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Narla Mohandas - One of the best experts on this subject based on the ideXlab platform.
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putative regulators for the continuum of erythroid differentiation revealed by single cell transcriptome of human bm and ucb cells
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Peng Huang, Yongzhong Zhao, Jianmei Zhong, Xinhua Zhang, Qifa Liu, Xiaoxia Qiu, Shaoke Chen, Hongxia Yan, Christopher D Hillyer, Narla MohandasAbstract:Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34-CD235a+) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L, TERF2IP, and RNF10, by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSCs to erythroid lineage and cell proliferation and delayed differentiation of colony-forming unit-erythroid (CFU-E) to the Proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation.
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the erythroid intron retention program encompasses developmentally stable and dynamic networks and regulates diverse gene classes
Blood, 2015Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Computational analysis of RNA-seq data from highly purified human erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in Proerythroblasts, but elevated intron retention in orthochromatic erythroblasts prior to enucleation. Dynamic regulation of late erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues. Disclosures No relevant conflicts of interest to declare.
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abnormal erythroid maturation leads to microcytic anemia in the tsap6 steap3 null mouse model
American Journal of Hematology, 2015Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Gargi Debnath, Narla MohandasAbstract:Genetic ablation of the ferrireductase STEAP3, also known as TSAP6, leads to severe microcytic and hypochromic red cells with moderate anemia in the mouse. However, the mechanism leading to anemia is poorly understood. Previous results indicate that TSAP6/Steap3 is a regulator of exosome secretion. Using TSAP6/Steap3 knockout mice, we first undertook a comprehensive hematologic characterization of the red cell compartment, and confirmed a dramatic decrease in the volume and hemoglobin content of these erythrocytes. We observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with these observations, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. However, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. Furthermore, there were no differences in red cell survival between wild type and knockout animals. However, when we monitored erythropoiesis, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3(-/-) animals. In addition, progression from the Proerythroblastic to the orthochromatic stage was affected, with accumulation of cells at the polychromatic stage. Altogether, our findings demonstrate that abnormal erythroid maturation is the main cause of anemia in these mice.
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ineffective erythropoiesis is the major cause of microcytic anemia in the tsap6 steap3 null mouse model
Blood, 2014Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Narla MohandasAbstract:STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p Disclosures No relevant conflicts of interest to declare.
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an erythroid specific intron retention program regulates expression of selected genes during terminal erythropoiesis
Blood, 2014Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human erythroblasts were determined via RNA-seq analysis of FACS-purified erythroblast populations. Comparison of IR among erythroblast populations and between erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of Proerythroblasts differed substantially from that in orthochromatic erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation. Disclosures No relevant conflicts of interest to declare.
Stuart H Orkin - One of the best experts on this subject based on the ideXlab platform.
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corepressor rcor1 is essential for murine erythropoiesis
Blood, 2014Co-Authors: Devorah C Goldman, Stuart H Orkin, Tamilla Nechiporuk, Sunita Kawane, Shannon Mcweeney, Jeffrey W Tyner, Marc A Kerenyi, William H Fleming, Gail MandelAbstract:The corepressor Rcor1 has been linked biochemically to hematopoiesis, but its function in vivo remains unknown. We show that mice deleted for Rcor1 are profoundly anemic and die in late gestation. Definitive erythroid cells from mutant mice arrest at the transition from Proerythroblast to basophilic erythroblast. Remarkably, Rcor1 null erythroid progenitors cultured in vitro form myeloid colonies instead of erythroid colonies. The mutant Proerythroblasts also aberrantly express genes of the myeloid lineage as well as genes typical of hematopoietic stem cells (HSCs) and/or progenitor cells. The colony-stimulating factor 2 receptor β subunit (Csf2rb), which codes for a receptor implicated in myeloid cytokine signaling, is a direct target for both Rcor1 and the transcription repressor Gfi1b in erythroid cells. In the absence of Rcor1, the Csf2rb gene is highly induced, and Rcor1−/− progenitors exhibit CSF2-dependent phospho-Stat5 hypersensitivity. Blocking this pathway can partially reduce myeloid colony formation by Rcor1-deficient erythroid progenitors. Thus, Rcor1 promotes erythropoiesis by repressing HSC and/or progenitor genes, as well as the genes and signaling pathways that lead to myeloid cell fate.
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the lim domain binding protein ldb1 and its partner lmo2 act as negative regulators of erythroid differentiation
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Jane E Visvader, Kyungmin Hahm, Yuko Fujiwara, Stuart H OrkinAbstract:The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER Proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.
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erythroid cell specific properties of transcription factor gata 1 revealed by phenotypic rescue of a gene targeted cell line
Molecular and Cellular Biology, 1997Co-Authors: Mitchell J Weiss, Stuart H OrkinAbstract: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.
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arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor gata 1
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Yuko Fujiwara, Carol P Browne, Kerrianne Cunniff, Sabra C Goff, Stuart H OrkinAbstract:Abstract The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the Proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early Proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.
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thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Mark W Kieran, Stuart H Orkin, Andrew C Perkins, Leonard I ZonAbstract:Abstract The interaction of the hormone erythropoietin and its receptor (EpoR) is though to be required for normal hematopoiesis. To define the role of EpoR in this process, the murine EpoR was disrupted by homologous recombination. Mice lacking the EpoR died in utero at embryonic day 11-12.5 with severe anemia. Embryonic erythropoiesis was markedly diminished, while fetal liver hematopoiesis was blocked at the Proerythroblast stage. Other cell types known to express EpoR, including megakaryocytes, mast, and neural cells were morphologically normal. Reverse transcription-coupled PCR analysis of RNA from embryonic yolk sac, peripheral blood, and fetal liver demonstrated near normal transcripts levels for EKLF, thrombopoietin (Tpo), c-MPL, GATA-1, GATA-2, and alpha- and embryonic beta H1-globin but non for adult beta maj-globin. While colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) colonies were not present in cultures derived from EpoR-/- liver or yolk sac cells, hemoglobin-containing BFU-E colonies were detected in cultures treated with recombinant Tpo and Kit ligand or with Tpo and interleukin 3 and 11. Rescued BFU-E colonies expressed adult beta-globin and c-MPL and appeared morphologically normal. Thus, erythroid progenitors are formed in vivo in mice lacking the EpoR, and our studies demonstrate that a signal transmitted through the Tpo receptor c-MPL stimulates proliferation and terminal differentiation of these progenitors in vitro.
Lionel Blanc - One of the best experts on this subject based on the ideXlab platform.
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abnormal erythroid maturation leads to microcytic anemia in the tsap6 steap3 null mouse model
American Journal of Hematology, 2015Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Gargi Debnath, Narla MohandasAbstract:Genetic ablation of the ferrireductase STEAP3, also known as TSAP6, leads to severe microcytic and hypochromic red cells with moderate anemia in the mouse. However, the mechanism leading to anemia is poorly understood. Previous results indicate that TSAP6/Steap3 is a regulator of exosome secretion. Using TSAP6/Steap3 knockout mice, we first undertook a comprehensive hematologic characterization of the red cell compartment, and confirmed a dramatic decrease in the volume and hemoglobin content of these erythrocytes. We observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with these observations, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. However, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. Furthermore, there were no differences in red cell survival between wild type and knockout animals. However, when we monitored erythropoiesis, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3(-/-) animals. In addition, progression from the Proerythroblastic to the orthochromatic stage was affected, with accumulation of cells at the polychromatic stage. Altogether, our findings demonstrate that abnormal erythroid maturation is the main cause of anemia in these mice.
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ineffective erythropoiesis is the major cause of microcytic anemia in the tsap6 steap3 null mouse model
Blood, 2014Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Narla MohandasAbstract:STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p Disclosures No relevant conflicts of interest to declare.
Julien Papoin - One of the best experts on this subject based on the ideXlab platform.
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abnormal erythroid maturation leads to microcytic anemia in the tsap6 steap3 null mouse model
American Journal of Hematology, 2015Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Gargi Debnath, Narla MohandasAbstract:Genetic ablation of the ferrireductase STEAP3, also known as TSAP6, leads to severe microcytic and hypochromic red cells with moderate anemia in the mouse. However, the mechanism leading to anemia is poorly understood. Previous results indicate that TSAP6/Steap3 is a regulator of exosome secretion. Using TSAP6/Steap3 knockout mice, we first undertook a comprehensive hematologic characterization of the red cell compartment, and confirmed a dramatic decrease in the volume and hemoglobin content of these erythrocytes. We observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with these observations, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. However, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. Furthermore, there were no differences in red cell survival between wild type and knockout animals. However, when we monitored erythropoiesis, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3(-/-) animals. In addition, progression from the Proerythroblastic to the orthochromatic stage was affected, with accumulation of cells at the polychromatic stage. Altogether, our findings demonstrate that abnormal erythroid maturation is the main cause of anemia in these mice.
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ineffective erythropoiesis is the major cause of microcytic anemia in the tsap6 steap3 null mouse model
Blood, 2014Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Narla MohandasAbstract:STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p Disclosures No relevant conflicts of interest to declare.
Adam Telerman - One of the best experts on this subject based on the ideXlab platform.
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abnormal erythroid maturation leads to microcytic anemia in the tsap6 steap3 null mouse model
American Journal of Hematology, 2015Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Gargi Debnath, Narla MohandasAbstract:Genetic ablation of the ferrireductase STEAP3, also known as TSAP6, leads to severe microcytic and hypochromic red cells with moderate anemia in the mouse. However, the mechanism leading to anemia is poorly understood. Previous results indicate that TSAP6/Steap3 is a regulator of exosome secretion. Using TSAP6/Steap3 knockout mice, we first undertook a comprehensive hematologic characterization of the red cell compartment, and confirmed a dramatic decrease in the volume and hemoglobin content of these erythrocytes. We observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with these observations, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. However, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. Furthermore, there were no differences in red cell survival between wild type and knockout animals. However, when we monitored erythropoiesis, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3(-/-) animals. In addition, progression from the Proerythroblastic to the orthochromatic stage was affected, with accumulation of cells at the polychromatic stage. Altogether, our findings demonstrate that abnormal erythroid maturation is the main cause of anemia in these mice.
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ineffective erythropoiesis is the major cause of microcytic anemia in the tsap6 steap3 null mouse model
Blood, 2014Co-Authors: Lionel Blanc, Julien Papoin, Michel Vidal, Robert Amson, Adam Telerman, Narla MohandasAbstract:STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of Proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p Disclosures No relevant conflicts of interest to declare.
