Aberrant iron accumulation and oxidized status of erythroid-specific δ-aminolevulinate synthase (ALAS2)–deficient definitive erythroblasts

Abstract Alas2 encodes the erythroid-specific δ-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with theAlas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking theAlas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype ofAlas2-null definitive erythroblasts was examined.Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes inAlas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate thatAlas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron,Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.

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Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽

10.1242/dev.116.supplement.157 ◽

1992 ◽

Vol 116 (Supplement) ◽

pp. 157-165 ◽

Cited By ~ 20

Author(s):

R. S. P. Beddington ◽

P. Rashbass ◽

V. Wilson

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Primitive Streak ◽

Coat Colour ◽

Es Cell ◽

Wild Type ◽

Mesoderm Cell ◽

Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

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GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽

10.1182/blood.v92.11.4108 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4108-4118 ◽

Cited By ~ 54

Author(s):

Naruyoshi Suwabe ◽

Satoru Takahashi ◽

Toru Nakano ◽

Masayuki Yamamoto

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Es Cell ◽

Globin Mrna ◽

Differentiated Cells ◽

Vitro Analysis ◽

Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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Emp Null Mice Are Non-Viable and Exhibit Erythroid Differentiation Defect.

Blood ◽

10.1182/blood.v106.11.806.806 ◽

2005 ◽

Vol 106 (11) ◽

pp. 806-806 ◽

Cited By ~ 1

Author(s):

Shivani Soni ◽

Shashi Bala ◽

Babette Gwynn ◽

Kenneth E. Sahr ◽

Luanne L. Peters ◽

...

Keyword(s):

Fetal Liver ◽

Direct Sequencing ◽

Embryonic Stem ◽

Gene Trap ◽

Physical Contact ◽

Erythroid Cells ◽

Wild Type ◽

Definitive Erythropoiesis

Abstract Emp, erythroblast macrophage protein, was originally detected in erythroblasts and macrophages, which form erythroblastic islands during erythropoiesis in the human bone marrow. The physical contact between erythroblasts and macrophages was suggested to promote the terminal maturation of erythroblasts, leading to their enucleation in vitro. To evaluate the function of Emp in vivo, we employed gene targeting studies to develop an Emp(−/−) mouse model. Mouse embryonic stem cells containing a gene-trap insertion in Emp were obtained from BayGenomics. Insertion of the gene-trap vector into Emp was verified by direct sequencing of cDNA obtained by 5′RACE. Chimeric mice generated by blastocyst microinjection were intercrossed, and the offspring were genotyped by PCR and Southern hybridization. The Emp (+/−) mice were healthy and fertile. However, no live Emp (−/−) mice were found among the progeny of the Emp (+/−) intercrosses. Analysis of timed pregnancies revealed that Emp (−/−) embryos were present at a frequency roughly consistent with Mendelian inheritance throughout the embryonal stages. Homozygous Emp (−/−) embryos were small and pale compared to their littermates, and they survived embryonic development but died at birth. To determine the effect, if any, of Emp gene deletion on definitive hematopoiesis, livers of +/+, +/−, and −/− embryos at E15.5 were examined after H&E and Giemsa staining of paraffin-embedded serial sections, and cytospins. We found few mature erythroid cells in the sinusoids of homozygotes, in contrast to those of either wild-type or heterozygotes, where abundant enucleated red blood cells were observed. Although nucleated erythrocytes were found in both wild-type and mutant embryos, their relative proportions were very different: the less mature forms (proerythroblasts) predominated in the −/− embryos whereas the more mature forms (polychromatophilic/orthochromatic and enucleated erythrocytes) were most common in +/+ and +/− embryos. Furthermore, erythroblastic islands consisting of a central macrophage surrounded by developing erythroblasts were seen in the cytospin preparations of wild-type and heterozygote livers but not in those of homozygous null livers. Since fetal liver macrophages (FLMs) are indispensable for definitive erythropoiesis, we investigated the effect that Emp’s absence might have on development of FLMs. The E15.5 fetal liver sections were stained with the macrophage-specific F4/80 antigen. Numerous F4/80-positive macrophages were present throughout the liver of normal embryos whereas, the number was substantially reduced in Emp (−/−) liver. In summary, in the absence of Emp, FLMs are significantly reduced and terminal maturation of erythroid cells is negatively affected. Thus, the availability of Emp(−/−) embryos will provide a unique experimental model to study the function of macrophages in definitive erythropoiesis.

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GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽

10.1182/blood.v92.11.4108.423k29_4108_4118 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4108-4118 ◽

Cited By ~ 4

Author(s):

Naruyoshi Suwabe ◽

Satoru Takahashi ◽

Toru Nakano ◽

Masayuki Yamamoto

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Es Cell ◽

Globin Mrna ◽

Differentiated Cells ◽

Vitro Analysis ◽

Definitive Hematopoiesis

Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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Telomerase-Associated Protein TEP1 Is Not Essential for Telomerase Activity or Telomere Length Maintenance In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8178-8184.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8178-8184 ◽

Cited By ~ 47

Author(s):

Yie Liu ◽

Bryan E. Snow ◽

M. Prakash Hande ◽

Gabriela Baerlocher ◽

Valerie A. Kickhoefer ◽

...

Keyword(s):

Telomere Length ◽

Rna Binding ◽

Es Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Telomerase Rna ◽

Wild Type ◽

Rnp Complex ◽

Successive Generations

ABSTRACT TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. ThemTep1-deficient (mTep1 −/−) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues frommTep1 −/− mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1 −/− mice. Telomere length, even in later generations ofmTep1 −/− mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

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Dp1 Is Largely Dispensable for Embryonic Development

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7197-7205.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7197-7205 ◽

Cited By ~ 20

Author(s):

Matthew J. Kohn ◽

Sandra W. Leung ◽

Vittoria Criniti ◽

Monica Agromayor ◽

Lili Yamasaki

Keyword(s):

Cell Cycle ◽

Embryonic Development ◽

Cell Cycle Progression ◽

Target Genes ◽

Es Cells ◽

Embryonic Stem ◽

Wild Type ◽

Cycle Progression ◽

Cell Cycle Genes

ABSTRACT E2F/DP complexes activate or repress the transcription of E2F target genes, depending on the association of a pRB family member, thereby regulating cell cycle progression. Whereas the E2F family consists of seven members, the DP family contains only two (Dp1 and Dp2), Dp1 being the more highly expressed member. In contrast to the inactivation of individual E2F family members, we have recently demonstrated that loss of Dp1 results in embryonic lethality by embryonic day 12.5 (E12.5) due to the failure of extraembryonic lineages to develop and replicate DNA properly. To bypass this placental requirement and search for roles of Dp1 in the embryo proper, we generated Dp1-deficient embryonic stem (ES) cells that carry the ROSA26-LacZ marker and injected them into wild-type blastocysts to construct Dp1-deficient chimeras. Surprisingly, we recovered mid- to late gestational embryos (E12.5 to E17.5), in which the Dp1-deficient ES cells contributed strongly to most chimeric tissues as judged by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining and Western blotting. Importantly, the abundance of DP2 protein does not increase and the expression of an array of cell cycle genes is virtually unchanged in Dp1-deficient ES cells or chimeric E15.5 tissues with the absence of Dp1. Thus, Dp1 is largely dispensable for embryonic development, despite the absolute extraembryonic requirement for Dp1, which is highly reminiscent of the restricted roles for Rb and cyclins E1/E2 in vivo.

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Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.626 ◽

1995 ◽

Vol 15 (2) ◽

pp. 626-633 ◽

Cited By ~ 57

Author(s):

G A Blobel ◽

M C Simon ◽

S H Orkin

Keyword(s):

Zinc Finger ◽

Binding Proteins ◽

Es Cells ◽

Zinc Fingers ◽

Embryonic Stem ◽

Transcription Activation ◽

Erythroid Cells ◽

Zinc Finger Region

Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.

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Limitation of adipose tissue by the number of embryonic progenitor cells

eLife ◽

10.7554/elife.53074 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Kristina Hedbacker ◽

Yi-Hsueh Lu ◽

Olof Dallner ◽

Zhiying Li ◽

Gulya Fayzikhodjaeva ◽

...

Keyword(s):

Adipose Tissue ◽

Es Cells ◽

Embryonic Stem ◽

Proliferative Potential ◽

Wild Type ◽

Tissue Mass ◽

Adipose Tissue Mass ◽

The Difference ◽

Turn Over

Adipogenesis in adulthood replaces fat cells that turn over and can contribute to the development of obesity. However, the proliferative potential of adipocyte progenitors in vivo is unknown (Faust et al., 1976; Faust et al., 1977; Hirsch and Han, 1969; Johnson and Hirsch, 1972). We addressed this by injecting labeled wild-type embryonic stem cells into blastocysts derived from lipodystrophic A-ZIP transgenic mice, which have a genetic block in adipogenesis. In the resulting chimeric animals, wild-type ES cells are the only source of mature adipocytes. We found that when chimeric animals were fed a high-fat-diet, animals with low levels of chimerism showed a significantly lower adipose tissue mass than animals with high levels of chimerism. The difference in adipose tissue mass was attributed to variability in the amount of subcutaneous adipose tissue as the amount of visceral fat was independent of the level of chimerism. Our findings thus suggest that proliferative potential of adipocyte precursors is limited and can restrain the development of obesity.

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Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells

Blood ◽

10.1182/blood-2004-02-0570 ◽

2004 ◽

Vol 104 (6) ◽

pp. 1873-1880 ◽

Cited By ~ 76

Author(s):

Sebastian Carotta ◽

Sandra Pilat ◽

Andreas Mairhofer ◽

Uwe Schmidt ◽

Helmut Dolznig ◽

...

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Basic Research ◽

Culture Conditions ◽

Erythroid Cells ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Mouse Es Cells ◽

Defined Culture

Abstract Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. Besides their characterization in colony assays, protocols exist for the cultivation of lymphoid, myeloid, and erythroid cells. With the possible exception of mast cells, however, long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here, we describe for the first time an efficient yet easy strategy to generate mass cultures of pure, immature erythroid progenitors from mouse ES cells (ES-EPs), using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived, adult, definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions, ES-EPs differentiated into mature, enucleated erythrocytes. Importantly, ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition, the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells.

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