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.

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.

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.

FGF-receptor signalling controls neural cell diversity in the zebrafish hindbrain by regulating olig2 and sox9.

The mechanisms underlying the generation of neural cell diversity are the subject of intense investigation, which has highlighted the involvement of different signalling molecules including Shh, BMP and Wnt. By contrast, relatively little is known about FGF in this process. In this report we identify an FGF-receptor-dependent pathway in zebrafish hindbrain neural progenitors that give rise to somatic motoneurons, oligodendrocyte progenitors and differentiating astroglia. Using a combination of chemical and genetic approaches to conditionally inactivate FGF-receptor signalling, we investigate the role of this pathway. We show that FGF-receptor signalling is not essential for the survival or maintenance of hindbrain neural progenitors but controls their fate by coordinately regulating key transcription factors. First, by cooperating with Shh, FGF-receptor signalling controls the expression of olig2, a patterning gene essential for the specification of somatic motoneurons and oligodendrocytes. Second, FGF-receptor signalling controls the development of both oligodendrocyte progenitors and astroglia through the regulation of sox9, a gliogenic transcription factor the function of which we show to be conserved in the zebrafish hindbrain. Overall, for the first time in vivo, our results reveal a mechanism of FGF in the control of neural cell diversity.

Metabolic Regulation of Inflammasome Activity Controls Embryonic Hematopoietic Stem and Progenitor Cell Production.

Embryonic hematopoietic stem and progenitor cells (HSPCs) robustly proliferate while maintaining multilineage potential in vivo; however, an incomplete understanding of spatiotemporal cues governing their generation has impeded robust production from human induced pluripotent stem cells (iPSCs) in vitro. Using the zebrafish model, we demonstrate that NLRP3 inflammasome-mediated interleukin-1-beta (IL1ß) signaling drives HSPC production in response to metabolic activity. Genetic induction of active IL1ß or pharmacologic inflammasome stimulation increased HSPC number as assessed by in situ hybridization for runx1/cmyb and flow cytometry. Loss of inflammasome components, including il1b, reduced CD41+ HSPCs and prevented their expansion in response to metabolic cues. Cell ablation studies indicated that macrophages were essential for initial inflammasome stimulation of Il1rl1+ HSPCs. Significantly, in human iPSC-derived hemogenic precursors, transient inflammasome stimulation increased multilineage hematopoietic colony-forming units and T cell progenitors. This work establishes the inflammasome as a conserved metabolic sensor that expands HSPC production in vivo and in vitro.

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

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 CBFß. 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.

HIF1α-induced PDGFRß signaling promotes developmental HSC production via IL-6 activation.

Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate each of the mature blood cell lineages and thereby reconstitute the entire blood system. Therefore, HSCs are therapeutically valuable for treatment of hematological malignances and bone marrow failure. We showed recently that transient glucose elevation elicited dose-dependent effects on HSCs through elevated metabolic activity and subsequent reactive oxygen species-mediated induction of Hypoxia-Inducible Factor 1α (Hif1α). Platelet-Derived Growth Factor B (pdgfb), a Hif1α-target, and its receptor, pdgfrb, were significantly upregulated in response to metabolic stimulation. Although the function of PDGF signaling is well established in vascular development, its role in hematopoiesis is less understood. Exposure to either a pan-PDGF inhibitor or a PDGFRß-selective antagonist in the context of Hif1α stimulation blocked elevations in hematopoietic stem and progenitor cell (HSPC) formation as determined by runx1;cmyb whole-mount in situ hybridization (WISH) and HSPC-reporter flow cytometry analysis. Similar results were observed for morpholino (MO) knockdown of pdgfrb or dominant-negative pdgfrb expression, indicating that PDGFRß signaling is a key downstream mediator of Hif1α-mediated induction of HSPCs. Notably, overexpression of Pdgfb ligand enhanced HSPC numbers in the aorta-gonado-mesonephros (AGM) at 36 hours postfertilization (hpf) and in the caudal hematopoietic tissue at 48 hpf. A survey of known PDGF-B/PDGFRß regulatory targets by expression analysis revealed a significant increase in inflammatory intermediates, including Interleukin 6 (IL-6) and its receptor (IL-6R). MO-mediated knockdown of il6 or chemical inhibition of IL-6R antagonized the effect of Pdgfb overexpression. Furthermore, epistatic analysis of IL-6/IL-6R function confirmed activity downstream of Hif1α. Together, these findings define a Hif1α-regulated signaling axis mediated through PBFGB/PDGFRß and IL-6/IL-6R that acts to control embryonic HSPC production.

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

The Central Nervous System Regulates Embryonic HSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling.

Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adapt to fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GR loss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers.

Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production.

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.

Prostaglandin E2 regulates liver versus pancreas cell-fate decisions and endodermal outgrowth.

The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver versus pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate, whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell-fate decisions and outgrowth of the embryonic endodermal anlagen.

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 17ß-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.