Tropomodulin3-Null Mice Are Embryonic Lethal with Anemia Due to Defects in Erythroblast Enucleation and Erythroblast-Macrophage Islands in the Fetal Liver

Abstract Abstract 84 Tropomodulin1 (Tmod1) binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild sphero-elliptocytic anemia due to mis-regulation of actin filament lengths and a disrupted membrane skeleton. The mild phenotype may be explained by the compensation of Tmod3, which is not found in wild-type RBCs but exists in Tmod1-null RBCs (one-fifth level of Tmod1). Our experiments with human erythroblasts show that the expression of Tmod1 increases during terminal differentiation while the level of Tmod3 is relatively constant, only decreasing at a very late stage. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) with a gene-trap vector insertion into intron 1 of Tmod3. Both RT-PCR and western-blot results show that the expression of Tmod3 is abolished in Tmod3−/− mice. Tmod3+/− mice are viable and fertile, while Tmod3−/− animals are embryonic lethal, with most nulls dying between E16.5-E17.5. Tmod3−/− embryos are pale and anemic with a smaller fetal liver, suggesting that the lethality might be due to defective definitive erythropoiesis. This is supported by analysis of peripheral blood, which shows fewer definitive enucleated erythroblasts in Tmod3-null embryos. Flow-cytometry of fetal liver erythroblasts labeled with Ter119 and CD71 indicates that the late stage R3 population is reduced by about one-third in absence of Tmod3, while R1-R2 populations are somewhat increased. In addition, Annexin V staining shows a two-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. These data indicate that definitive erythropoiesis is defective due to impaired erythroblast terminal differentiation in absence of Tmod3. To determine the underlying mechanisms, we used histology and confocal fluorescence microscopy to investigate the morphology and actin cytoskeleton of erythroblasts in process of enucleation. These experiments show abnormal nuclear morphology in orthochromatic Tmod3-null fetal liver erythroblasts, as well as defective F-actin contractile ring assembly in Tmod3−/− erythroblasts in process of nuclear expulsion, suggesting a role for Tmod3 in enucleation. Since macrophages are required for production of definitive erythroblasts and enucleation in vivo, we explored the role of macrophages in the Tmod3−/− phenotype. Immunofluorescence staining of fetal liver cryosections with F4/80, Ter119 and Hoechst reveals that macrophages display strikingly less dendritic morphologies in the Tmod3−/− mice, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Isolation of native erythroblast-macrophage islands from fetal liver demonstrates that islands isolated from Tmod3−/− fetal livers contain fewer erythroblasts compared with those from wild-type fetal liver. Further, reconstitution experiments indicate that erythroblasts from Tmod3−/− fetal liver are unable to form normal islands, indicating that Tmod3 function is required in erythroblasts. In conclusion, our study shows that knockout of Tmod3 leads to defective definitive erythropoiesis and embryonic lethality in mice, due to defects in island formation and abnormal enucleation. These data suggest that Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion as well as contractile ring assembly during erythroblast enucleation. Disclosures: No relevant conflicts of interest to declare.

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Tropomodulin3-Null Mice Are Embryonic Lethal with Anemia and Impaired Definitive Erythropoiesis in the Fetal Liver

Blood ◽

10.1182/blood.v118.21.1036.1036 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1036-1036 ◽

Cited By ~ 1

Author(s):

Roberta B. Nowak ◽

Andrea Bacconi ◽

Zhenhua Sui ◽

Nancy E. Kim ◽

Velia M. Fowler

Keyword(s):

Fetal Liver ◽

Es Cells ◽

Insertion Site ◽

Terminal Differentiation ◽

Proliferative Potential ◽

Wild Type ◽

Mild Phenotype ◽

Embryonic Lethal ◽

Definitive Erythropoiesis

Abstract Abstract 1036 Tropomodulin1 binds tropomyosin and caps the pointed ends of the short actin filaments in the spectrin-actin network of red blood cells (RBCs). Tmod1-null mice display a mild compensated, sphero-elliptocytic anemia with fragile RBCs, due to mis-regulation of actin filament lengths and a disrupted membrane skeleton lattice (Moyer et al. 2010. Blood 116:2590–2599). The mild phenotype may be explained by the appearance of Tmod3, which is not found in wild-type RBCs but is in Tmod1-null RBCs, albeit at only 1/5th of normal Tmod1 levels. Tmod3 is ubiquitously expressed, including in erythroblasts, and thus the presence of Tmod3 in Tmod1-null RBCs is likely due to aberrant persistence of Tmod3 during terminal differentiation and maturation. To investigate the role of Tmod3 in RBCs, we created a Tmod3 knockout mouse from ES cells (#RRF004, BayGenomics) in which intron 1 of the Tmod3 locus was disrupted by retroviral-mediated insertion of a gene-trap vector. The insertion creates a novel fusion transcript joining sequences from exons 5' to the insertion site to the beta-galactosidase marker. Tmod3+/− mice are viable and fertile, but Tmod3−/− animals are embryonic lethal, with 94% of nulls dying between E13.5 – E15.5 (175 total embryos, E12.5-E18.5). Tmod3−/− embryos are pale and anemic starting at E13.5, with much smaller livers. Tmod3 mRNA and protein are reduced in Tmod3+/− and absent in Tmod3−/−, demonstrating that this is a true null. Tmod1 mRNA and protein are also reduced, consistent with anemia, fewer erythroblasts and RBCs, indicating that Tmod1 expression does not compensate for absence of Tmod3. The peripheral blood of wild-type E13.5-E15.5 embryos contains abundant large primitive and smaller definitive RBCs, but few or no definitive enucleated RBCs are observed in Tmod3-null embryos. Fetal livers from Tmod3−/− embryos were about ½ the size of wild-type livers, and histology showed altered cellularity. However, the proliferative potential of erythroid progenitors appears not to be impaired based on similar numbers of BFU-E and CFU-E colonies from Tmod3+/+ and Tmod3−/− fetal livers. Cytospins of dissociated fetal liver cells from E13.5-E14.5 embryos reveal the presence of erythroblast/macrophage islands in both genotypes, but with some macrophages containing abundant ingested cellular debris in the Tmod3−/− cytospins. High magnification images show increased erythroblast blebbing in absence of Tmod3, suggesting a weaker cortex, and/or apoptosis of erythroblasts. Flow cytometry of E14.5 fetal liver erythroblasts labeled with Ter119 and CD71 indicate that the late stage R3 population is reduced by about 40–50% in absence of Tmod3, while R1-R2 populations are somewhat increased (possibly as a compensatory response to fetal anemia). In addition, Annexin V staining shows a 2-fold increase in apoptotic cells in the fetal liver, most of which are in the R1 population. Measurement of enucleation frequency in R populations shows a marked reduction of enucleated cells as the erythroblasts mature through the R3-R5 populations. To explore the role of macrophages in the Tmod3−/− phenotype, we performed immunofluorescence staining and confocal microscopy of fetal liver cryosections stained with F4/80 (macrophages), Ter119 (erythroblasts) and Hoechst (nuclei). Macrophages displayed strikingly less dendritic morphologies in the Tmod3−/−, with macrophages sometimes containing Ter119-positive nucleated erythroblasts. Our data show that definitive erythropoiesis is impaired during terminal differentiation in absence of Tmod3, and we speculate that this is due to defective erythroblast-macrophage interactions during terminal differentiation. Experiments are in progress to determine whether Tmod3 function is required in erythroblasts or macrophages, and to identify the molecular pathway in which Tmod3 functions in erythropoiesis. Supported by NIH/NHLBI grant R01HL083464 to V.M.F. Disclosures: No relevant conflicts of interest to declare.

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The role of anillin in meiotic cytokinesis of Drosophila males

Journal of Cell Science ◽

10.1242/jcs.112.14.2323 ◽

1999 ◽

Vol 112 (14) ◽

pp. 2323-2334 ◽

Cited By ~ 10

Author(s):

M.G. Giansanti ◽

S. Bonaccorsi ◽

M. Gatti

Keyword(s):

Narrow Band ◽

Actin Binding ◽

Cleavage Furrow ◽

Ph Domain ◽

Wild Type ◽

Contractile Ring ◽

Ring Assembly ◽

Actin Binding Domain ◽

Carboxy Terminal

Anillin is a 190 kDa actin-binding protein that concentrates in the leading edges of furrow canals during Drosophila cellularization and in the cleavage furrow of both somatic and meiotic cells. We analyzed anillin behavior during D. melanogaster spermatogenesis, and focused on the relationships between this protein and the F-actin enriched structures. In meiotic anaphases anillin concentrates in a narrow band around the cell equator. Cytological analysis of wild-type meiosis and examination of mutants defective in contractile ring assembly (chickadee and KLP3A), revealed that the formation of the anillin cortical band occurs before, and does not require the assembly of the F-actin based contractile ring. However, once the acto-myosin ring is assembled, the anillin band precisely colocalizes with this cytokinetic structure, accompanying its contraction throughout anaphase and telophase. In chickadee and KLP3A mutant ana-telophases the cortical anillin band fails to constrict, indicating that its contraction is normally driven by the cytokinetic ring. These findings, coupled with the analysis of anillin behavior in twinstar mutants, suggested a model on the role of anillin during cytokinesis. During anaphase anillin would concentrate in the cleavage furrow before the assembly of the contractile ring, binding the equatorial cortex, perhaps through its carboxy-terminal pleckstrin homology (PH) domain. Anillin would then interact with the actin filaments of the acto-myosin ring through its actin-binding domain, anchoring the contractile ring to the plasma membrane throughout cytokinesis.

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

Blood ◽

10.1182/blood.v106.11.806.806 ◽

2005 ◽

Vol 106 (11) ◽

pp. 806-806 ◽

Cited By ~ 1

Author(s):

Shivani Soni ◽

Shashi Bala ◽

Babette Gwynn ◽

Kenneth E. Sahr ◽

Luanne L. Peters ◽

...

Keyword(s):

Fetal Liver ◽

Direct Sequencing ◽

Embryonic Stem ◽

Gene Trap ◽

Physical Contact ◽

Erythroid Cells ◽

Wild Type ◽

Definitive Erythropoiesis

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

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A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.3149.3149 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3149-3149

Author(s):

Yoko Ogawara ◽

Takuo Katsumoto ◽

Takeshi Uchiumi ◽

Kimitoshi Kohno ◽

Issay Kitabayashi

Keyword(s):

Acute Myeloid Leukemia ◽

Colony Formation ◽

Myeloid Leukemia ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Wild Type ◽

Embryonic Lethal ◽

Hoxa Genes ◽

Fetal Liver Cells ◽

Acute Myeloid

Abstract Abstract 3149 Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established. Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis. To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

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Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Blood ◽

10.1182/blood-2021-146004 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 21-21

Author(s):

Mayuri Tanaka-Yano ◽

Dahai Wang ◽

Eleanor Meader ◽

Melissa A. Kinney ◽

Vivian Morris ◽

...

Keyword(s):

Young Adult ◽

Developmental Stage ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Polycomb Group ◽

Wild Type ◽

Hematopoietic Stem ◽

Protein Levels ◽

Developmental Maturation

Abstract Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis. First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7. Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels. As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold). To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors. Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. Disclosures No relevant conflicts of interest to declare.

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Temporally Distinct Developmental Waves of Erythropoiesis from Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v126.23.1170.1170 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1170-1170

Author(s):

Orna Steinberg Shemer ◽

Marta Byrska-Bishop ◽

Jacob C Ulirsch ◽

Osheiza Abdulmalik ◽

Yu Yao ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Fetal Liver ◽

Terminal Differentiation ◽

Embryoid Bodies ◽

Hematopoietic Stem ◽

Erythroid Precursors ◽

Definitive Erythropoiesis ◽

Human Ipsc

Abstract Mammalian erythropoiesis during embryogenesis occurs in several distinct stages or "waves" that vary according to timing, site of production, gene expression and physiology. The ontogeny of mammalian erythropoiesis is most thoroughly studied in mice where the earliest circulating erythroblasts released from the yolk sac are termed primitive. Later, the first definitive erythroid lineage is established by erythro-myeloid progenitors (EMPs) that originate in the yolk sac and migrate to the fetal liver for terminal differentiation. A second wave of definitive erythropoiesis is established from hematopoietic stem/progenitor cells that originate in the dorsal aorta and migrate to later stage fetal liver for terminal differentiation. Finally around birth, definitive erythropoiesis shifts to the bone marrow. The ontogeny of erythropoiesis overlaps in mice and humans, although less is known about the latter, as hematopoietic tissues from precisely staged early human embryos are difficult to obtain. We hypothesized that the initial steps of human erythroid ontogeny could be recapitulated by induced pluripotent stem cells (iPSCs) induced to undergo hematopoietic differentiation. We used a serum- and feeder-free protocol to differentiate iPSCs into embryoid bodies (EBs) that produced two sequential waves of distinctly different erythroid precursors. At day 8 of differentiation, EBs began to release hematopoietic precursors. Thereafter, erythroid precursors were released from the EBs in the presence of stem cell factor (SCF), erythropoietin (EPO) and insulin-like growth factor 1 (IGF-1). Erythroid precursors produced during wave 1 (days 12-23 of differentiation) were relatively large and expressed embryonic-type globins (zeta and epsilon), resembling those produced during primitive erythropoiesis. In contrast, wave 2 erythroblasts (day 27 and later) were smaller and expressed mainly gamma and alpha globins with some beta globin, suggestive of fetal-type definitive erythropoiesis. To investigate further the similarity of wave 1 and wave 2 erythroblasts to cells at the primitive and definitive stages of ontogeny, respectively, we used Affymetrix Genechips to analyze the global transcriptomes of stage-matched (CD235+ CD71high) cells. As primary human primitive erythroblasts were not available for comparison, we compared the transcriptomes from the iPSC-derived erythroblasts with those of primary murine definitive and primitive erythroblasts that were flow cytometry-purified from embryonic day 15.5 (E15.5) fetal liver and E10.5 bloodstream, respectively. The comparisons showed that wave 1 erythroblasts from human pluripotent cells resembled more closely the erythroid primitive lineage from mice, while wave 2 erythroblasts from the human cells resembled the erythroid definitive lineage of mice (P-value < 0.05 by a modified Kolmogorov-Smirnov test). For example, SOX6 and BCL11A, preferentially expressed during definitive erythropoiesis, were expressed at relatively high levels in wave 2 erythroblasts. In addition, gene set enrichment analysis (GSEA) demonstrated that wave 2 human iPSC-derived erythroblasts and primary murine definitive erythroblasts expressed numerous genes related to immune/inflammatory pathways that were shown previously to be important for the formation of definitive hematopoietic stem and progenitor cells in zebrafish and mouse embryos. Our findings demonstrate that human iPSC-derived embryoid bodies recapitulate early stages of erythroid ontogeny with respect to the timing of emerging lineages and their gene expression. Additionally, gene expression studies of human iPSC-derived primitive and definitive erythroblasts indicate inflammatory signaling as a potential regulator of the later stage of erythroid development. Disclosures No relevant conflicts of interest to declare.

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Epor Stimulates Rapid Cycling and Larger Red Cells during Mouse and Human Erythropoiesis

Blood ◽

10.1182/blood-2021-154403 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 852-852

Author(s):

Daniel Hidalgo ◽

Jacob Bejder ◽

Ramona Pop ◽

Kyle Gellatly ◽

Yung Hwang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Size ◽

Fetal Liver ◽

Red Cells ◽

Red Cell ◽

Rapid Cycling ◽

Wild Type ◽

The Relationship

Abstract Erythroid terminal differentiation (ETD) entails cell divisions coupled to decreasing cell size. The tight link between the number of cell divisions and red cell size is apparent in nutritional deficiencies or genetic variants in which fewer cycles result in larger red cells. Here we investigated novel EpoR functions, finding that EpoR signaling disrupts the relationship between cell cycle number and cell size, simultaneously promoting rapid cycling and the formation of larger red cells. EpoR is essential for erythroblast survival, but it is unclear whether it has other non-redundant functions. To address this, we developed a genetic system in which we rescue mouse Epor -/- fetal liver progenitors from apoptosis by transduction with the anti-apoptotic protein Bcl-x L, and compare their ensuing differentiation with that of Epor -/- progenitors rescued with EpoR (Fig 1a). We found that the Bcl-x L survival signal, in the absence EpoR, supported formation of enucleated red cells. However, key ETD features were abnormal. First, Bcl-x L-transduced Epor -/- erythroblasts underwent slower and fewer cell cycles (Figure 1b), differentiating prematurely into enucleated red cells. Premature induction of the cyclin-dependent-kinase inhibitor p27 KIP1 was in part responsible for the fewer cycles in the absence of EpoR signaling. We confirmed that EpoR also stimulates rapid cycling in wild-type erythroblasts in vivo, using a mouse transgenic for a live-cell reporter of cell cycle speed. Second, using imaging flow cytometry, we found that Bcl-x L-transduced Epor -/- erythroblasts were smaller than EpoR-transduced Epor -/- cells (Fig 1c,d). By doubly transducing Epor -/- erythroblasts with both Bcl-x L and EpoR, we verified that EpoR absence, and not Bcl-x L overexpression, is responsible for the smaller size of Bcl-x L-transduced Epor -/- erythroblasts and reticulocytes. Bcl-x L-transduced Epor -/- erythroblasts failed to upregulate the transferrin receptor, suggesting that iron deficiency may be responsible for their smaller size. However, neither iron supplementation, nor transduction with the transferrin receptor, rescued their smaller size. Iron regulates cell size through Heme-regulated eIF2α kinase (HRI). To definitively test the role of iron and HRI, we generated mice doubly deleted for both EpoR and HRI. We then rescued both Epor -/- and Epor -/-Hri -/- -fetal liver cells in parallel, by transduction with either Bcl-x L or EpoR. In agreement with the known role of HRI as a negative regulator of erythroblast size, both Bcl-x L- transduced and EpoR-transduced erythroblasts were larger on the Epor -/-Hri -/- genetic background. However, the difference in size between Bcl-x L and EpoR-rescued erythroblasts persisted in Epor -/-Hri -/- erythroblasts and reticulocytes (Fig 1c,d), conclusively showing that EpoR signaling regulates cell size independently of the HRI pathway. EpoR promoted increased erythroblast and reticulocyte cell size in wild-type mice in vitro and in vivo, in response to Epo concentrations ranging from 10 to 10,000 mU/ml. We also evaluated the effect of Epo on red cell size in humans, in two independent studies, where healthy volunteers were administered Epo for either 3 weeks (20 IU /kg every 48 hours, 25 subjects, Study #1) or for 7 weeks (weekly Epo dosing that increased hemoglobin by 10 -15%; 24 subjects, Study #2). In a third intervention, 21 subjects participated in a randomized double-blind placebo-controlled crossover study in which 900 ml of whole blood was withdrawn from the treatment group by venipuncture. In all three studies, the increase in MCV in the treatment groups persisted long after Epo and reticulocyte levels returned to baseline (Figure 2). There was no correlation between MCV and the reticulocyte count, whose time courses were clearly divergent (r < 0.1, Pearson's product-moment correlation). Further, computational simulation suggests that the extent and duration of the increase in MCV is unlikely to be the result of skewing of the circulating red cell population in favor of younger, larger red cells. Our work reveals a paradoxical EpoR-driven increase in erythroblast cycling simultaneously with increased erythroblast and red cell size. It suggests that EpoR alters the relationship between cell cycle and biomass in erythroblasts. It further suggests that hypoxia, anemia and other high-Epo syndromes are new diagnostic interpretations of increased MCV in the clinic. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The -566 Aγ-Globin HPFH Mutation Disrupts Temporal Repression of Fetal Hemoglobin Synthesis During Fetal Liver Definitive Erythropoiesis.

Blood ◽

10.1182/blood.v114.22.2019.2019 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2019-2019

Author(s):

Kenneth R Peterson ◽

Halyna Fedosyuk ◽

Flavia C Costa

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Wild Type ◽

Repressor Complex ◽

Definitive Erythropoiesis ◽

Gata Motif ◽

Globin Gene Expression

Abstract Abstract 2019 Poster Board I-1041 Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T>G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1201 ◽

2009 ◽

Vol 20 (8) ◽

pp. 2160-2173 ◽

Cited By ~ 57

Author(s):

Colleen T. Skau ◽

Erin M. Neidt ◽

David R. Kovar

Keyword(s):

Fission Yeast ◽

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Actin Assembly ◽

Animal Cells ◽

Contractile Ring ◽

Direct Role ◽

Ring Assembly

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

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Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver

Blood ◽

10.1182/blood-2013-03-492710 ◽

2014 ◽

Vol 123 (5) ◽

pp. 758-767 ◽

Cited By ~ 32

Author(s):

Zhenhua Sui ◽

Roberta B. Nowak ◽

Andrea Bacconi ◽

Nancy E. Kim ◽

Hui Liu ◽

...

Keyword(s):

Cell Cycle ◽

Fetal Liver ◽

Terminal Differentiation ◽

Cell Types ◽

Cell Cycle Exit ◽

Erythroid Progenitors ◽

Island Formation ◽

Erythroid Terminal Differentiation ◽

Embryonic Lethal ◽

Key Points

Key Points Tmod3 deletion leads to reduced erythroid progenitors and impaired erythroblast survival, cell-cycle exit, and enucleation. Erythroblast-macrophage islands are reduced in the absence of Tmod3, which is required in both cell types for island formation.

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