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Balancing hematopoietic cell fate and endothelial identity in hemogenic endothelium
Oral Short Talk Presentations/ Experimental Hematology 42 (2014) S13–S21
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Oral Short Talk Presentations O1001 - BALANCING HEMATOPOIETIC CELL FATE AND ENDOTHELIAL IDENTITY IN HEMOGENIC ENDOTHELIUM Carlos Lizam1, John Hawkins1, Frank Bos1, Joan Zape1, Joshua Wythe2, Mary Donohoe3, and Ann Zovein1,4 1 Cardiovascular Research Institute, University of California, San Francisco, California, USA; 2Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California, USA; 3Burke Medical Research Institute Weill Cornell Medical College, White Plains, New York, USA; 4Department of Pediatrics, Division of Neonatology, University of California San Francisco School of Medicine, San Francisco, California, USA Hematopoietic stem and progenitor cells (HP/SCs) first emerge from the embryonic endothelium during development to establish what will eventually become the adult hematopoietic system, in a process termed the endothelial to hematopoietic transition (EHT). Factors proven critical for EHT include Runx1, Sox17, and Notch1. However, early endothelial loss of either notch1 or sox17 also disrupts vascular specification, while runx1 impacts hematopoiesis without an overt vascular consequence. Here we present data that Notch1 and Sox17 are actually dispensable for hematopoietic emergence from the endothelium during EHT, and that endothelial loss after vascular specification results in augmented hematopoietic emergence. Using in vivo and in vitro timed genetic deletion and rescue, we demonstrate that endothelial and hematopoietic fates are determined by Sox17 which acts as a transcriptional gatekeeper of downstream endothelial (Notch1) and hematopoietic (Runx1) genes. Sox17 prevents EHT in mature endothelium by directly promoting endothelial Notch1 signaling, while directly repressing Runx1 expression. Thus, the timing of Sox17 or Notch1 endothelial loss has a compelling impact on hematopoietic outcome, as early deletion prevents EHT while later deletion promotes it.
O1002 - EMERGENCE OF THE NEUTROPHIL LINEAGE IN THE MAMMALIAN EMBRYO Kathleen E. McGrath, Katherine Fegan, Seana Catherman, and James Palis University of Rochester, Rochester, New York, USA Hematopoietic stem cells (HSCs) are responsible for the maintenance of circulating neutrophils in the adult. However, the onset and developmental origin of neutrophils in the embryonic bloodstream is poorly understood. In order to examine early neutrophil emergence in murine embryos, we mated GFP+ male mice with wild-type females, which allowed us to distinguish embryonic from maternal cells. By flow cytometry, we identified rare GFP+ Gr1+Mac1+ cells circulating in embryonic day 11.5 (E11.5) that increased in numbers from E12.5 to E14.5. These sorted GFP+ cells had typical neutrophil morphology. Interestingly, we also found maternal GFP- Gr1+Mac1+ cells in embryonic blood at levels that could not be accounted for by maternal contamination during dissection. This suggests that the early embryonic circulation contains neutrophils of both embryonic and maternal origin. Increasing numbers of GFP+ Gr1+Mac1+ cells were also identified in the fetal liver at E12.5 through E14.5. Characteristic morphologic features of neutrophil maturation, including changes in cell size and nuclear morphology, were confirmed by imaging flow cytometry. The identification of maturing embryonic neutrophils at E11.5 suggests that their developmental origin precedes HSC emergence. We had previously identified granulocyte-macrophage progenitors beginning at E8.5 in the yolk sac of mouse embryos (Palis, 1999) and their subsequent spatial and temporal kinetics suggest that they are a component of the erythro-myeloid wave of hematopoietic progenitors (EMPs) that precedes the emergence of HSCs. Clonal analysis of individual EMP sorted from E9.5-10.5 mouse embryos confirm that they contain both definitive erythroid and granulocyte potential. In liquid cultures, EMPs are capable of differentiating into a broad array of myeloid lineages, including eosinophils and basophils as well as neutrophils and macrophages. Taken together, these data indicate both maternal- and embryonic EMP-derived neutrophils circulate in the embryo by E11.5, before HSC-derived hematopoiesis is established.
O1003 - INTERACTIONS BETWEEN THE CHROMATIN REMODELLER CHD1 AND THE SPLICEOSOME ARE CRITICAL FOR HEMATOPOIETIC STEM AND PROGENITOR CELL EMERGENCE Adriana De La Garza-Sauceda, Rosannah Cameron, Sara Payne, and Teresa Bowman Albert Einstein College of Medicine, Bronx, New York, USA Defects in hematopoietic stem cells (HSCs) lead to hematological diseases, such as myelodysplastic syndromes (MDS) and leukemia. Recurrent mutations in spliceosomal components were recently identified in these diseases, suggesting an underappreciated role for splicing regulation in HSC biology. Based on this hypothesis, we explored the importance of splicing on hematopoietic stem and progenitor cell (HSPC) specification during zebrafish development. Zebrafish mutants in U2 snRNP components, specifically sf3a3hi1950 and sf3b1hi3394, have diminished runx1 expression within their aorta by 28 hours post fertilization (hpf), while expression of the endothelial marker flk1/kdrl is mostly unaffected, indicating a defect at the level of HSPC formation. Using these mutants, we defined chd1 as a genetic interaction partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chromodomain Helicase DNA Binding Protein 1) is a chromatin remodeler that interacts with SF3A and SF3B spliceosomal complexes in human cells and is needed for their recruitment to actively transcribed genes. Transient morpholino-mediated knockdown of chd1 in zebrafish embryos has no effect on HSPC development. Similarly, embryos heterozygous for sf3a3hi1950 have no noticeable hematopoietic phenotype, however, in combination, diminished chd1 levels in sf3a3hi1950 heterozygotes leads to a significant decline in HSPC levels. These results demonstrate a clear in vivo connection between splicing and epigenetic regulation within HSPCs. As these two processes are the most frequently mutated in MDS, our data might also have implications for understanding the origins of MDS at the HSC level.
O1004 - VITAMIN D REGULATES HEMATOPOIETIC STEM CELL MAINTENANCE BY TWO DISTINCT MECHANISMS Mauricio Cortes3,4, Michael Chen2,4, Sarah Liu3, David Stachura1, Thorsten Schlaeger2,4, David Traver1, George Daley2,4, Wolfram Goessling5,4, and Trista North3,4 1 UCSD, La Jolla, California, USA; 2Boston Children’s Hospital, Boston, Massachusetts, USA; 3Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA; 4Harvard Medical School, Boston, Massachusetts, USA; 5 Brigham and Women’s Hospital, Boston, Massachusetts, USA Vitamin D was identified via a chemical screen in zebrafish as a modulator of expression of the hematopoietic stem cell (HSC) markers runx1/cmyb in the aorta-gonad mesonephros, the site of definitive hematopoiesis. Treatment with the vitamin D precursor choleclaciferol (D3) during HSC niche formation resulted in decreased runx1/cmyb expression; however, D3 treatment during HSC induction and expansion, had no effect, suggesting D3 affects vascular niche formation. In contrast, treatment with active vitamin D (1,25OH-D3) during either time window resulted in increased runx1/cmyb expression. FACS analysis of hematopoietic stem cell progenitors cells (HSPCs) quantified and confirmed our findings. Hedgehog (Hh) signaling is essential for artery/vein specification via regulation of notch signaling. Treatment with D3 resulted in decreased Hh signaling as determined by FACS using an Hh reporter line. Knockdown of CYP2R1, the enzyme required for D3 25-hydroxylation, also resulted in decreased Hh signaling and reduced HSPCs. Consistent with defects in Hh signaling, D3 treated embryos had diminished notch activity and abnormal artery/vein specification. In contrast, FACS analysis of HSCs exposed to 1,25OH-D3 showed a significant increase in cell number, with a 2-fold increase in cell proliferation. Knockdown of the vitamin D receptor (VDRA) decreased runx1 expression marked by a reduction in HSC number, indicating that these effects were receptor mediated. Colony forming assays (CFUs) of FACS-sorted adult HSCs treated with 1,25OH-D3 showed a significant increase in total CFUs indicating direct action of 1,25OH-D3 on HSCs. Similarly, CFU-C assays of CD34+ human umbilical cord blood cells (hUBCs) exposed to 1,25OH-D3 elicited a 2-fold increase in total CFUs. In sum, our studies have characterized two mechanisms by which vitamin D can modulate hematopoiesis. In addition, we have shown conservation in hUBCs, suggesting that active vitamin D may have clinical utility for the expansion of human HSCs for transplantation.
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Oral Short Talk Presentations O1001 - BALANCING HEMATOPOIETIC CELL FATE AND ENDOTHELIAL IDENTITY IN HEMOGENIC ENDOTHELIUM Carlos Lizam1, John Hawkins1, Frank Bos1, Joan Zape1, Joshua Wythe2, Mary Donohoe3, and Ann Zovein1,4 1 Cardiovascular Research Institute, University of California, San Francisco, California, USA; 2Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California, USA; 3Burke Medical Research Institute Weill Cornell Medical College, White Plains, New York, USA; 4Department of Pediatrics, Division of Neonatology, University of California San Francisco School of Medicine, San Francisco, California, USA Hematopoietic stem and progenitor cells (HP/SCs) first emerge from the embryonic endothelium during development to establish what will eventually become the adult hematopoietic system, in a process termed the endothelial to hematopoietic transition (EHT). Factors proven critical for EHT include Runx1, Sox17, and Notch1. However, early endothelial loss of either notch1 or sox17 also disrupts vascular specification, while runx1 impacts hematopoiesis without an overt vascular consequence. Here we present data that Notch1 and Sox17 are actually dispensable for hematopoietic emergence from the endothelium during EHT, and that endothelial loss after vascular specification results in augmented hematopoietic emergence. Using in vivo and in vitro timed genetic deletion and rescue, we demonstrate that endothelial and hematopoietic fates are determined by Sox17 which acts as a transcriptional gatekeeper of downstream endothelial (Notch1) and hematopoietic (Runx1) genes. Sox17 prevents EHT in mature endothelium by directly promoting endothelial Notch1 signaling, while directly repressing Runx1 expression. Thus, the timing of Sox17 or Notch1 endothelial loss has a compelling impact on hematopoietic outcome, as early deletion prevents EHT while later deletion promotes it.
O1002 - EMERGENCE OF THE NEUTROPHIL LINEAGE IN THE MAMMALIAN EMBRYO Kathleen E. McGrath, Katherine Fegan, Seana Catherman, and James Palis University of Rochester, Rochester, New York, USA Hematopoietic stem cells (HSCs) are responsible for the maintenance of circulating neutrophils in the adult. However, the onset and developmental origin of neutrophils in the embryonic bloodstream is poorly understood. In order to examine early neutrophil emergence in murine embryos, we mated GFP+ male mice with wild-type females, which allowed us to distinguish embryonic from maternal cells. By flow cytometry, we identified rare GFP+ Gr1+Mac1+ cells circulating in embryonic day 11.5 (E11.5) that increased in numbers from E12.5 to E14.5. These sorted GFP+ cells had typical neutrophil morphology. Interestingly, we also found maternal GFP- Gr1+Mac1+ cells in embryonic blood at levels that could not be accounted for by maternal contamination during dissection. This suggests that the early embryonic circulation contains neutrophils of both embryonic and maternal origin. Increasing numbers of GFP+ Gr1+Mac1+ cells were also identified in the fetal liver at E12.5 through E14.5. Characteristic morphologic features of neutrophil maturation, including changes in cell size and nuclear morphology, were confirmed by imaging flow cytometry. The identification of maturing embryonic neutrophils at E11.5 suggests that their developmental origin precedes HSC emergence. We had previously identified granulocyte-macrophage progenitors beginning at E8.5 in the yolk sac of mouse embryos (Palis, 1999) and their subsequent spatial and temporal kinetics suggest that they are a component of the erythro-myeloid wave of hematopoietic progenitors (EMPs) that precedes the emergence of HSCs. Clonal analysis of individual EMP sorted from E9.5-10.5 mouse embryos confirm that they contain both definitive erythroid and granulocyte potential. In liquid cultures, EMPs are capable of differentiating into a broad array of myeloid lineages, including eosinophils and basophils as well as neutrophils and macrophages. Taken together, these data indicate both maternal- and embryonic EMP-derived neutrophils circulate in the embryo by E11.5, before HSC-derived hematopoiesis is established.
O1003 - INTERACTIONS BETWEEN THE CHROMATIN REMODELLER CHD1 AND THE SPLICEOSOME ARE CRITICAL FOR HEMATOPOIETIC STEM AND PROGENITOR CELL EMERGENCE Adriana De La Garza-Sauceda, Rosannah Cameron, Sara Payne, and Teresa Bowman Albert Einstein College of Medicine, Bronx, New York, USA Defects in hematopoietic stem cells (HSCs) lead to hematological diseases, such as myelodysplastic syndromes (MDS) and leukemia. Recurrent mutations in spliceosomal components were recently identified in these diseases, suggesting an underappreciated role for splicing regulation in HSC biology. Based on this hypothesis, we explored the importance of splicing on hematopoietic stem and progenitor cell (HSPC) specification during zebrafish development. Zebrafish mutants in U2 snRNP components, specifically sf3a3hi1950 and sf3b1hi3394, have diminished runx1 expression within their aorta by 28 hours post fertilization (hpf), while expression of the endothelial marker flk1/kdrl is mostly unaffected, indicating a defect at the level of HSPC formation. Using these mutants, we defined chd1 as a genetic interaction partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chromodomain Helicase DNA Binding Protein 1) is a chromatin remodeler that interacts with SF3A and SF3B spliceosomal complexes in human cells and is needed for their recruitment to actively transcribed genes. Transient morpholino-mediated knockdown of chd1 in zebrafish embryos has no effect on HSPC development. Similarly, embryos heterozygous for sf3a3hi1950 have no noticeable hematopoietic phenotype, however, in combination, diminished chd1 levels in sf3a3hi1950 heterozygotes leads to a significant decline in HSPC levels. These results demonstrate a clear in vivo connection between splicing and epigenetic regulation within HSPCs. As these two processes are the most frequently mutated in MDS, our data might also have implications for understanding the origins of MDS at the HSC level.
O1004 - VITAMIN D REGULATES HEMATOPOIETIC STEM CELL MAINTENANCE BY TWO DISTINCT MECHANISMS Mauricio Cortes3,4, Michael Chen2,4, Sarah Liu3, David Stachura1, Thorsten Schlaeger2,4, David Traver1, George Daley2,4, Wolfram Goessling5,4, and Trista North3,4 1 UCSD, La Jolla, California, USA; 2Boston Children’s Hospital, Boston, Massachusetts, USA; 3Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA; 4Harvard Medical School, Boston, Massachusetts, USA; 5 Brigham and Women’s Hospital, Boston, Massachusetts, USA Vitamin D was identified via a chemical screen in zebrafish as a modulator of expression of the hematopoietic stem cell (HSC) markers runx1/cmyb in the aorta-gonad mesonephros, the site of definitive hematopoiesis. Treatment with the vitamin D precursor choleclaciferol (D3) during HSC niche formation resulted in decreased runx1/cmyb expression; however, D3 treatment during HSC induction and expansion, had no effect, suggesting D3 affects vascular niche formation. In contrast, treatment with active vitamin D (1,25OH-D3) during either time window resulted in increased runx1/cmyb expression. FACS analysis of hematopoietic stem cell progenitors cells (HSPCs) quantified and confirmed our findings. Hedgehog (Hh) signaling is essential for artery/vein specification via regulation of notch signaling. Treatment with D3 resulted in decreased Hh signaling as determined by FACS using an Hh reporter line. Knockdown of CYP2R1, the enzyme required for D3 25-hydroxylation, also resulted in decreased Hh signaling and reduced HSPCs. Consistent with defects in Hh signaling, D3 treated embryos had diminished notch activity and abnormal artery/vein specification. In contrast, FACS analysis of HSCs exposed to 1,25OH-D3 showed a significant increase in cell number, with a 2-fold increase in cell proliferation. Knockdown of the vitamin D receptor (VDRA) decreased runx1 expression marked by a reduction in HSC number, indicating that these effects were receptor mediated. Colony forming assays (CFUs) of FACS-sorted adult HSCs treated with 1,25OH-D3 showed a significant increase in total CFUs indicating direct action of 1,25OH-D3 on HSCs. Similarly, CFU-C assays of CD34+ human umbilical cord blood cells (hUBCs) exposed to 1,25OH-D3 elicited a 2-fold increase in total CFUs. In sum, our studies have characterized two mechanisms by which vitamin D can modulate hematopoiesis. In addition, we have shown conservation in hUBCs, suggesting that active vitamin D may have clinical utility for the expansion of human HSCs for transplantation.