scholarly journals Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 798-805
Author(s):  
Hideo Harigae ◽  
Naruyoshi Suwabe ◽  
Peter H. Weinstock ◽  
Mayumi Nagai ◽  
Hiroyoshi Fujita ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Aminolevulinate Synthase ◽  
E Gene ◽  
In The Wild ◽  
Mutant Cells

The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

scholarly journals Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 798-805 ◽  
Author(s):  
Hideo Harigae ◽  
Naruyoshi Suwabe ◽  
Peter H. Weinstock ◽  
Mayumi Nagai ◽  
Hiroyoshi Fujita ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Aminolevulinate Synthase ◽  
E Gene ◽  
In The Wild ◽  
Mutant Cells

Abstract The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

Primitive Erythropoiesis and Osteogenesis Are Differentially Impaired In Rpl5 and Rps19 Mutant Murine Embryonic Stem Cell Models Of Diamond Blackfan Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3704-3704
Author(s):  
Brian M. Dulmovits ◽  
Sharon A. Singh ◽  
Tracie Goldberg ◽  
Sebastien Didier ◽  
Jeffrey M. Lipton ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Bone Marrow Failure ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Alizarin Red ◽  
Diamond Blackfan Anemia ◽  
Mutant Cells

Abstract Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red blood cell hypoplasia, congenital anomalies and cancer predisposition. In addition, short stature and poor skeletal growth are found in a subset of DBA patients, suggesting similar developmental abnormalities in erythropoiesis and osteogenesis in that subset. Furthermore it has been shown recently that osteoblasts secrete erythropoietin, linking the marrow niche to the modulation of erythropoiesis. DBA has been shown in the majority of cases to result from haploinsufficiency of large or small ribosomal subunit proteins. The p53 pathway, known to be activated by abortive ribosome assembly, contributes to the erythroid failure of DBA. We studied two DBA genotypes in vitro using murine embryonic stem (ES) cell lines harboring gene trap mutations in ribosomal proteins RPS19 and RPL5, respectively. Both mutants had decreased embryoid body (EB) formation, decreased definitive erythroid colony formation and similar p53-dependent primitive erythroid differentiation defects (see Figure A). Cell cycle analyses were normal in the Rps19 mutant ES cells, but there was a significant G2/M arrest in the Rpl5 mutant ES cells, which was unaffected by p53 knockdown. In addition, the Rpl5 mutant cells had a more pronounced growth defect in culture compared to the Rps19 mutant cells (Figure B). ES cells were differentiated, in vitro, to osteoblasts using established culture conditions, and confirmed both by morphology and molecular characterization (e.g. RUNX2 and Osteopontin). Following 14 days of osteogenic differentiation, bone mineralization was confirmed via Alizarin Red staining. A marked reduction in Alizarin Red staining was seen in the Rpl5 mutant cells while there was only a slight diminution of staining in the Rps19 mutant ES cultures (see Figure C). Therefore the erythroid differentiation defect appears similar in both the Rps19 and Rpl5 mutant ES cells. However the Rpl5 mutant appears to have a more severe phenotype at the ES stage, as evidenced by a pronounced p53-independent G2/M arrest and slower growth rate and subsequently during osteogenic differentiation. These data suggest an explanation for the more severe non-erythroid phenotype seen in a subset of DBA patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

1997 ◽  
Vol 17 (3) ◽  
pp. 1642-1651 ◽  
Author(s):  
M J Weiss ◽  
C Yu ◽  
S H Orkin
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Zinc Finger ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Stage Of Development ◽  
Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1857-1863 ◽  
Author(s):  
Kenji Kitajima ◽  
Makoto Tanaka ◽  
Jie Zheng ◽  
Hilo Yen ◽  
Ayuko Sato ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Dependent Manner ◽  
Hematopoietic Differentiation ◽  
Macrophage Differentiation ◽  
Conditional Gene Expression

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

scholarly journals ImpairedIn VitroErythropoiesis following Deletion of theScl(Tal1) +40 Enhancer Is Largely Compensated forIn Vivodespite a Significant Reduction in Expression

2013 ◽  
Vol 33 (6) ◽  
pp. 1254-1266 ◽  
Author(s):  
Rita Ferreira ◽  
Dominik Spensberger ◽  
Yvonne Silber ◽  
Andrew Dimond ◽  
Juan Li ◽  
...  
Keyword(s):  
Germ Line ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Definitive Erythropoiesis

TheScl(Tal1) gene encodes a helix-loop-helix transcription factor essential for hematopoietic stem cell and erythroid development. TheScl+40 enhancer is situated downstream ofMap17, the 3′ flanking gene ofScl, and is active in transgenic mice during primitive and definitive erythropoiesis. To analyze thein vivofunction of theScl+40 enhancer within theScl/Map17transcriptional domain, we deleted this element in the germ line.SclΔ40/Δ40mice were viable with reduced numbers of erythroid CFU in both bone marrow and spleen yet displayed a normal response to stress hematopoiesis. Analysis ofSclΔ40/Δ40embryonic stem (ES) cells revealed impaired erythroid differentiation, which was accompanied by a failure to upregulateSclwhen erythropoiesis was initiated.Map17expression was also reduced in hematopoietic tissues and differentiating ES cells, and theScl+40 element was able to enhance activity of theMap17promoter. However, onlySclbut notMap17could rescue theSclΔ40/Δ40ES phenotype. Together, these data demonstrate that theScl+40 enhancer is an erythroid cell-specific enhancer that regulates the expression of bothSclandMap17. Moreover, deletion of the +40 enhancer causes a novel erythroid phenotype, which can be rescued by ectopic expression ofSclbut notMap17.

scholarly journals Gene editing in rat embryonic stem cells to producein vitromodels andin vivoreporters

2017 ◽  
Author(s):  
Yaoyao Chen ◽  
Sonia Spitzer ◽  
Sylvia Agathou ◽  
Ragnhildur Thora Karadottir ◽  
Austin Smith
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Glycogen Synthase ◽  
Clonal Selection ◽  
Es Cells ◽  
Embryonic Stem ◽  
Brain Slices ◽  
Patch Clamp Recording ◽  
Mutant Cells

SummaryRat embryonic stem (ES) cells offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions inLef1.Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knock-in ofDsRedat theSox10locus. Two clones produced germline chimaeras. Comparative expression of DsRed and Sox10 validated the fidelity of the reporter. To illustrate utility, oligodendrocyte lineage cells were visualised by live imaging of DsRed in neonatal brain slices and subjected to patch clamp recording. Overall these results show that CRISPR/Cas9 gene editing technology in germline competent rat ES cells is enabling forin vitrostudies and for generating genetically modified rats.

scholarly journals Biased Suppression of Hematopoiesis and Multiple Developmental Defects in Chimeric Mice Containing Shp-2 Mutant Cells

1998 ◽  
Vol 18 (10) ◽  
pp. 6075-6082 ◽  
Author(s):  
Cheng-Kui Qu ◽  
Wen-Mei Yu ◽  
Biagio Azzarelli ◽  
Scott Cooper ◽  
Hal E. Broxmeyer ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Lumbar Vertebrae ◽  
Chimeric Mice ◽  
Developmental Defects ◽  
Homozygous Mutant ◽  
Mutant Cells

ABSTRACT Shp-2 is a cytoplasmic tyrosine phosphatase that contains two Src homology 2 (SH2) domains at the N terminus. Biochemical data suggests that Shp-2 acts downstream of a variety of receptor and cytoplasmic tyrosine kinases. A targeted deletion mutation in the N-terminal SH2 (SH2-N) domain results in embryonic lethality of homozygous mutant mice at midgestation. In vitro embryonic stem (ES) cell differentiation assays suggest that Shp-2 might play an important role in hematopoiesis. By aggregating homozygous mutant (Shp-2−/−) ES cells and wild-type (WT) embryos, we created Shp-2−/−-WT chimeric animals. We report here an essential role of Shp-2 in the control of blood cell development. Despite the widespread contribution of mutant cells to various tissues, no Shp-2−/− progenitors for erythroid or myeloid cells were detected in the fetal liver and bone marrow of chimeric animals by using the in vitro CFU assay. Furthermore, hematopoiesis was defective in Shp-2−/− yolk sacs. In addition, the Shp-2 mutation caused multiple developmental defects in chimeric mice, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lungs, intestines, and skin. These results demonstrate a functional involvement of Shp-2 in the differentiation of multiple tissue-specific cells and in body organization. More importantly, the requirement for Shp-2 is more stringent in hematopoiesis than in other systems.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
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

The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Carrie A. Adelman ◽  
Subrata Chattopadhyay ◽  
James J. Bieker
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Smad Pathway ◽  
Cellular Expression ◽  
Novel Approach

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

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