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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.

While the genetic control of hematopoietic stem cell (HSC) function is increasingly understood, less is known about factors that pattern the embryonic hematopoietic niche and specify the location of HSC emergence. 17beta-estradiol (E2) and related estrogenic compounds were identified in a zebrafish chemical genetic screen as modifiers of the number of runx1+ HSCs in the Aorta-Gonad-Mesonephros region. Exposure to exogenous E2 during hematopoietic specification significantly decreased production of runx1+ HSCs by in situ hybridization (ISH) and qPCR (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1268 The intrinsic signaling pathways regulating hematopoietic stem cells (HSC) are increasingly understood; in contrast, less is known about the potential effect of exposure to environmental factors, such as xenoestrogens, on the formation of HSCs. RUNX1 (AML1) is a highly conserved transcription factor that is required for definitive HSC induction and is also the target of many chromosomal alterations in leukemia. Through a chemical genetic screen, estrogen-related compounds were identified as modifiers of runx1 expression in the zebrafish. Exposure to 17b-estradiol (E2) throughout hematopoietic development (5 somites (som) to 36 hours post fertilization (hpf)) significantly decreased the number of runx1+ HSCs in the zebrafish Aorta-Gonad-Mesonephros Region (AGM) compared to controls (n≥25–50 embryos /condition). Use of the nonspecific estrogen receptor inhibitor fulvestrant confirmed that estrogen was required for HSC regulation and functioned through classical estrogen receptors. Microarray analysis of FACS-sorted cell populations during zebrafish development demonstrated differential spatio-temporal regulation and expression of esr1 (esrα) and esr2a/b (esrβ) in vascular and hematopoietic cell types; use of an ERE-GFP reporter fish verified that estrogen signaling is active during this stage of embryonic development. During the primitive wave of hematopoiesis, exposure to E2 and the esr1-agonist PPT significantly enhanced red blood cell number as seen by in situ hybridization for embryonic globin (hbbe3) and quantified by fluorescent microscopy and FACS analysis of the Tg(globin:GFP) line. Conversely, the esr2-specific agonist DPN diminished definitive HSC formation after exposure from 5 som to 24 hpf; this phenotype was mediated by disruption of vessel formation, as indicated by flk1 (kdrl) expression, and alteration in the assignment of artery-vein identity. Alterations in arterial specification appear to be mediated by the Notch/VEGF pathway. E2 exposure from 5 somites to 36 hpf decreased GFP expression in notch reporter fish as well as expression of deltaC and notch5 by in situ hybridization. Interestingly, when exposure to E2 or DPN occurred from 24 – 36 hpf, after arterial establishment and initiation of blood flow, estrogen treatment enhanced HSC formation; this was confirmed by FACS analysis and fluorescent microscopy using the Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP) HSC reporter lines. E2 treatment was found to elicit both pro-apoptotic (TUNEL+ and acridine orange) and pro-proliferative (BrdU+) effects on HSCs and the vascular niche depending on the timing of exposure. Morpholino-mediated gene knockdown of esr1 and the two esr2 alleles alone and in combination with E2 confirmed that esr2 was responsible for the effects on definitive hematopoiesis. Using the Tg(TOP:GFP)w25 line, modifications in Wnt activity were seen post-E2 exposure from 24 – 36 hpf. To determine whether environmental estrogens could mediate similar alterations in HSC specification and proliferation, we exposed embryos to the phytoestrogen genistein, the synthetic estrogen ethinylestradiol, and the xenoestrogen bisphenol A (BPA) and found that all decreased formation of HSCs; using fulvestrant and the ERE-GFP reporter, we confirmed that the phenotype elicited by each was at least partially dependent on estrogen receptor stimulation. In an adult zebrafish marrow injury model, E2 significantly enhanced stem and progenitor cell regeneration in males and females by day 10 post irradiation (n≥10 /condition). Intriguingly, we found that females, with higher circulating estrogen levels, recovered better after injury than male siblings, both in the presence and absence of exogenous estrogen. Finally, murine bone marrow treated with E2 or DPN produced significantly (n=10/condition, p Disclosures: Goessling: Fate Therapeutics: Consultancy. North:Fate Therapeutics: Consultancy.

Abstract 1266 During development, the embryo is exposed to rapidly changing metabolic conditions and demands. The impact of elevated glucose levels on the developing hematopoietic system is not well characterized. Intriguingly, children born to mothers with gestational diabetes as well as those diagnosed with diabetes themselves have a higher risk of developing childhood leukemia, suggesting increased blood sugar concentrations may have lasting impact on hematopoiesis. To evaluate the consequences of elevated glucose levels on hematopoietic stem cell (HSC) development, zebrafish embryos were exposed to glucose and modifiers of glucose metabolism during embryonic blood cell specification from 12 to 36 hours post fertilization (hpf). HSCs in the Aorta-Gonad-Mesonephros (AGM) region were analyzed by in situ hybridization for the conserved markers runx1 and cmyb (n≥25–50 embryos/condition). D-glucose expanded HSCs in a dose-responsive manner, while the metabolically inactive enantiomer L-glucose did not affect HSCs. Quantitation by qPCR and FACS analysis of fluorescent HSC reporter embryos runx1:eGFP, cmyb:eGFP and CD41:GFP revealed a ∼3-fold increase in HSCs. Enhanced cellular proliferation (BrdU) was detected in the AGM in response to increased glucose levels. The effects of glucose on HSCs were sustained without any block in differentiation potential in larvae, and in adults after marrow injury. AGM time course analysis and ultrastructural assessment by electron microscopy (EM) revealed accelerated runx1 expression and hematopoietic cluster formation. EM and in situ hybridization for scl, gata1, and globin also demonstrated enhanced red cell numbers in response to elevated glucose levels. Genetic and chemical modulations of the metabolic hormone insulin did not alter the effects of glucose on HSC number. Activity of the glucose transporter glut1 was required to observe enhanced HSC induction, and glucose exposure increased glucose, ATP and lactate concentrations in the embryo. Additionally, incubation with inhibitors of glycolysis (lonidamine, ethyl-3-bromopyruvate) and oxidative phosphorylation (cyanide, oxaloacetate) reversed the beneficial effects of glucose, demonstrating that glucose affects HSCs specifically through energy metabolism. Enhanced oxidative phosphorylation produces excess reactive oxygen species (ROS), which can directly serve as hematopoietic signaling factors; treatment with the antioxidants N-acetylcysteine and MitoQ decreased HSC formation and blocked the effect of concomitant glucose exposure, while H2O2 expanded HSCs. Similarly, genetic enhancement of ROS levels by knockdown of peroxiredoxin 1 increased HSCs. ROS were visualized in vivo in erythrocytes and CD41+ cells using the fluorescent sensor peroxyfluorescein 2; increased glucose levels significantly enhanced ROS in the AGM as determined by FACS analysis of the lmo2:dsRed hematopoietic precursor and vascular reporter. ROS directly affects the stability of the cellular hypoxia sensor, hypoxia inducible factor 1α (hif1α), which can regulate expression of hematopoietic genes, such as vegf and epo. Stabilization of hif1α by cobalt chloride or morpholino knockdown of vhl enhanced HSCs and rescued the block in HSC induction following inhibition of energy metabolism or ROS production. Furthermore, by microarray and qPCR gene expression analysis, glucose exposure greatly impacted hematopoietic related transcripts, in addition to eliciting significant changes in the hif1α gene network: glucose increased expression of epo and epor during the primitive wave of hematopoiesis, confirming the effects on erythrocytes seen in vivo. In addition to vegf, nos and igf were induced during the definitive wave of hematopoiesis in a dose- and time-dependent fashion, providing signals to support HSC induction and expansion. These data indicate that fluctuations in glucose levels, and subsequent metabolic activity initially occurring in the dorsal aorta, are sensed by hif1α, which then functions to genetically enhance hematopoietic supply in advance of the anticipated demands of the growing organism during development. Our work provides direct evidence that the developing embryo responds dynamically to metabolic challenges by accelerating and expanding blood formation and demonstrates a connection between glucose metabolism and hif1α signaling in regulating HSC induction. Disclosures: Vander Heiden: Agios Pharmaceuticals: Consultancy, Equity Ownership. Goessling:Fate Therapeutics: Consultancy. North:Fate Therapeutics: Consultancy.

Abstract 1617 The intrinsic signaling pathways regulating hematopoietic stem cells (HSC) are increasingly well recognized. However, less is known about how in utero exposure to common environmental xenobiotic compounds may alter HSC development and increase the risk of carcinogenesis. RUNX1 (AML1), required for definitive HSC induction in all vertebrates, is the target of frequent chromosomal alterations associated with leukemia. Through a chemical genetic screen for modifiers of runx1 expression in the zebrafish, estrogen-related compounds were identified. Here, we found that exposure to 17β-estradiol (E2) throughout the initial waves of hematopoietic development (5 somites (som) to 36 hours post fertilization (hpf)) significantly altered the number of runx1+ HSCs in the zebrafish Aorta-Gonad-Mesonephros Region (AGM) compared to controls (n≥25-50 embryos /condition). Other physiological estrogens, such as estrone and estriol, elicited a similar hematopoietic response. However, treatment with either the isomer 17α-estradiol, or the related steroid hormones testosterone or progesterone, could not mimic the effect of E2 on HSCs. Use of the aromatase inhibitor anastrozole and the pan-estrogen receptor inhibitor fulvestrant confirmed that estrogen was both required for nascent HSC regulation and functioned through classical estrogen receptor (esr) signaling. Microarray analysis of FACS-sorted cell populations during zebrafish development demonstrated differential spatio-temporal regulation of esr1 (esrα) and esr2a/b (esrβ) in vascular and hematopoietic cell types. During the primitive wave of hematopoiesis, exposure to E2 and the esr1-agonist PPT significantly enhanced red blood cell number as seen by in situ hybridization for embryonic globin (hbbe3) and quantified by fluorescent microscopy and FACS analysis of the Tg(globin:GFP) line. Conversely, the esr2-specific agonist DPN diminished definitive HSC formation after exposure from 5 som to 24 hpf; this phenotype was mediated by disruption of vessel formation, as indicated by flk1 (kdrl) expression, and alteration in the assignment of artery-vein identity. Interestingly, when exposure to E2 or DPN occurred from 24 – 36 hpf, after the establishment of ephb2+ arteries and the initiation of blood flow, estrogen treatment enhanced HSC formation; this was confirmed by FACS analysis and fluorescent microscopy using the Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP) HSC-reporter lines. E2 treatment was found to elicit both pro-apoptotic (TUNEL+) and pro-proliferative (BrdU+) effects on HSCs and the vascular niche depending on the timing of exposure, but independent of the concentration of E2 over the physiological range and above (10nM to 10mM). Morpholino-mediated gene knockdown of esr1 and the two esr2 alleles alone and in combination with E2 confirmed that esr2 was responsible for the effects on definitive hematopoiesis. Using the Tg(TOP:GFP)w25 line, alterations in estrogen signaling were shown to mediate effects on wnt activity. To determine whether exposure to environmental estrogens could mediate similar alterations in HSC specification and proliferation, we exposed embryos to the phytoestrogen genistein, the synthetic estrogen ethinylestradiol, and the xenoestrogen bisphenol A (BPA) and found results reminiscent of E2; using fulvestrant, we confirmed that the phenotype elicited by each was dependent on estrogen receptor stimulation. In an adult zebrafish marrow injury model, E2 significantly enhanced stem and progenitor cell regeneration in males and females by day 10 post irradiation (n≥10 /condition). Intriguingly, we found that females, with higher circulating estrogen levels, recovered better after injury than male siblings, both in the presence and absence of exogenous estrogen. Finally, murine bone marrow treated with E2 or DPN produced significantly (n=10 /condition, p Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. North:Fate Therapeutics: Consultancy, Patents & Royalties.

Abstract 408 Metabolic disorders, including obesity, diabetes, and their related complications have become the leading cause of preventable death in the U.S. While effects of increased blood sugar on the cardiovascular system and other organs are well established, consequences for the hematopoietic system are less well characterized. Specifically, the effects of gestational diabetes on embryonic blood formation have not been elucidated in detail. To determine the impact of elevated glucose concentrations on hematopoietic stem cell (HSC) induction and expansion, we treated zebrafish embryos with D-glucose from 12 to 36 hours post fertilization (hpf). HSCs in the Aorta-Gonad-Mesonephros (AGM) region were expanded in a dose-responsive manner, as assessed by in situ hybridization for the conserved HSC markers, runx1 and cmyb; this effect was verified by analysis of CD41 expression (n≥25–50 embryos/condition). A significant 3-fold enhancement in HSC number was observed after 1% glucose treatment by qPCR for runx1 and FACS analysis of fluorescent HSC reporter embryos, Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP). Other mono-, di-, and tri-saccharides exerted similar effects, expanding the number of HSCs. However, L-glucose, the inactive enantiomer, did not affect runx1 expression, implying that the effect of the metabolizable saccharides is independent of osmotic dynamics. Furthermore, treatment with glucose from 12 to 120 hpf resulted in a sustained increase in HSC number throughout the duration of treatment. Increased cellular proliferation was observed in the AGM region following 1% glucose treatment, as assessed by elevated BrdU incorporation and cyclin D1 expression. Additionally, acridine orange staining revealed a decrease in apoptotic cell number. HSC formation was impaired by morpholino knockdown of the glucose transporter glut1, indicating the requirement of glucose uptake for HSC formation. Exposure to chemical inhibitors of glycolysis (ethyl-3-bromopyruvate) and of oxidative phosphorylation (cyanide and oxaloacetate) reversed the beneficial effects of glucose on HSCs. In contrast, pharmacological and genetic modulations of the metabolic endocrine hormones IGF and insulin did not alter the effects of glucose treatment. Treatment with the glycolytic intermediate pyruvate expanded HSCs, while exposure to glucosamine, the first component of the hexosamine biosynthetic pathway, had no effect on blood stem cells. These results suggest that glucose catabolism specifically is responsible for HSC expansion. A potential consequence of heightened glucose metabolism is increased generation of reactive oxygen species (ROS), which may serve as important signaling factors mediating the observed effects on HSCs. In support of this hypothesis, treatment with the antioxidant N-acetylcysteine, which alone decreased HSC formation, could not be rescued by concomitant glucose exposure, while the oxidant, 1-phenyl-2-thiourea, elicited an expansion in HSC number. Using the fluorescent ROS sensor, dihydroethidium, we observed ROS in circulating erythrocytes, a subpopulation of CD41+ cells and in the adjacent somitic muscle. ROS are known inducers of the hypoxia sensor, hif1α, which regulates important hematopoietic genes including vegf and epo, as well as the glut1 glucose transporter, indicating a potential feedback loop controlling HSC induction. Chemical induction of hypoxia by cobalt chloride similarly enhanced HSC formation. Intriguingly, excess glucose can overcome the reduction of HSC number normally observed in silent heart mutants lacking blood circulation, possibly by ROS/hif1α-mediated induction of vegf and NO in the hematopoietic niche. The beneficial effect of glucose on hematopoiesis was conserved in adult zebrafish: FACS analysis of kidney marrow following sublethal irradiation revealed a more rapid recovery of the progenitor population after glucose exposure. Our data suggest that, in conjunction with the cardiovascular system, energy metabolism plays a key role in regulating hematopoetic induction and homeostasis. These results could lead to novel therapeutic approaches for HSC modulation, and may unveil specific risks of obesity and diabetes for hematopoiesis during gestation and in the adult. (equal contribution: WG, TEN). Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. North:Fate Therapeutics: Consultancy, Patents & Royalties.

Abstract 1496 Poster Board I-519 Obesity and subsequent diabetes have emerged as major health problems in the U.S. While the consequences of elevated blood glucose levels on the cardiovascular system and other organs are well known, the direct effects on the hematopoietic system are more elusive. Similarly, the impact of gestational diabetes on embryonic hematopoiesis has not been examined in detail. The zebrafish has emerged as an important model system to study conserved regulators of organ development and homeostasis. In order to evaluate the role of elevated glucose levels on hematopoietic stem cell (HSC) production, zebrafish embryos were exposed to increasing doses of D-glucose from 5 somites to 36 hours post fertilization (hpf); HSC number, as indicated by in situ hybridization for the conserved markers runx1 and cmyb in the Aorta-Gonad-Mesonephros (AGM) region, was increased at 0.5, 1% and 2% glucose; results were confirmed by in analysis of CD41 expression. Quantification using FACS analysis of fluorescent HSC reporter embryos and qPCR revealed a 2-3-fold enhancement following 1% glucose treatment. Other mono, di-, and trisaccharide sugars had similar effects, causing increased numbers of HSCs, however, L-glucose had no impact. BrdU incorporation in the AGM region was elevated after 1% glucose treatment, while acridine orange staining revealed an inhibitory effect on apoptosis. To evaluate potential mediators of these glucose-responsive effects, embryos were injected with antisense morpholino oligonucleotides (MO) against both the insulin (insr), and insulin-like growth factor receptors (igfr); insr and igfr receptors can each bind insulin, released following elevations in blood sugar levels. MO knockdown of insra or igfrb, but not igfra, influenceded runx1+ HSCs substantially, indicating an important role of these endocrine regulatory signaling pathways in HSC formation. However, D-glucose completely reversed these effects, implying either functional redundancy, or a multi-step, multi-effector process of HSC regulation by endocrine factors. To further clarify when insr- and/or igfr-mediated activity was influencing HSC formation and to correlate that effect with elevated glucose exposure, embryos were treated for defined periods with either 1% glucose, insulin, or IGF; exposure from 10 somites to 24 hpf influences the formation and arterial/venous specification of dorsal aorta, the conserved site of initial definitive HSC production, while exposure from 24 to 36 hpf regulates HSC induction. IGF exerted a positive effect on HSCs only after the establishment of the hematopoietic niche (>24hpf). Glucose treatment, however, positively influenced HSC formation at all time points examined, suggesting it works not only in the HSC niche to induce HSCs, but also prior to HSC formation. MO knockdown of the glucose transporter glut1 resulted in diminished HSC production, confirming a direct role of glucose in this process. To determine whether the effect of glucose elevation was mediated by changes in cellular energy production, embryos were exposed to chemical inhibitors of oxidative phosphorylation. Cyanide and oxaloacetate reversed the beneficial effects of D-glucose, indicating that energy production can modulate HSC formation. Investigation into the functional redundancy and cross-regulation of insulin and IGF on HSC self-renewal and the evolutionary conservation of the effects of energy metabolism on HSC production are ongoing; further studies will be needed to determine if glucose maintains an influential role on HSC homeostasis or bone marrow recovery following injury. These results could have an impact on methods for HSC modulation for therapeutic purposes, and may further unveil specific risks of obesity and diabetes for hematopoiesis and HSC homeostasis during gestation and in the adult. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. North: Fate Therapeutics: Consultancy, Patents & Royalties.