Yolk Sac Erythroid Cells Research Articles

Krüppel-like factor 2 regulated gene expression in mouse embryonic yolk sac erythroid cells

KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell and erythroid development. KLF2−/− mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. In KLF2−/− embryos, β-like globin gene expression is reduced, and E10.5 erythroid cells exhibit abnormal morphology. In this study, other genes regulated by KLF2 were identified by comparing E9.5 KLF2−/− and wild-type (WT) yolk sac erythroid precursor cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression between KLF2−/− and WT; eighty-nine of these are downregulated in KLF2−/−. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. The SOX2 gene, encoding a pluripotency factor, is regulated by KLF2 in both ES and embryonic erythroid cells. Previous work had identified genes with erythroid-enriched expression in the yolk sac. The erythroid-enriched genes reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2−/− compared to WT and are therefore candidates for controlling primitive erythropoiesis. Each of these genes contains a putative KLF2 binding site(s) in its promoter and/or an intron. Reelin has an established role in neuronal development. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter in erythroid cells, validating this approach to identify KLF2 target genes.

Ikaros interacts with P-TEFb and cooperates with GATA-1 to enhance transcription elongation

Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human β-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.

Comparing genetic profiles of embryonic day 9 (E9) mouse yolk sac erythroid and erythroid and epithelial cells isolated by microdissection

Comparing genetic profiles of embryonic day 9 (E9) mouse yolk sac erythroid and erythroid and epithelial cells isolated by microdissection

Hypoxia-inducible Factor-1 Deficiency Results in Dysregulated Erythropoiesis Signaling and Iron Homeostasis in Mouse Development

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

Comparing the genetic profiles of E9 mouse yolk sac erythroid and epithelial cells isolated by microdissection

Comparing the genetic profiles of E9 mouse yolk sac erythroid and epithelial cells isolated by microdissection

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice.

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.

PRIMITIVE ERYTHROPOIESIS IN YOLK SAC EXPLANTS IS INHIBITED BY ANTISENSE ERYTHROPOIETIN-RECEPTOR, c-RAF, AND JAK2 OLIGODEOXYNUCLEOTIDES (ODN). • 940

Erythropoietin (EPO) binds its specific receptor (EPO-R) to mediate survival and proliferation of definitive (bone marrow/fetal liver) red cell precursors. In contrast, little is known about cytokine signaling inprimitive (yolk sac) erythroblasts. We detected EPO-R mRNA by in situ hybridization at early stages of murine yolk sac blood island development(E7.5-E8.5). Because the size and location of the mammalian embryo makes experimental manipulation difficult, we developed a serum-free murine yolk sac explant culture system {Blood 86:156, 1995}, and an antisense experimental approach to examine the functional role of EPO/EPO-R during the initial development of primary primitive erythroblasts from extra-embryonic mesoderm cells. The addition of EPO to E7.5 yolk sac explants increased both the accumulation of βH1-globin mRNA and the number of differentiating yolk sac erythroblasts compared to untreated explants. Antisense EPO-R ODN decreased βH1-globin accumulation 95% (p 50% (p<0.05), compared to missense EPO-R ODN-treated and untreated explants. A similar experimental approach was used to investigate putative EPO-R intracellular signaling cascades. Antisense c-raf and Jak2 ODN each decreased the number of primitive erythroblasts 40% (p<0.01), but only antisense c-raf ODN significantly decreased erythroid progenitors. We conclude that EPO/EPO-R signaling is functionally active during the initial differentiation of primary yolk sac erythroid cells, and suggest that this signaling may involve both the ras/raf and the Jak2/Stat pathways.

Existence and differential changes of peptidylarginine deiminase type II in mouse yolk-sac erythroid cells.

Peptidylarginine deiminase (PAD) catalyzes the conversion of arginyl residues in proteins to citrullyl residues in the presence of Ca2+. Recently, we obtained a monoclonal antibody, EH7, which reacted only with mouse PAD type II. Here, we describe immunohistochemical findings on the cellular localization of PAD type II in mouse fetus by using the monoclonal antibody. PAD type II is expressed in yolk-sac erythroid cells and the level of the enzyme in these cells decreases as the cells differentiate.

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.

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro.

The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.

Erythropoietin: Receptor characteristics during the ontogeny of hamster yolk sac erythroid cells

Erythropoietin (Epo) binds specifically to receptors on the surface membrane of responsive erythroid cells. In search of ontogenic changes in Epo receptor behavior, we studied characteristics of specific binding to hamster yolk sac erythroid cells during hamster ontogeny. We detected receptors specific for Epo on these cells throughout the duration of their intravascular existence (hamster gestational days 8 through 13). These receptors are saturable at an Epo concentration of 1.2 nM in the incubation medium. Attainment of equilibrium of binding prior to hormone internalization, a requirement for receptor binding assays, was possible at 10 degrees C but not at 37 degrees C. Hence, all incubations of cells with Epo were carried out at 10 degrees C. Data on specific binding analyzed by the method of Scatchard demonstrated that yolk sac erythroid cells possess a single class of Epo receptors at each stage of gestation examined. Binding affinity and numbers of receptors per cell change as ontogeny progresses: Kd (the dissociation constant) increases, a phenomenon observed in other differentiating cell systems, whereas the number of receptors per cell peaks on gestational day 10. The variability in number of receptors per cell is consonant with up and down regulation controlled by Epo availability. We propose that the progressive increase in Kd might be best explained by ontogenic changes in cell membrane structure contiguous to the receptors themselves.

Ontogeny of hamster hemoglobins in yolk-sac erythroid cells in vivo and in culture.

During mammalian hemoglobin ontogeny, synthesis of the earliest globin chains (embryonic) is ultimately replaced by synthesis of globin chains (adult) characteristic of the fully formed organism. Elements of control of initiation, progression, and completion of globin-chain ontogeny are poorly understood. In search of a cell culture system in which ontogeny might be studied under closely controlled experimental conditions, we chose erythroid cells of the hamster embryo. First, the ontogeny of globin chains was defined in these yolk-sac-derived erythroid cells from day 10 through day 13 in gestation. Amounts of individual embryonic and adult globin chains were quantified, as were their rates of synthesis. Next, analogous studies were performed on yolk-sac erythroid cells from day 10 in gestation (prior to the appearance of fetal liver) grown in culture for 3 days, corresponding to days 10-13 in vivo. The ontogenic program in culture was virtually identical to that in vivo. Approximately 70% of active globin synthesis was embryonic at day 10 in gestation (day 0 of culture), declining to 30% by day 13 in gestation (day 3 of culture). Whereas only trace synthesis of the adult non-alpha chains (beta major and beta minor) were initially observed, their combined active synthesis achieved a level of approximately 30% 3 days later both in vivo and in culture. Cell hemoglobin content and cell morphology were similar in both systems. We conclude that an ontogenic program for globin-chain synthesis exists in these primitive erythroid cells, overriding possible influences of cell environment. Further, we suggest that these cells in culture provide a means of examining cell mechanisms associated with globin-gene ontogeny under controlled experimental conditions.

Simultaneous expression of globin genes for embryonic and adult hemoglobins during mammalian ontogeny.

The dominant hemoglobin of the adult hamster was detected in yolk-sac erythroid cells, and its identity was confirmed by peptide mapping and by analysis of relevant peptides. Both the presence and active synthesis of two embryonic hemoglobins presumed to exist only in yolk-sac erythroid cells were detected in neonatal liver and spleen. Thus the time span of expression of both embryonic and adult globin genes during mammalian ontogeny may be considerably broader than presently believed.

STRAIN-SPECIFIC APPEARANCE OF A POSSIBLE FETAL α-GLOBIN POLYPEPTIDE CHAIN IN MICE.

DDD mouse embryos at 12-16 days of gestation have a putative 'fetal'α-globin polypeptide chain, detected by its lower electrophoretic mobility than the authentic α-globin chain on acidic urea-Triton gel. When hepatic erythroid cells of DDD embryos were contaminated with yolk sac erythroid cells, two α-bands were visible, but only 'fetal'α seemed to be synthesized, because in embryos of strain ddY which share a common ancestry with DDD, only 'fetal'α was detectable by autoradiography when the hepatic erythroid cells of embryos of more than 15 days of gestation showed an authentic adult α-globin chain other than this 'fetal'α-globin chain. If this 'fetal'α-globin chain is structurally different from the yolk sac α and adult peripheral blood α-globin chains, then switching over of the transcription of α-globin polypeptide chain occurs in hepatic erythroid cells of mid-late DDD embryos. However, it is still possible that this 'fetal'α-globin chain is formed by post-translational modification of the authentic α-globin chain. The α-globin chains of hepatic erythroid cells of C57BL/6 and BALB/c embryos had similar mobilities to those of yolk sac and adult erythroid cells.

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

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

Terminal stages of erythroid differentiation: persistent production of globin messenger RNA is necessary to sustain globin synthesis

The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

Terminal Stages of Erythroid Differentiation: Persistent Production of Globin Messenger RNA is Necessary to Sustain Globin Synthesis

The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

Control of ribosomal RNA maturation in differentiating yolk sac erythroid cells

The processing of nucleolar pre-ribosomal RNA to cytoplasmic ribosomal RNA has been studied in differentiating yolk sac erythroid cells from mouse fetuses at the 10th and 13th day of gestation. The transcription of ribosomal genes is 20 times faster in less differentiated erythroblasts at day 10 than in mature erythroid cells at day 13; conversely, the processing of the methylated portion of 45S RNA is completely conservative in erythroid cells at day 13, but is conservative only at 20% in erythroblasts at day 10. In day-10 erythroblasts the processing of 32S RNA to 28S RNA is inhibited and both 32S RNA and 45S RNA accumulate in nucleoli; at the same time 18S RNA in excess over 28S RNA is destroyed before reaching the stage of mature ribosomes. The inhibition of 32S RNA processing is not due to a deficit of methylation, but may be related to a deficient complementation of proteins to newly made nucleolar RNA.

Arrest of cell division and hemoglobin synthesis in differentiating yolk sac erythroid cells

Yolk sac derived erythroid cells of C57B1/Cas fetal mice which are X irradiated with 120 rads at the 10th day of fetal development perform three normal cell cycles and synthesize hemoglobin in a normal fashion until day 12. Between day 12 and 13 normal cells perform one final cell cycle and reduce to half the rate of hemoglobin synthesis per cell; conversely, irradiated erythroid cells do not divide and maintain the full rate of hemoglobin formation. At day 13 irradiated non-divided erythroid cells contain polyribosomes in the same amount and with the same size distribution as normally divided cells and yet synthesize hemoglobin at a two-fold faster rate. This difference of hemoglobin synthesizing capacity is not due to new formation of messenger RNA for hemoglobin, neither it is due to a faster rate of translation, but rather because at day 13 non-divided erythroid cells have maintained the full complement of messenger RNA-ribosomal complexes active for hemoglobin synthesis, while in normally divided erythroid cells the number of messenger RNA-ribosomal complexes active for hemoglobin synthesis is cleaved in the course of cell division. In the irradiated erythroid population the total amount of each globin chain produced in the course of differentiation is not decreased in spite of the arrest of cell division. These data suggest that the total amount of hemoglobin produced during the development of the progeny of a single erythroid cell is programmed in early stages of erythroid differentiation.

Protein synthesis: its control in erythropoiesis.

Erythropoiesis in the fetal mouse provides a model to study several important aspects of the regulation of cell differentiation and differentiated protein synthesis. Changes in the patterns of hemoglobins formed during fetal and postfetal development are shown to be associated with the substitution of the liver erythroid cell line. In the course of differentiation of yolk sac erythroid cells there are at least two classes of proteins distinguishable with respect to dependence on continued RNA formatoin. The bulk of nuclear proteins, "nondifferentiated" proteins, appear to be dependent on relatively short-lived messenger RNA while synthesis of differentiated proteins, the hemoglobins, proceeds on relatively stable molecules of messenger RNA. Hemoglobin formation occurs in those cells which are actively synthesizing DNA and dividing. On the average, two to three cell divisions may occur after the formation and stabilization of the messenger RNA for globin. Yolk sac erythropoiesis, at least from day 10 of gestation, is unresponsive to erythropoietin. By comparison, in fetal liver erythropoiesis, the hormone, erythropoietin, acts selectively on the most immature erythroid cell precursor to induce differentiation, cell replication, and hemoglobin formation. The erythropoietin responsive cell in the liver is apparently differentiated from the progenitor, pluripotential stem cell and committed to erythroblast formation and hemoglobin synthesis on exposure to the hormone. The initial effects of erythropoietin on macromolecular synthesis are to stimulate RNA synthesis, which temporally is followed by cell replication and the increase in hemoglobin formation. During liver erythropoiesis, there appears to be a transition from hemoglobin synthesis dependent on RNA formation to hemoglobin synthesis directed by relatively stable messenger RNA.