S13Oral Short Talk Presentations/ Experimental Hematology 42 (2014) S13–S21

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

1Cardiovascular 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 endo-

thelium during development to establish what will eventually become the adult hemato-

poietic system, in a process termed the endothelial to hematopoietic transition (EHT).

Factors proven critical for EHT includeRunx1, Sox17, andNotch1. However, early endo-

thelial loss of either notch1 or sox17 also disrupts vascular specification, while runx1 im-

pacts hematopoiesis without an overt vascular consequence. Here we present data that

Notch1 and Sox17 are actually dispensable for hematopoietic emergence from the endo-

thelium 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)

andhematopoietic (Runx1) genes.Sox17preventsEHTinmature endotheliumbydirectly

promoting endothelial Notch1 signaling, while directly repressing Runx1 expression.

Thus, the timing of Sox17 orNotch1 endothelial loss has a compelling impact on hemato-

poietic outcome, as early deletion prevents EHTwhile 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 neu-

trophils in the adult. However, the onset and developmental originof neutrophils in the em-

bryonic bloodstream is poorly understood. In order to examine early neutrophil emergence

in murine embryos, we mated GFP+male micewith wild-type females, which allowed us

to distinguish embryonic frommaternal cells. By flow cytometry, we identified rare GFP+

Gr1+Mac1+ cells circulating in embryonic day 11.5 (E11.5) that increased in numbers

fromE12.5 toE14.5. These sortedGFP+ cells had typical neutrophilmorphology. Interest-

ingly, 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 ofGFP+Gr1+Mac1+cellswere 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 gran-

ulocyte-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 pre-

cedes 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 poten-

tial. 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 neutro-

phils 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 spliceoso-

mal components were recently identified in these diseases, suggesting an underappre-

ciated 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 interac-

tion partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chro-

modomain 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 knock-

down 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 impli-

cations 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

1UCSD, 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;5Brigham and Women’s Hospital, Boston, Massachusetts, USA

Vitamin D was identified via a chemical screen in zebrafish as a modulator of expres-

sion of the hematopoietic stem cell (HSC) markers runx1/cmyb in the aorta-gonad

mesonephros, the site of definitive hematopoiesis. Treatment with the vitamin D pre-

cursor 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 re-

sulted 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 decreasedHh signaling and reduced HSPCs. Consistent with defects in Hh signaling,

D3 treated embryos had diminished notch activity and abnormal artery/vein specifica-

tion. 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 expressionmarked 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 in-

crease in total CFUs indicating direct action of 1,25OH-D3 onHSCs. 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 Dmay have clinical util-

ity for the expansion of human HSCs for transplantation.