Differences in Steady-State Erythropoiesis in Different Mouse Bones and Postnatal Spleen

Adult erythropoiesis is a highly controlled sequential differentiation of hematopoietic stem cells (HSCs) to mature red blood cells in the bone marrow (BM). The bones which contain BM are diverse in their structure, embryonic origin, and mode of ossification. This has created substantial heterogeneity in HSCs function in BM of different bones, however, it is not known if this heterogeneity influences erythropoiesis in different bones and different regions of the same bone. In this study, we examined steady state BM erythroid progenitors and precursors from different bones – the femur, tibia, pelvis, sternum, vertebrae, radius, humerus, frontal, parietal bone, and compared all to the femur. Trabecular and cortical regions of the femur were also compared for differences in erythropoiesis. In addition, mouse spleen was studied to determine at which age erythropoietic support by the spleen was lost postnatally. We report that total erythroid cells, and erythroid precursors in the femur are comparable to tibia, pelvis, humerus and sternum, but are significantly reduced in the vertebrae, radius, frontal, and parietal bones. Erythroid progenitors and multipotential progenitor numbers are comparable in all the bones except for reduced number in the parietal bone. In the femur, the epiphysis and metaphysis have significantly reduced number of erythroid precursors and progenitors, multipotential progenitors and myeloid progenitors compared to the diaphysis region. These results show that analysis of erythroid precursors from diaphysis region of the femur is representative of tibia, pelvis, humerus and sternum and have significant implications on the interpretation of the steady-state erythropoiesis finding from adult BM. Postnatal spleen supports erythroid precursors until 6 weeks of age which coincides with reduced number of red pulp macrophages. The residual erythroid progenitor support reaches the adult level by 3 months of age. In conclusion, our findings provide insights to the differences in erythropoiesis between different bones, between trabecular and cortical regions of the femur, and developmental changes in postnatal spleen erythropoiesis.

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EPO-Dependent Recovery of Late-Stage Erythroid Progenitors in the Marrow Precedes Splenic Expansion: Insights From a Sublethal Radiation Model

Blood ◽

10.1182/blood.v118.21.180.180 ◽

2011 ◽

Vol 118 (21) ◽

pp. 180-180

Author(s):

Scott A Peslak ◽

Jesse Wenger ◽

Amali P Epa ◽

Jeffrey C Bemis ◽

Paul D Kingsley ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Late Stage ◽

Erythroid Cell ◽

Functional Evaluation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Irradiation Injury ◽

Radiation Induced ◽

Sublethal Irradiation

Abstract Abstract 180 Erythropoiesis is a robust process of cellular expansion and maturation that occurs in the bone marrow and spleen of mice. Following clastogenic injury such as total body irradiation (TBI), erythroblasts are severely depleted in these organs, resulting in loss of reticulocyte output and the development of a mild anemia (Peslak et al., Exp. Hematol. 2011). However, the mechanistic and microenvironmental factors underlying erythroid recovery following sublethal TBI are poorly understood. To this end, we utilized colony assays to quantify erythroid progenitors, which consist of immature d7 erythroid burst-forming units (BFU-E) and more mature d3 BFU-E and erythroid colony forming units (CFU-E). Imaging flow cytometry was used to quantify erythroblast precursors. We found that d7 BFU-E undergo a slow, incomplete recovery during the first 10 days post-4 Gy TBI of C57Bl/6 mice. In contrast, d3 BFU-E exhibit a robust recovery beginning at 4 days post-TBI that is immediately followed by a rapid increase in CFU-E numbers to over 200 percent of steady-state levels. This initial erythroid progenitor recovery is followed by a wave of erythroid precursor maturation and red cell formation that occurs in close association with macrophages in the bone marrow. These erythroblast islands undergo a rapid synchronous expansion that peaks at 6 days post-TBI, suggesting that the bone marrow microenvironment plays a role in the recovery of the erythron from sublethal TBI. We hypothesized that erythropoietin (EPO), the primary regulator of erythroid survival and proliferation, mediates the rapid, specific expansion of late-stage erythroid progenitors following radiation injury. We found that plasma EPO levels increase 13-fold 4 days after 4 Gy TBI, temporally correlated with expansion of d3 BFU-E. Furthermore, maintenance of steady-state hematocrit levels following TBI prevented EPO induction and blocked expansion of late-stage erythroid progenitors, while exogenous EPO administered at 1 hour post-radiation specifically advanced recovery of late-stage progenitors. These data indicate that EPO is required for expansion of d3 BFU-E and CFU-E following radiation-induced marrow depletion. During times of acute hypoxia, such as the severe anemia induced by bleeding or phenylhydrazine exposure, EPO production is rapidly upregulated and splenic stress erythropoiesis is induced. Surprisingly, splenic erythropoiesis is absent during the rapid initial recovery of erythropoiesis in the bone marrow at 4–6 days post-TBI. However, a massive expansion of CFU-E begins at 7–8 days post-4 Gy TBI in spleen. EPO administration at 4 days following 4 Gy TBI significantly enhances late-stage progenitor recovery exclusively in the marrow, indicating that erythroid progenitors are not present in spleen at the time of rapid bone marrow expansion and that late-stage erythroid progenitor recovery initiates in the marrow and subsequently proceeds to the spleen. Furthermore, we found that erythroid progenitors transiently emerge in the bloodstream at 6–8 days post-TBI, following marrow recovery and prior to initiation of splenic erythropoiesis. These data are consistent with endogenous migration of the erythron from the bone marrow to the spleen during recovery from radiation-induced erythroid injury. Taken together, our data indicate that recovery from sublethal irradiation injury is regulated primarily by the EPO-induced expansion of late-stage erythroid progenitors in the bone marrow. This form of clastogenic injury is critically different from bleeding or hemolysis, which preserve bone marrow and splenic erythroblasts and induce expansion of splenic erythroid stress progenitors. Sublethal irradiation injury thus provides a unique model for the in vivo study of endogenous erythroid recovery. This model may be clinically useful for the functional evaluation of therapeutic factors that regulate or modulate erythroid cell maturation. Disclosures: Bemis: Litron Laboratories: Employment, Patents & Royalties.

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Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus

Blood ◽

10.1182/blood-2010-07-299743 ◽

2011 ◽

Vol 117 (9) ◽

pp. 2708-2717 ◽

Cited By ~ 72

Author(s):

Samantha J. England ◽

Kathleen E. McGrath ◽

Jenna M. Frame ◽

James Palis

Keyword(s):

Stem Cell ◽

Glucocorticoid Receptors ◽

Ex Vivo ◽

Fetal Liver ◽

Erythroid Progenitors ◽

Transfusion Therapy ◽

Self Renewal ◽

Hematopoietic Stem ◽

Erythroid Precursors ◽

Definitive Erythropoiesis

Abstract In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (102- to 105-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (106- to 1060-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.

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SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2021-153814 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2189-2189

Author(s):

Mark C Wilkes ◽

Aya Shibuya ◽

Vanessa M Scanlon ◽

Hee-Don Chae ◽

Anupama Narla ◽

...

Keyword(s):

Cord Blood ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Culture Media ◽

Hsp70 Expression ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Enhancer Element ◽

Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

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Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽

10.1182/blood-2021-152898 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 288-288

Author(s):

Suhita Ray ◽

Linda Chee ◽

Nicholas T. Woods ◽

Kyle J Hewitt

Keyword(s):

Steady State ◽

Hemolytic Anemia ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Interacting Proteins ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Capping Protein ◽

Sam Domain ◽

Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

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Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2019-129570 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 455-455

Author(s):

Mark C Wilkes ◽

Jacqueline D Mercado ◽

Mallika Saxena ◽

Jun Chen ◽

Kavitha Siva ◽

...

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Healthy Controls ◽

In Vitro Activity ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program

Blood ◽

10.1182/blood.v130.suppl_1.7.7 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 7-7

Author(s):

Charu Mehta ◽

Kirby D Johnson ◽

Xin Gao ◽

Irene Ong ◽

Koichi Ricardo Katsumura ◽

...

Keyword(s):

Target Gene ◽

Fetal Liver ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Erythroid Precursors ◽

A Cell ◽

Erythroid Maturation ◽

Myeloid Progenitors

Abstract GATA-2 levels must be stringently regulated to ensure normal hematopoiesis, and human GATA-2 mutations cause hematologic disorders. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells and are essential for hematopoiesis and embryonic development. Mechanisms underlying how the enhancers control Gata2 expression and GATA-2 instigated genetic networks in a cell-specific manner are not completely understood. Targeted deletion of an intronic Gata2 enhancer 9.5 kb downstream of the transcription start site (+9.5) abrogates HSC genesis in the aorta-gonad-mesonephros (AGM) region (Gao et al., JEM, 2013). By contrast, the -77 kb enhancer (-77) activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation (Johnson et al., Science Advances, 2015). To dissect relationships between the enhancers, we developed a compound heterozygous (CH) mouse model bearing +9.5 and -77 enhancer mutations on different Gata2 alleles. While the CH embryos were alive at E13.5, nearly all died by E14.5 (p = 3.58 x 10-5). Flow cytometric analyses and embryo confocal imaging demonstrated that CH embryos have modestly reduced HSC numbers in the fetal liver (2.9-fold) and the AGM (41%, p = 7.8 x 10-5), which was comparable to +9.5+/- embryos. Thus, -77 does not genetically interact with +9.5 to control HSC emergence. Flow cytometric analysis revealed that Lin-Sca1-Kit+ myelo-erythroid progenitors were 6.6-fold lower in CH vs. WT embryos (p = 1.8 x 10-11), with the difference involving disproportionate losses of GMP (8.6-fold; p = 3.7 x 10-6) and MEP (379-fold; p = 3.2 x 10-9). By contrast, +9.5+/- fetal livers had 2-fold fewer myeloid progenitors, which involved similar reductions of CMP (2.1-fold; p = 1 x 10-6), GMP (2.6-fold; p = 0.0007) and MEP (1.9-fold; p = 0.002). Consistent with the myelo-erythroid progenitor reductions and MEP depletion, CH fetal livers lacked BFU-E (p < 0.001) and CFU-GEMM (p < 0.001) in a colony assay. These results illustrate a genetic interaction between +9.5 and -77 in progenitors, but not HSCs, and a new paradigm in which both enhancers must reside on a single allele to generate MEPs. As erythroid precursor cells express GATA-2, we tested whether the -77 deletion impairs erythroid maturation due to a reduction in myelo-erythroid progenitors or due to a cell-autonomous requirement of the enhancer in erythroid precursors. -77-/- E14.5 fetal livers were pale and smaller than WT counterparts, and -77-/- fetal liver cellularity was reduced 7.2-fold (5.3 x 10-4). When liver size was taken into account, there was little difference in the number of E14.5 R1 cells in -77-/- liver vs. WT littermates (p = 0.31). However, -77-/- R2-R5 cells declined sharply (R2, 8.2-fold, p = 0.004; R3, 14-fold, p < 10-5; R4, 9.7-fold, p = 0.002; R5, 14-fold, p = 0.087). The mutant R1 cells were defective in forming BFU-Es and CFU-Es. Analysis of transcriptomes of purified 77-/- and WT R1 cells from E14.5 fetal livers revealed 2805 and 2519 upregulated and downregulated (p < 0.05) genes, respectively, in -77-/- R1 cells. The -77 enhancer conferred GATA-2 expression, which strongly upregulated GATA-1 and therefore a large GATA-1 target gene cohort. A comparison of WT and -77-/- R1 cell transcriptomes with those of early (Tgbfr3low) and late (Tgbfr3high) BFU-Es (Gao et al., Blood, 2016) revealed a -77-/- R1 signature that correlated with the early BFU-E signature (r = 0.73, p < 10-4) and negatively correlated with the late BFU-E signature (r = -0.42, p = 4 x 10-4) differing from WT cells. In addition to GATA-1 target gene alterations, 253 of the -77-activated genes were not GATA-1-regulated in the G1E-ER-GATA-1 system. These genes included Ryk, which encodes a non-canonical Wnt receptor, and had not been studied in erythroid cells. Two Ryk shRNAs significantly decreased BFU-Es and CFU-GMs in lineage-depleted fetal liver cells. Ongoing studies are integrating Ryk function into signaling circuits that control erythroid maturation and analyzing other -77-regulated targets predicted to constitute new regulators of erythroid cell maturation/function. Thus, loss of the -77 enhancer creates multi-faceted defects in erythroid precursors, involving deficiencies of constituents of signaling and transcriptional circuitry required to enable and drive erythroid maturation. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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The triple origin of skull in higher vertebrates: a study in quail-chick chimeras

Development ◽

10.1242/dev.117.2.409 ◽

1993 ◽

Vol 117 (2) ◽

pp. 409-429 ◽

Cited By ~ 50

Author(s):

G.F. Couly ◽

P.M. Coltey ◽

N.M. Le Douarin

Keyword(s):

Neural Crest ◽

Sella Turcica ◽

Parietal Bone ◽

Paraxial Mesoderm ◽

Avian Embryo ◽

Evolutionary Context ◽

Embryonic Origin ◽

Neural Crest Origin ◽

Parietal Bones ◽

Double Origin

We have used the quail-chick chimera technique to study the origin of the bones of the skull in the avian embryo. Although the contribution of the neural crest to the facial and visceral skeleton had been established previously, the origin of the vault of the skull (i.e. frontal and parietal bones) remained uncertain. Moreover formation of the occipito-otic region from either the somitic or the cephalic paraxial mesoderm had not been experimentally investigated. The data obtained in the present and previous works now allow us to assign a precise embryonic origin from either the mesectoderm, the paraxial cephalic mesoderm or the five first somites, to all the bones forming the avian skull. We distinguish a skull located in front of the extreme tip of the notochord which reaches the sella turcica and a skull located caudally to this boundary. The former ('prechordal skull') is derived entirely from the neural crest, the latter from the mesoderm (cephalic or somitic) in its ventromedial part ('chordal skull') and from the crest for the parietal bone and for part of the otic region. An important point enlighten in this work concerns the double origin of the corpus of the sphenoid in which basipresphenoid is of neural crest origin and the basipostsphenoid is formed by the cephalic mesoderm. Formation of the occipito-otic region of the skeleton is particularly complex and involves the cooperation of the five first somites and the paraxial mesoderm at the hind-brain level. The morphogenetic movements leading to the initial puzzle assembly could be visualized in a reproducible way by means of small grafts of quail mesodermal areas into chick embryos. The data reported here are discussed in the evolutionary context of the ‘New Head’ hypothesis of Gans and Northcutt (1983, Science, 220, 268–274).

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An erythroid to myeloid cell fate conversion is elicited by LSD1 inactivation

Blood ◽

10.1182/blood.2021011682 ◽

2021 ◽

Author(s):

Lei Yu ◽

Greggory Myers ◽

Chia-Jui Ku ◽

Emily Schneider ◽

Yu Wang ◽

...

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Myeloid Cell ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Cell Fate Conversion ◽

Bac Transgene

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of b-globinopathies (sickle cell disease and b-thalassemia) since it is a component of g-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 BAC transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of GMP-like cells, converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells (HSPC), coincident with induction of myeloid transcription factors (e.g. PU.1 and CEBPa). Finally, blocking the activity of transcription factors PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors, and that inhibition of the myeloid differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

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RUNX1 and NF-E2 Transcriptional Upregulation Is Not Specific For Myeloproliferative Neoplasms But Is Seen In Those Polycythemic Disorders With Augmented HIFs Signaling

Blood ◽

10.1182/blood.v122.21.2177.2177 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2177-2177

Author(s):

Katarina Kapralova ◽

Lucie Lanikova ◽

Felipe R Lorenzo V ◽

Monika Horvathova ◽

Vladimir Divoky ◽

...

Keyword(s):

Stem Cells ◽

Myeloproliferative Neoplasms ◽

Cell Mass ◽

Homozygous Mutation ◽

Erythroid Progenitors ◽

Gain Of Function ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Exon 2 ◽

Secondary Polycythemia

Abstract RUNX1 and NF-E2 are transcription factors that regulate hematopoietic stem cell homeostasis. It has been reported that increased RUNX1 expression in the granulocytes is present in all three classical myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis (Wang et al, Blood 2010), and that elevated NF-E2 promotes erythropoietin (EPO)-independent erythroid maturation of hematopoietic stem cells in vitro (Bogeska et al, Stem Cells Transl Med 2013). A mouse model overexpressing the NF-E2 transgene in hematopoietic cells was reported to be a new model of myeloproliferative neoplasms (Kaufmann et al, J Exp Med 2012). Polycythemic states can be divided into primary polycythemias, characterized by intrinsically hyperproliferative erythroid progenitors that are hypersensitive to EPO, and secondary polycythemias, wherein erythroid progenitors respond normally to EPO but circulating EPO is elevated or inappropriately normal for the level of increased red cell mass. Some congenital disorders including those with mutations in the hypoxia sensing pathway may share features of both primary and secondary polycythemias. We considered the possibility that increased transcripts of RUNX1 and NF-E2 might be the feature of other primary polycythemic states as well. We report a study of 19 polycythemic patients with primary or secondary polycythemia with diverse etiologies including mutations in positive and negative regulators of hypoxia sensing pathway. RUNX1 and NF-E2 transcripts were quantitated in granulocytes and BFU-E colonies by qPCR. All primary polycythemic patients had erythroid progenitors hypersensitive to or independent to EPO; all secondary polycythemic subjects had normal erythroid progenitor response to EPO. RUNX1 and NF-E2 gene transcripts were increased in granulocytes and BFU-E colonies in all PV patients, two unrelated subjects with the VHLR200W homozygous mutation (Chuvash polycythemia), one polycythemic patient homozygous for the VHLP138L exon 2 mutation, and a patient with the HIF2αM535V gain-of-function mutation. We also found upregulated expression of RUNX1 and NF-E2 in granulocytes and BFU-Es from a polycythemic patient (with no detectable EPOR, JAK2V617F or JAK2 exon 12 mutations and low level of EPO < 1 mU/mL) who was heterozygous for a SNP in exon 3 (rs147341899) in the LNK gene. We examined transcripts of RUNX1 and NF-E2 genes in granulocytes from two Croatian polycythemic patients with a homozygous VHLH191D exon 3 mutation whose erythroid progenitors were not hypersensitive to EPO and whose RUNX1, but not NF-E2, transcript was increased. We found similar results in two compound heterozygotes for VHLT124A exon 2 and VHLL188V exon 3 mutations. These two polycythemic siblings had hypersensitive erythroid colonies, increased RUNX1 transcripts and decreased NF-E2 transcripts in granulocytes. RUNX1 and NF-E2 transcripts were normal in two subjects with primary polycythemia due to the EPOR gain-of-function EPORQ434Xmutation, and in five unrelated subjects with secondary polycythemia. We next examined granulocyte transcripts of HIF-regulated genes: TFRC, SLC2A1, VEGF, BNIP3 and HK1, and found them to be increased in all PV patients and all studied polycythemic patients with VHL, HIF2α or LNK mutations, but not in polycythemic EPORQ434Xpatients or five patients with secondary polycythemia. Increased transcripts of HIF regulated genes are compatible with the previously unappreciated Warburg effect in PV (see S. Sana's Abstract at this ASH meeting). We propose that increased expression of RUNX1 and NF-E2 is not specific for myeloproliferative neoplasms but also is not universal for primary polycythemic disorders. Therefore, increased expression of RUNX1 and NF-E2 do not seem to be underlying mechanism for MPNs development but rather represent factors associated with diverse primary polycythemia states with augmented HIF signaling. (Note: KK and LL contributed equally to this work.) This work was supported by 1P01CA108671-O1A2 (NCI) Myeloproliferative Disorders (MPD) Consortium (PI Ron Hoffman) project#1 (PI Prchal) and the Leukemia & Lymphoma Society. Work by KK, LL, MH and VD was in part supported by the European Structural Funds (project CZ.1.07/2.3.00/20.0164 and CZ.1.07/2.3.00/30.0041), grant LF_2013_010 and by Czech Science Foundation (Project-P301/12/1503). Disclosures: No relevant conflicts of interest to declare.

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CAM Kinase IV Regulates Lineage Commitment and Survival of Erythroid Progenitors in a Non-Cell–Autonomous Manner

The Journal of Cell Biology ◽

10.1083/jcb.151.4.811 ◽

2000 ◽

Vol 151 (4) ◽

pp. 811-824 ◽

Cited By ~ 9

Author(s):

Gary A. Wayman ◽

Melinda J. Walters ◽

Kathryn Kolibaba ◽

Thomas R. Soderling ◽

Jan L. Christian

Keyword(s):

Apoptotic Cell ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Dependent Protein Kinase ◽

Hematopoietic Stem ◽

Erythroid Precursors ◽

Dependent Protein ◽

Strict Regulation ◽

Cell Commitment ◽

Primitive Erythropoiesis

Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

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