Vertebrate Erythropoiesis Research Articles

Evolutionary conservation of a regulative pathway of erythropoiesis in Poikilothermic vertebrates

Apoptosis, programmed cell death, plays a central role in haematopoiesis. Mature erythrocytes of non-mammalian vertebrates maintain a permanent nucleus; these cells can undergo apoptosis (eryptosis), as do other somatic cells of a given non-mammalian vertebrate. In this study, we have investigated the expression and subcellular distribution of Bcl-2, Bcl-XL and Bax proteins in the maturation phases and after X-ray irradiation of nucleated erythrocytes of Torpedo marmorata and Caretta caretta and the effect of X-ray irradiation on nucleated circulating erythrocytes of Torpedo marmorata. The cellular distribution of proteins was detected in erythrocytes by using immunocytochemistry at light microscopy and immunoelectron microscopy. The electrophoretic separation and immunoblotting of pro- and anti-apoptotic proteins of immature and mature erythroid cells was performed too, after X-ray irradiation of torpedoes. The results of the immunocytochemical analyses show an increase, in the expression level of Bax in mature as compared to young erythrocytes and a corresponding decrease of Bcl-2 and Bcl-XL. This maturation pattern of Bax, Bcl-2 and Bcl-XL was abrogated in X-ray irradiated torpedo erythrocytes. On the basis of these observations, Bax, Bcl-2 and Bcl-XL seems to play a role in the erythropoiesis of Torpedo marmorata Risso and in Caretta caretta. In conclusion, the same apoptotic proteins of somatic cells appear to be conserved in circulating nucleated erythrocytes thus suggesting to play a role in the maturation of these cells.

Tpr Deficiency Disrupts Erythroid Maturation With Impaired Chromatin Condensation in Zebrafish Embryogenesis.

Vertebrate erythropoiesis involves nuclear and chromatin condensation at the early stages of terminal differentiation, which is a unique process to distinguish mature erythrocytes from erythroblasts. However, the underlying mechanisms of chromatin condensation during erythrocyte maturation remain elusive. Here, we reported a novel zebrafish mutantcas7 with erythroid maturation deficiency. Positional cloning showed that a single base mutation in tprb gene, which encodes nucleoporin translocated promoter region (Tpr), is responsible for the disrupted erythroid maturation and upregulation of erythroid genes, including ae1-globin and be1-globin. Further investigation revealed that deficient erythropoiesis in tprbcas7 mutant was independent on HIF signaling pathway. The proportion of euchromatin was significantly increased, whereas the percentage of heterochromatin was markedly decreased in tprbcas7 mutant. In addition, TPR knockdown in human K562 cells also disrupted erythroid differentiation and dramatically elevated the expression of globin genes, which suggests that the functions of TPR in erythropoiesis are highly conserved in vertebrates. Taken together, this study revealed that Tpr played vital roles in chromatin condensation and gene regulation during erythroid maturation in vertebrates.

Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

AbstractKit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

Relationship between the Induced Iron Overload Model and Hepatic Erythropoiesis in Xenopus laevis.

Iron is an essential element for hemoglobin synthesis during erythropoiesis. Iron overload, in contrast, adversely affects erythropoiesis and causes organ dysfunction. Research using various animal models may help to elucidate pathophysiological mechanisms induced by excess iron. In the present study, we evaluated the relationship between iron metabolism and erythropoietic activity in the African clawed frog, Xenopus laevis. In X. laevis, both erythropoiesis and iron metabolism occur in the liver. First, we developed a method to quantify iron levels in the liver and plasma using 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl) amino] phenol (Nitroso-PSAP). We then measured iron levels and analyzed hematopoietic parameters in frogs that were orally administered sodium ferrous citrate (SFC). The hepatic iron level increased in the SFC group, but the number of erythrocytes, hematocrit, and hemoglobin concentration did not change, suggesting that the regulation of the production and release of mature erythrocytes in the liver was not directly affected by dietary iron. At four days after administration of 2 mg/kg SFC, the number of immature erythrocytes decreased in the liver. Interestingly, atypical blood cells with hyper-segmented nuclei were observed, identified by acridine orange cell staining; these atypical blood cells were hardly detectable under the steady state. Compared with previously reported results in mice, the increase in the hepatic iron levels was small, but our results indicate that SFC affects hematopoietic activity. These results establish a novel model for iron metabolism and provide new insights into the relationship between iron metabolism and erythropoiesis in vertebrates.

RASA3 Is Essential for Early Mammalian Hematopoiesis and Modulates ERK Signaling in Terminal Erythropoiesis

The Ras GTPase-activating protein, RASA3, plays a critical non-redundant role in vertebrate erythropoiesis. The autosomal recessive scat mouse model carries a missense (G125V) mutation in Rasa3 that results in severe anemia, thrombocytopenia and leukopenia with markedly reduced survival. Terminal erythroid differentiation is dysregulated in scat bone marrow (BM) and spleen with delayed cell cycle progression, decreased apoptosis, and increased reactive oxygen species. To further study the role of RASA3 in hematopoiesis, we created a conditional Rasa3 allele. Germline null mice die at E12.5-13.5 of severe hemorrhage, pallor and reduced fetal liver size. Specific ablation of Rasa3 in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs) driven by Vav-Cre and Tek-Cre recapitulates the null phenotype, highlighting the specific requirement of RASA3 in hematopoietic and vascular compartments. To study RASA3 deficiency specifically in terminal erythropoiesis, we utilized EpoR-Cre. Surprisingly, EpoR-Cre driven deletion does not produce an abnormal erythroid phenotype, and normal levels of RASA3 protein are seen on western blots of Ter119+ spleen and BM cells despite efficient deletion of the floxed Rasa3 allele, suggesting that RASA3 produced early in HSPCs is sufficient to maintain normal terminal erythroid differentiation. Indeed, deletion of Rasa3 in the entire hematopoietic system at 8 weeks of age via inducible Mx1-Cre results in death within 2 weeks of induction with profound anemia, thrombocytopenia, and leukopenia, recapitulating the scat phenotype. While HSC numbers are normal in the RASA3 depleted BM of this model, the frequency and absolute numbers of all progenitors are significantly decreased, and spleen stress erythropoiesis is significantly blunted. Altered Ras activity is associated with delayed erythropoiesis. ERK signaling, a key downstream Ras effector, is hyperactivated 3-fold by western blotting in scat BM CD45-depleted cells vs. wild type (WT). To extend RASA3 studies to human cells, we utilized cord blood CD34+ cells in a three-phase erythroid differentiation culture system. Similar to the mouse models, shRNA knockdown of RASA3 leads to delayed erythroid differentiation at culture day 11 based on glycophorin A, Band 3 and a4-integrin expression levels. Dysregulated cell cycle progression with accumulation in the S-phase is also evident at day 12 of culture. Unlike the mouse models, however, ERK activation was decreased (67-72%) in serum-starved sorted proerythroblasts and later day 11 populations of RASA3 knockdown cells treated with erythropoietin (Epo), stem cell factor (SCF), or both. To elucidate additional molecular mechanisms downstream of RASA3, we performed RNAseq on total spleen and BM, as well as sorted stem and myeloid progenitors (SMPs) of scat mice and WT littermate controls. Principle component analysis confirms that genotype is the primary contributing factor to differential gene expression. In preliminary analyses, MAPK signaling, including negative regulators of ERK, phosphatidyl-inositol signaling, cell cycle regulation, and transcriptional regulation emerge from pathways and GO term analyses of differentially expressed genes. In conclusion, we show that (1) absence of RASA3 results in embryonic lethality with evidence of severe HSPC/EC defects; (2) RASA3 is required for hematopoiesis starting at the HPC stage or earlier; (3) missense mutations (scat) or loss of RASA3 in adults cause pancytopenia and premature death; (4) mechanistically, aberrant ERK signaling and cell cycle defects contribute to the RASA3 deficient erythroid phenotype, while RNAseq studies provide further potential mechanistic targets. Disclosures No relevant conflicts of interest to declare.

Alternative Erythropoietin Receptors in the Nervous System.

In addition to its regulatory function in the formation of red blood cells (erythropoiesis) in vertebrates, Erythropoietin (Epo) contributes to beneficial functions in a variety of non-hematopoietic tissues including the nervous system. Epo protects cells from apoptosis, reduces inflammatory responses and supports re-establishment of compromised functions by stimulating proliferation, migration and differentiation to compensate for lost or injured cells. Similar neuroprotective and regenerative functions of Epo have been described in the nervous systems of both vertebrates and invertebrates, indicating that tissue-protective Epo-like signaling has evolved prior to its erythropoietic function in the vertebrate lineage. Epo mediates its erythropoietic function through a homodimeric Epo receptor (EpoR) that is also widely expressed in the nervous system. However, identification of neuroprotective but non-erythropoietic Epo splice variants and Epo derivatives indicated the existence of other types of Epo receptors. In this review, we summarize evidence for potential Epo receptors that might mediate Epo’s tissue-protective function in non-hematopoietic tissue, with focus on the nervous system. In particular, besides EpoR, we discuss three other potential neuroprotective Epo receptors: (1) a heteroreceptor consisting of EpoR and common beta receptor (βcR), (2) the Ephrin (Eph) B4 receptor and (3) the human orphan cytokine receptor-like factor 3 (CRLF3).

RASA3 Deficiency Contributes to Anemia By Multiple Mechanisms

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage.The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p<0.05).Studies exploring the influence of the galectin family on erythropoiesis and Ras signaling in the context of scat are underway.Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis.Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. DisclosuresNo relevant conflicts of interest to declare.

Using the Zebrafish as an Approach to Examine the Mechanisms of Vertebrate Erythropoiesis.

The zebrafish, Danio rerio, is a powerful model for the study of erythropoiesis and defining the genetic basis of hematological diseases. The mechanisms of erythroid differentiation are highly conserved in the zebrafish, permitting translational research studies and the modeling of erythropoiesis in higher vertebrates. An advantage of the system is the ability to manipulate gene expression and observe the effect on erythroid development in vivo, with relative ease and rapidity. The production of optically transparent embryos also makes it an attractive tool for visual analysis of circulating erythrocytes that can be used to study erythropoiesis. Through large-scale chemical mutagenesis screens, a variety of zebrafish blood mutants have been identified that are used for gene discoveries and the recapitulation of human diseases. Experimental techniques including in situ hybridization, o-dianisidine staining, flow cytometry, and microinjection are now commonly employed to study red blood cell biochemistry and erythropoiesis in the zebrafish. These techniques have been applied for identifying novel genes required for the hemoglobin synthesis, isolating blood cell lineages, visualizing genetic expression within erythroid tissues, and characterizing the phenotype of blood disorders. The applications of zebrafish methodology to the study of erythropoiesis and optimized step-by-step protocols are discussed in this chapter.

Erythroid-Specific Variant of the Nuclear Exportin Xpo7 Conserved Only in Mammals May Explain Functional Differences Between Mammalian Definitive and Lower Vertebrate (or Primitive) Erythropoiesis

Exportin 7 (Xpo7) was previously found to be crucial for late murine erythroid nuclear maturation when its knockdown significantly inhibited chromatin condensation and ultimate enucleation in a primary mouse erythroblast culture [1]. Additionally, Xpo7 was found to have an erythroid-specific isoform, transcribed from an erythroid-specific transcription start site (TSS) in the first intron of Xpo7 (named Xpo7-1B in contrast to the ubiquitously expressed variant -1A) [1]. Xpo7-1B differs from Xpo7-1B in 8 amino acid residues in the first exon and 9 residues in the 5th exon, and these differences are not conserved either in lower vertebrates or in primitive mouse erythropoiesis. It is intriguing that both lower vertebrate and primitive mammalian erythroblasts do not enucleate.In order to understand the in vivo function of Xpo7 and determine if these differences in the two isoforms confer differential functions to the proteins or are simply regulatory, we examined the definitive fetal liver erythropoiesis (considered adult "stress erythropoiesis") and adult steady state erythropoiesis of transgenic mouse models lacking either Xpo7-1A, Xpo7-1B, or both. We had previously knocked down Xpo7 in zebrafish using morpholinos (zebrafish only express the Xpo7-1A homologue) and found no effect on adult erythroid differentiation or proliferation. Adult Xpo7-1A knockout (KO) mice are born in normal Mendelian ratios, are not anemic, and express normal Xpo7-1B only in the blood. Closer examination reveals that fetal liver erythroid proliferation is actually higher in Xpo7-1A KO mice than either heterozygous (HET) or wild type (WT) while differentiation is normal. The double KO (Xpo7-1AB KO) appears to be embryonic lethal as only 4 live knockout pups were born out of 71 live births and even these 4 pups appear to be partial chimeras. We characterized the Xpo7-1AB HET mice in more detail and fetal liver erythroblasts of HET mice showed normal proliferation and differentiation compared to WT, and adults showed no differences in red blood cell (RBC) counts, hematocrit or hemoglobin. No complete live Xpo7-1AB KO has been born so we are now evaluating embryos at E3, E9.5, and E14.5. Given the possibility of embryonic lethality, in order to evaluate the erythroid-specific isoform Xpo7-1B, we have crossed a mouse floxed for the Xpo7-1B TSS/1st exon with a tamoxifen-inducible Cre-recombinase mouse. These mice have been given tamoxifen after reaching adulthood and we are currently evaluating their hematopoietic parameters. We have also set up HET-HET timed matings and administered tamoxifen to pregnant females to evaluate fetal liver erythropoiesis in Xpo7-1B HET, WT, and KO embryos.Not only will this work further our understanding of the process of global chromatin condensation during erythropoiesis, data from this in vivo study may reveal differences in erythropoiesis between mammals and lower vertebrates, which may also reflect intrinsic differences in mammalian primitive versus definitive erythropoiesis. Based on our in vitro knockdown studies, we anticipate that the phenotype of the Xpo7-1B KO mice may be similar to congenital dyserythropoietic anemia (CDA), a human disorder involving abnormalities in erythroid progenitor chromatin condensation and nuclear membrane substructure, so examination of these mice may also uncover a novel mechanism for this form of ineffective erythropoiesis.[1] Hattangadi, SM, et al. Blood (2014) 124, 1931-40. DisclosuresNo relevant conflicts of interest to declare.

Erythropoiesis in vertebrates: from ontogeny to clinical relevance.

Erythropoiesis is a complex process that starts in the course of embryo formation and it is maintained throughout the life of an organism. During the fetal development, erythropoiesis arises from different body sites and erythroblast maturation occurs in the fetal liver. After birth, erythropoiesis and erythroblast maturation take place exclusively in the bone marrow, generating a lifetime reservoir of red blood cells (RBCs), which are responsible for transporting oxygen through the bloodstream to tissues and organs. Several transcription factors and cytokines, such as GATA-1, GATA-2, FOG-1 and erythropoietin (EPO), constitute an elaborated molecular network that regulates erythropoiesis as they are involved in the differentiation and maturation of RBCs. The profound understanding of erythropoiesis is fundamental to avoid, treat or even soften the effects of erythropoietic clinical disorders and may be useful to improve patients' well-being.

Erythropoiesis in vertebrates from ontogeny to clinical relevance

Erythropoiesis in vertebrates from ontogeny to clinical relevance

RASA3 Is Involved in Cell Cycle Progression, Hemoglobinization and Generation of Reactive Oxygen Species during Mammalian Erythropoiesis

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. Defective RASA3 in zebrafish and mice results in severe anemia and thrombocytopenia. Indeed, in the mouse model scat (severe combined anemia and thrombocytopenia), a G125V mutation in Rasa3 leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Previous studies showed that this mutation in Rasa3 causes severely delayed erythroid differentiation at the polychromatophilic stage and decreased hemoglobinization due, at least in part, to mislocalization of RASA3 to the cytosol and resultant increased Ras activity. Here, we provide evidence that RASA3 plays a pivotal role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels in erythropoiesis during crisis episodes. First, we analyzed the cell cycle progression in scat erythroblasts during crisis episodes. Using propidium iodide and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, p<0.001) while S phase was decreased (40.2% ± 2.9% vs. 49.6% ± 3.3% p<0.01) and G2 was not affected in scat proerythroblasts. These data suggest that Rasa3 is involved in the G1 checkpoint. In addition, we observed an increase in the ROS levels in scat throughout differentiation including proerythroblasts (1.03% ± 0.3% vs. 0.25% ± 0.1% in controls, p < 0.01), EryA (1.5% ± 0.21% vs. 0.25% ± 0.05%, p < 0.01), EryB (2.6% ± 1.41% vs. 0.25% ± 0.01%, p < 0.01), EryC (2.0% ± 0.45% vs. 0.25% ± 0.02%, p < 0.01) and mature red cells (1.15% ± 0.1% vs. 0.9% ± 0.03%, p < 0.05); the highest levels of ROS were observed in the basophilic-polychromatic erythroblast (i.e. EryB) containing populations. In addition, we consistently found increased levels of ROS in both reticulocytes and red blood cells from scat peripheral blood. Therefore, the cell cycle arrest and increased ROS likely contribute to the erythropoietic defect associated with scat. No mutations involving RASA3 have been involved in human bone marrow failure syndromes yet. However, in more than 30% of the cases, the etiology remains unexplained. To understand the putative role of RASA3 in the pathophysiology of hematopoiesis, and based on our data obtained in the mouse and the zebrafish, we investigated the role of RASA3 in human erythropoiesis using si and shRNA knockdowns in cord blood-derived CD34+ cells. Knockdown efficiencies were evaluated by western blot, and revealed that RASA3 protein levels were reduced by greater than 50% using two different shRNA constructs and siRNA. Cells were differentiated using an adapted 3-phase liquid culture system that fully recapitulates erythropoiesis. Similar to the scat mouse, we observed delayed differentiation by flow cytometry using glycophorin A, band3 and α4-integrin as markers of terminal differentiation with 62.0% of RASA3 knockdown erythroblasts α4-integrinhi (compared to 27.5% in controls) at day 16. In addition, the nucleus was substantially less pyknotic and the nucleocytoplasmic ratio remained elevated during differentiation, as compared to control cells. Indeed, 64 ± 7% of the cells had an uncondensed nucleus in the RASA3 knockdown samples, compared to 30 ± 3% in the scramble controls. Moreover as in scat, hemoglobinization was defective; qRT-PCR analyses revealed a 25% reduction in γ-globin and a 20% reduction in β-globin expression. These findings strengthen and emphasize the notion that RASA3 has a conserved function during vertebrate terminal erythropoiesis. Experiments investigating cell cycle progression, apoptosis and ROS production in human CD34+ cells are underway. Together, our studies demonstrate that RASA3 plays and important role in hemoglobinization, cell cycle progression and cell survival during terminal erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

Genome-wide analysis of the zebrafish Klf family identifies two genes important for erythroid maturation

Krüppel-like transcription factors (Klfs), each of which contains a CACCC-box binding domain, have been investigated in a variety of developmental processes, such as angiogenesis, neurogenesis and somatic-cell reprogramming. However, the function and molecular mechanism by which the Klf family acts during developmental hematopoiesis remain elusive. Here, we report identification of 24 Klf family genes in zebrafish using bioinformatics. Gene expression profiling shows that 6 of these genes are expressed in blood and/or vascular endothelial cells during embryogenesis. Loss of function of 2 factors (klf3 or klf6a) leads to a decreased number of mature erythrocytes. Molecular studies indicate that both Klf3 and Klf6a are essential for erythroid cell differentiation and maturation but that these two proteins function in distinct manners. We find that Klf3 inhibits the expression of ferric-chelate reductase 1b (frrs1b), thereby promoting the maturation of erythroid cells, whereas Klf6a controls the erythroid cell cycle by negatively regulating cdkn1a expression to determine the rate of red blood cell proliferation. Taken together, our study provides a global view of the Klf family members that contribute to hematopoiesis in zebrafish and sheds new light on the function and molecular mechanism by which Klf3 and Klf6a act during erythropoiesis in vertebrates.

Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis

The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is 5-fold reduced in yeast lacking mtClpX activity and that total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX, therefore, is a widely conserved stimulator of an essential biosynthetic pathway and uses a previously unrecognized mechanism for AAA+ unfoldases.

Diverse of Erythropoiesis Responding to Hypoxia and Low Environmental Temperature in Vertebrates.

Erythrocytes are responsible for transporting oxygen to tissue and are essential for the survival of almost all vertebrate animals. Circulating erythrocyte counts are tightly regulated and respond to erythrocyte mass and oxygen tension. Since the discovery of erythropoietin, the erythropoietic responses to environment and tissue oxygen tension have been investigated in mice and human. Moreover, it has recently become increasingly clear that various environmental stresses could induce the erythropoiesis via various modulating systems, while all vertebrates live in various environments and habitually adapt to environmental stress. Therefore, it is considered that investigations of erythropoiesis in vertebrates provide a lead to the various erythropoietic responses to environmental stress. This paper comparatively introduces the present understanding of erythropoiesis in vertebrates. Indeed, there is a wide range of variations in vertebrates' erythropoiesis. This paper also focused on erythropoietic responses to environmental stress, hypoxia, and lowered temperature in vertebrates.

RASA3 Plays a Critical, Conserved Role in Erythroid Differentiation

AbstractAbstract 3186Identification of new key players in erythropoiesis can lead to a better understanding of the etiology of anemia of unknown origin. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. Crisis episodes are characterized by severe anemia and thrombocytopenia while, remarkably, the phenotype reverts to normal in the remission phase. Remissions are transient, however, and 94% of scat/scat mice die by P30 during a second crisis episode. We recently demonstrated a mechanism contributing to crisis episodes. The G125V mutation in Rasa3 is a loss of function mutation causing the protein to be mislocalized to the cytosol in scat reticulocytes. This results in loss of GAP activity and, as a consequence, increased levels of active GTP-bound Ras and a severe block in erythroid differentiation. Morpholino knockdowns of rasa3 in zebrafish result in profound anemia, confirming a conserved and non-redundant role for Rasa3 in vertebrate erythropoiesis. Here, we report that the cell cycle is affected in scat erythroid progenitors and extend studies to human primary erythroid cells. Using propidium iodine and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, × ± SD p<0.001) while S phase was decreased (40.2% ± 2.9% vs. 49.6% ± 3.3% p<0.01) and G2 was not affected in scat proerythroblasts. These data suggest that Rasa3 is involved in the G1 checkpoint. To determine if RASA3 function is conserved in human erythropoiesis, we isolated CD34+ cells from human cord blood and cultured them using the recently described three-phase culture system that recapitulates human erythropoiesis over a period of approximately 21 days. By western blotting, we found that RASA3 protein levels increase during the early stages of differentiation, peaking at Day 11, before decreasing continuously as terminal erythroid differentiation proceeds. We also used siRNA to transiently knock-down RASA3 during differentiation. Cells were transfected at D8 or D11, and effects of RASA3 silencing were evaluated after a 72 hours incubation period, at D11 or D14. The transfection efficiency was close to 90% and knock-down of RASA3 was verified at the protein level. While we did not see any apparent changes in terms of proliferation, as judged by cell counts and total protein content, the siRNA treated cells presented with a delay in differentiation as compared to control (scramble siRNA). Specifically, morphologic analyses of the cells by Giemsa and benzidine staining demonstrated that nuclear condensation and hemoglobinization were clearly affected, especially at D14. In the RASA3 siRNA transfected cells, the nucleus was substantially less pyknotic and the nucleocytoplasmic ratio did not decrease during differentiation, as compared to cells before transfection or transfected with scramble siRNA, and benzidine staining revealed a significant decrease in the extent of hemoglobinization. A lack of nuclear condensation and defects in hemoglobinization are consistent with a delay in differentiation, and extend the paradigm observed in the scat mouse model to human erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Failure of Erythropoiesis and Megakaryocytopoiesis in RASA3 Mutant Scat Mice

Abstract 680Scat (severe combined anemia and thrombocytopenia) is a spontaneous, autosomal recessive mutation coisogenic with the BALB/cBy inbred mouse strain. Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. As its name implies, crisis episodes are characterized by severe anemia and thrombocytopenia, but significant lymphocyte depletion occurs as well. The first crisis episode begins in utero, lasts until postnatal day (P) 9 on average, and is associated with 10–15% mortality. Remarkably, in homozygotes that survive the first crisis, a remission phase occurs wherein the disease phenotype reverts to normal. This remission is transient, however, and is followed by a second crisis episode during which 94% of scat/scat mice die by P30. Previously we showed that the scat phenotype is transferrable via the hematopoietic stem cells and is also recapitulated in scat/scat, Hox11−/− double homozygotes in which a spleen does not develop, indicating that the splenic micro-environment plays little or no role in disease appearance or progression. Positional cloning of scat revealed a missense mutation in Rasa3 encoding a GTPase activating protein (GAP) that negatively regulates Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. We further showed that Rasa3 is a conserved gene critical to vertebrate erythropoiesis via morpholino knockdowns in zebrafish which resulted in profound anemia. Here we report data that shed further light on RASA3 function during hematopoiesis. Overall, the data indicate that defects in RASA3 profoundly and negatively impact erythropoiesis and megakaryocytopoieis through, at least in part, a Ras-mediated mechanism. FACS analyses of scat spleen and bone marrow erythroid populations reveal a severe block in erythropoiesis during crisis periods. In the spleen, despite an initial increase in size due to expansion of Ter-119+ cells, there is ultimately a loss of compensatory erythropoiesis resulting in a return to normal cellularity and a striking loss of hemoglobinized cells as the crisis phenotype deepens. In addition, the bone marrow shows loss of Ter-119+ cells and overall cell depletion during crisis. Megakaryocyte numbers are increased in scat crisis BM and spleen. By transmission electron microscopy, scat crisis megakaryocytes display features characteristic of a significant developmental delay: a disorganized demarcation membrane system with no platelet forming areas and few granules with hypersegmented nuclei and excess rough endoplasmic reticulum. In addition to the severe anemia and thrombocytopenia, a significant lymphopenia occurs in scat crisis mice. However, the scat phenotype is not lymphocyte mediated, as the scat phenotype is completely recapitulated in mice doubly homozygous for scat and the immunodeficient mutations, scid and Rag1tm1Mom, in which B- and T-lymphocytes are completely depleted. Together these results suggest that lymphopenia is a secondary phenomenon in scat, and the severe anemia and thrombocytopenia aspect of the phenotype neither follows from nor is dependent upon loss of lymphocytes. Despite the delay observed in erythroid differentiation, some mature red cells are produced although ∼50% of these are reticulocytes. By confocal microscopy, we show that RASA3 protein localizes to the plasma membrane as well as internal membrane compartments in wild type reticulocytes, where it partially colocalizes with CD71. Western blot analyses of reticulocytes after Percoll gradient purification show that RASA3 is lost during the maturation step, both in vivo and in vitro. Interestingly, in scat, RASA3 is present in reticulocytes, but appears to be mislocalized, the protein being found in the cytosol. Preparation of ghosts from wild type and scat reticulocytes confirms that RASA3 is not attached to the membrane in scat animals during crisis. In pull-down assays active GTP-bound Ras is increased in scat crisis reticulocytes when compared to wild type, suggesting that scat is a RASA3 loss of function mutation due to its mislocalization and demonstrating a critical role for the RASA3-Ras axis during mammalian erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Analysis of TIF1gamma Conditional Knockout Establishes a Requirement for the Differentiation of Multiple Hematopoietic Lineages

AbstractAbstract 744Vertebrate hematopoiesis is regulated by cell-specific transcription factors that couple to RNA polymerase-associated basal machinery. Mutation of the chromatin factor TIF1gamma (TIF1g) gene in the zebrafish moonshine mutant causes a profound decrease in expression of most erythroid genes. Our previous work in the zebrafish system established an essential role of TIF1g in transcription elongation by coupling the SCL transcription factor complex to the transcription elongation machinery, and erythroid-specific transcription is paused in moonshine mutant (Bai et al., Cell 2010). Here we examined the role of TIF1g in murine hematopoiesis by studying conditional knockout (KO) models. Deletion of TIF1g was either induced in adult mice by Mx-Cre or during mouse development by vav-Cre. Both Cre systems induce excision at the HSC level and in all hematopoietic lineages. We observed the same trend of multi-lineage defects in both Cre lines, including decreased erythropoiesis in the bone marrow, loss of mature B cells and expansion of granulocytes. Bone marrow analysis of TIF1g-deleted mice revealed a block in erythroid differentiation starting as early as the BFU-E stage, consistent with the erythroid defect in the zebrafish moonshine mutant, confirming an evolutionarily conserved role for TIF1g in vertebrate erythropoiesis. A subset of the vav-Cre induced KO mice developed a MPD (myeloproliferative disease)-like phenotype at 1–3 months after birth, including a dramatic increase of dysplastic granulocytes in the peripheral blood and massive extramedullary hematopoiesis in spleen and liver, suggesting a requirement for TIF1g in myeloid differentiation and proliferation. Consistent with the KO mouse phenotype, we observed an increase of definitive myelopoiesis in the zebrafish TIF1g mutant. In addition, mature B cells were absent in KO mice, and this loss was observed as early as the pre-B stage, suggesting a defect occurring at the pro-B to pre-B transition. At the progenitor level, we observed an increase of GMP, MPP, and HSCs in bone marrow, suggesting that TIF1g may be required to control proliferation at the stem/progenitor cell stage or in the committed myeloid lineage. Our study reveals an essential function of TIF1g in regulating the differentiation and proliferation of multiple hematopoietic lineages.ReferenceBai X, Kim J, Yang Z, Jurynec M, Akie T, et al. (2010) TIF1gamma controls erythroid cell fate by regulating transcription elongation. Cell. 142:133-43. Disclosures:Zon:FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Positional Cloning of the Mouse Scat Mutation Reveals a Critical Role for RASA3, a Ras GTPase Activating Protein, in Vertebrate Erythropoiesis.

Abstract 776The spontaneous recessive mutation scat (severe combined anemia and thrombocytopenia) arose on the inbred BALB/cBy (BALB) mouse strain. The phenotype of scat is cyclic. All homozygotes are severely anemic and thrombocytopenic at birth. Leukocytes are also significantly depleted (Table). Approximately 13% die during this first “crisis” episode that lasts, on average, until the 9th postnatal day. Remarkably, a spontaneous remission ensues in those surviving the neonatal crisis period wherein all peripheral blood values revert to normal. A second crisis follows, and 94% of the mice die by 30 days of age. The recessive ENU (N-ethyl-N-nitrosourea)-induced mutation, hlb381, on the C57BL/6J (B6) background, is characterized by severe thrombocytopenia and leukopenia with mild anemia. Unlike scat, hlb381 is not cyclical. The phenotype is present at birth and persists throughout life. Despite the phenotypic differences, scat and hlb381 interact genetically; double heterozygotes show non-cyclical severe thrombocytopenia and leukopenia, and mild anemia (Table). This interaction implies that the scat and hlb381 genetic defects affect the same gene or distinct genes within the same pathway. Both scat and hlb381 mapped to overlapping intervals on mouse chromosome 8. Sequence analysis of genes within the interval identified Rasa3 (GAPIII, GAP1IP4BP) as a strong candidate gene for both scat and hlb381. In scat, Rasa3 carries a missense mutation near the N-terminus (G125V, exon 5) and, in hlb381, a missense mutation near the C-terminus (H794L, exon 23). RASA3 is a GTPase activating protein (GAP) that negatively regulates p21 Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. Analysis in Panther and SIFT predicts that both residues are highly conserved and substitutions are likely to be deleterious. Rasa3 is widely expressed throughout embryonic and fetal development in mice, and is ubiquitously expressed in zebrafish 24 hours post fertilization (hpf). RASA3 protein is detected in erythroid tissues and platelets in the adult mouse. Analysis of scat spleen and bone marrow erythrocyte populations by FACS (dual staining for Ter119 and CD71 followed by forward scatter of the Ter119 high population) reveals a severe block in erythropoiesis during crisis periods. The proerythroblast, EryA (basophilic erythroblasts), and EryB (late basophilic and polychromatophilic erythroblasts) populations are significantly increased in frequency vs. wild type, and the EryC (orthochromatophilic erythroblasts and reticulocytes) population is markedly decreased. Annexin V staining revealed no significant differences in any of these populations. Notably, a similar delay in erythroid maturation, albeit much milder, is also seen in hlb381. In pull-down assays using the Ras-binding domain of Raf1 to affinity purify active GTP-bound Ras followed by detection by western blotting with pan-Ras antibody, active GTP-bound Ras is deficient in scat crisis red cells but recovers during remission. Finally, injection of two independent splice-blocking morpholinos designed to disrupt exon 5 and induce disruption of rasa3 mRNA processing resulted in a major decrease in the number of hemoglobinized cells when stained with o-dianisidine at 48 and 72 hpf in zebrafish. Over 90% of morphants showed no hemoglobinized cells at all, or vastly reduced numbers (<20) of hemoglobinized cells. We conclude that RASA3 plays a critical role in vertebrate erythropoiesis. Differences in the scat and hlb381 phenotypes likely result from allele-specific interactions mediated by the different genetic backgrounds (B6 vs. BALB) or domain-specific functions of the RASA3 protein.Hematological ValuesWBC × 103/μLRBC × 106/μLHb g/dLHct %Reticulocytes %Platelets × 103/μLSpleen weight (% body weight)BALB-scat homozygotes in crisis and remissionBALB-nl5.28.713.241.911.79550.74scat crisis2.4*2.8*4.2*14.5*51.6*145*2.69*scat remission4.17.912.541.022.95611.22BALB,B6-hlb381 homozygotes and scat/hlb381 double heterozygotesBALB,B6-nl5.19.113.744.08.612190.28hlb381/hlb3812.1*9.113.343.512.0*24*0.50*hlb381/scat2.4*8.4*12.3*39.9*12.1*14*NAAll mice 18-30 days old. All values X ± SD; nl, normal littermates, NA, no data available.*P < 0.05 vs. normal littermates Disclosures:No relevant conflicts of interest to declare.

A New Animal Model for Anemia Induced by Environmental Low-Temperature: Physiology of Erythrocyte Production and Circulation

To survive, organisms must adapt to changes in the ambient environment. Here, we describe a new model of anemia based on exposure of African clawed frog, Xenopus laevis to low-temperature. Frogs exposed at low-temperature (5ºC) for five days had decreased numbers of peripheral blood erythrocytes, leukocytes, and thrombocytes as well as low hemoglobin levels. By contrast, spleen erythrocytes increased in number. Cell counts returned to normal in frogs re-warmed at ambient temperature (22ºC) for two days. To confirm these observations in vivo, we labeled peripheral blood cells with fluorescent reagent CFSE. During five days at 5ºC, labeled erythrocytes in peripheral blood decreased in number while those in spleen increased. When the temperature was raised to 22ºC, however, their numbers increased in peripheral blood. The findings suggested that exposure to low-temperature resulted in splenic pooling of peripheral erythrocytes. Accordingly, we looked at recovery from anemia induced by phenylhydrazine (PHZ) in this model. PHZ-treated frogs maintained at 22ºC decreased numbers of peripheral erythrocytes that were minimal on day 8, and increased gradually thereafter. In the liver, we found erythrocyte progenitors expressing erythropoietin receptor and GATA1-A detected by reverse transcription polymerase chain reactions and immunocytochemical staining but no mature forms. In PHZ-treated frogs exposed to 5ºC, peripheral erythrocyte counts remained minimal from day 8, and reversibly recovered when temperature returned to 22ºC. Erythrocyte progenitors were present in liver on day 8 but absent on day 12. Conversely, mature erythrocytes were absent in liver on day 8 but present on day 12. Finally, to learn whether the progenitors proliferate and differentiate without migrating from liver to peripheral blood, we treated frogs with thymidine analog bromodeoxyuridine (BrdU). In frogs kept at 22 ºC, BrdU-labeled erythrocytes were abundant in both liver and peripheral blood. However, frogs cooled at 5ºC had labeled cells in liver but few in peripheral blood. The findings suggest low-temperature exposure cause this anemia by impairing migration of mature/immature erythrocytes from the liver. In summary, this amphibian model offers a new perspective for investigating physiological effects of environmental temperature on vertebrate erythropoiesis.