hemoglobins at all stages of maturation and definitive cells contain adult and minor embryonic hemoglobins, but no major embryonic hemoglobins, not even in trace amounts. These results support a model for hemoglobin switch in the chicken embryo based on cell line substitution.     

A global role for zebrafish klf4 in embryonic erythropoiesis

There are two waves of erythropoiesis, known as primitive and definitive waves in mammals and lower vertebrates including zebrafish. The founding member of the Kruppel-like factor (KLF) family of CACCC-box binding proteins, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. Morpholino knockdown experiments have shown a role for zebrafish klf4 in primitive erythropoiesis and hatching gland formation. In order to generate a global understanding of how klf4 might influence gene expression and differentiation, we have performed expression profiling of klf4 morphants, and then performed validation of many putative target genes by qRT-PCR and whole mount in situ hybridization. We found a critical role for klf4 in embryonic globin, heme synthesis and hatching gland gene expression. In contrast, there was an increase in expression of definitive hematopoietic specific genes such as larval globin genes, runx1 and c-myb from 24 hpf, suggesting a selective role for klf4 in primitive rather than definitive erythropoiesis. In addition, we show klf4 preferentially binds CACCC box elements in the primitive zebrafish beta-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions.     

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

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

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.