Virginie Esain

Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.

Glucose metabolism impacts the spatiotemporal onset and magnitude of HSC induction in vivo

Many pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrial-derived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability.

Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production

SUMMARY Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8 (IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.

Estrogen Defines the Dorsal-Ventral Limit of VEGF Regulation to Specify the Location of the Hemogenic Endothelial Niche

Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17β-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.

Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial‐to‐hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch‐mediated EHT. In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT. Global or endothelial‐specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1–pAKT as a novel molecular pathway that, in conjunction with the shh–vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.

Cannabinoid Receptor-2 Regulates Embryonic Hematopoietic Stem Cell Development via Prostaglandin E2 and P-Selectin Activity

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB‐signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta‐gonad‐mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb+ HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2‐dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P‐selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2‐regulated P‐selectin activity. Together, these data suggest CNR2‐signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways. Stem Cells 2015;33:2596—2612

A tool compound targeting the core binding factor Runt domain to disrupt binding to CBFβ in leukemic cells

Abstract The core binding factor (CBF) gene RUNX1 is a target of chromosomal translocations in leukemia, including t(8;21) in acute myeloid leukemia (AML). Normal CBF function is essential for activity of AML1-ETO, product of the t(8;21), and for survival of several leukemias lacking RUNX1 mutations. Using virtual screening and optimization, we developed Runt domain inhibitors which bind to the Runt domain and disrupt its interaction with CBFβ. On-target activity was demonstrated by the Runt domain inhibitors’ ability to depress hematopoietic cell formation in zebrafish embryos, reduce growth and induce apoptosis of t(8;21) AML cell lines, and reduce progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. Runt domain inhibitors had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines. Our results confirmed that Runt domain inhibitors might prove efficacious in various AMLs and in T-ALL.

Abstract A33: Role for the tumor suppressor phf6 in hematopoiesis.

Plant Home domain Finger 6 (PHF6) is a tumor suppressor of unknown function for blood malignancies such as T-cell Acute Lymphoblastic Leukemia (T-ALL), Acute Myeloid leukemia (AML), and Chronic Myeloid Leukemia (CML). PHF6 contains two zinc finger-like PH domains and interacts with the Nucleosome Remodeling and Deacetylation (NuRD) complex, suggesting a role in chromatin remodeling. Although PHF6 loss-of-function mutations are found in nearly 40% of T-ALL patients, little is known about how PHF6 mutations contribute to blood development and leukemogenesis. To understand the function of PHF6, beginning with its role in hematopoiesis, we have undertaken developmental studies in zebrafish to discover how loss of phf6 affects blood development and to determine which pathways are regulated by phf6. Zebrafish will be used to study hematopoiesis due to the remarkable conservation of molecular pathways that regulate blood development, genetic tractability, and ability to observe embryonic development over a short window of time. RNA in situ hybridization studies of zebrafish embryos showed that phf6 is expressed broadly during zebrafish development, and especially in the dorsal aorta, a site analogous to the aorta-gonad-mesonephros (AGM) in mammals, from which hematopoietic stem cells (HSCs) arise. Further, phf6 is highly expressed in lymphocytes of adult zebrafish, reminiscent of the expression patterns found in human and mouse. To determine the effect of phf6 loss on hematopoiesis, phf6 expression was knocked down by morpholino injection. We find that phf6 morphants have increased numbers of HSCs by RNA in situ hybridization of runx1/cmyb in the AGM and caudal hematopoietic tissue ((CHT) analogous to mammalian fetal liver), sites of HSC emergence and migration. Later in development, phf6 morphants demonstrate increased lymphocytes by RNA in situ hybridization of rag1 at the thymus. Similar phenotypes were observed in homozygous phf6-null mutants generated by TALEN-mediated knockout. Phf6-null mutant zebrafish survive to Mendelian ratios and are fertile as adults. In total, we found a new role for phf6 in regulation of HSC formation. Citation Format: Finola E. Moore, Virginie Esain, Riadh Lobbardi, Jessica S. Blackburn, Trista E. North, David M. Langenau. Role for the tumor suppressor phf6 in hematopoiesis. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A33.