ALBERT

Description: Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

Description: Abstract 2430 Poster Board II-407 After transplantation, hematopoietic stem/progenitor cells (HSPCs) home to the marrow, where they engraft and self-renew. To explore the mechanism of this multi-step and dynamic repopulation process, we performed the first in vivo adult vertebrate chemical screen aimed at identifying novel chemical modulators of HSPC repopulation using a novel competitive marrow transplantation assay in zebrafish. To distinguish between the donors, we used ubiquitous GFP or DsRed2 transgenic fish, Tg(β-actin:GFP) and Red GloFish®, for marrow cell isolation. 20,000 GFP+ cells were treated with a chemical and mixed with 80,000 untreated DsRed2+ marrows. This pool of cells was injected retro-orbitally into a transparent adult zebrafish. After a recovery period, the fish was then anesthetized and the region of the kidney (the adult site of hematopoiesis) was examined by fluorescence microscopy. The competition between the two donors was determined by analyzing the ratios of GFP and DsRed2 fluorescence intensity with ImageJ software. Using this assay, we demonstrated that dmPGE2 and/or GSK-3β inhibitor treatment of GFP+ marrows for 3 hrs could dramatically increase repopulation in fish. A chemical library of 480 chemicals with known bioactivities was screened using this in vivo assay. GFP+ marrows were incubated with different chemicals for 3 hrs and ten recipient fish were transplanted for each chemical. By examining engraftment at 4 weeks, we found 10 chemicals that improved HSPC repopulation. Based on the known bioactivity, these chemicals were categorized into several signaling pathways, including prostaglandin metabolism and retinoic acid pathways. Several of the compounds also increased HSC formation in zebrafish embryos, indicating that some pathways might be shared by different developmental stages. To examine whether the bioactivities of these hits are conserved in mammals, CD45.1 mouse whole bone marrow cells were treated with hit compounds for 3 hrs and competitively transplanted into CD45.2 recipients. Peripheral blood was sampled at 3, 6, 12, and 20 week post transplant. Several hits were confirmed to increase long-term chimerism in mice. The retinoic acid pathway has been shown to play an important role in hematopoiesis. Among the six retinoic acid receptor (RAR) agonists in the chemical library, which includes all-trans retinoic acid (ATRA), only two structurally highly related compounds, AM-580 and TTNPB scored positive in the screen. These two compounds have distinctive chemical moieties from ATRA. This structural difference likely leads to stronger agonistic effects on RAR than ATRA and resists degradation. In conclusion, the in vivo chemical screening using zebrafish competitive marrow transplantation provides a successful example of phenotypic screening in whole adult vertebrates. The discovery of novel repopulation modulators should provide a better understanding of signaling events that regulate homing and self-renewal, and may have clinical application in marrow or cord blood transplantation. Disclosures: Zon: FATE Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Stemgent: Consultancy.

Description: Introduction In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive allogeneic transplant approach that has the potential to treat a number of congenital disorders, including hemoglobinopathies and immunodeficiencies. Donor cell engraftment at levels high enough to induce donor specific immune tolerance or to treat a target disease has been elusive and remains a major limitation to the clinical application of IUHCT. One of the most significant barriers to high levels of donor cell engraftment is competition with endogenous fetal hematopoietic stem cells (HSCs) for limited hematopoietic niches following transplant. 16,16-dimethyl-prostaglandin E2 (PGE2) and the epoxyeicosatrienoic acids (EETs) 11,12-EET and 14,15-EET have been shown to enhance donor HSC homing, survival, and cell cycling in postnatal murine and zebrafish models of HSC transplantation. We hypothesized that a single ex vivo treatment of donor cells with these eicosanoids would improve donor cell homing and survival following IUHCT,resulting in higher levels of postnatal donor engraftment. Methods Ten million bone marrow (BM) mononuclear cells from C57Bl/6-GFP mice (H2Kb) were injected intravenously into embryonic day (E14) Balb/c fetuses (H2Kd) via the vitelline vein. Donor cells were treated with vehicle, PGE2, 11,12-EET, or 14,15-EET immediately prior to IUHCT. Early homing to and engraftment of the fetal liver (FL) and spleen (FS) of PGE2 and vehicle treated BM cells at 4, 24 and 72 hours after IUHCT was assessed by flow cytometry. Donor cell survival and apoptosis was also assessed in the FL 96 hours post-IUHCT in these two treatment groups by flow cytometric analysis of intracellular expression of survivin (anti-apoptotic) and anti-caspase 3 (pro-apoptotic). Long-term peripheral blood donor cell engraftment was assessed monthly up to 6 months of age and multilineage engraftment (donor T cells, B cells, granulocytes, and macrophages) was determined at 6 months of age in recipients of all donor cell treatment groups. Statistical analysis was performed using ANOVA with BonferroniÕs multiple comparison test or Kruskal-Wallis with DunnÕs multiple comparison test for normal and non-normal data, respectively. Data reported as mean +/- SEM. Results PGE2 pre-treatment produced a significant increase in FL and FS engraftment at 72 hours post-IUHCT compared to vehicle treated donor cells (FL: 37.5 +/- 3.1% vs 21.6 +/- 1.3%; FS: 52.2 +/- 3.7% vs 39.2 +/- 1.8%; p 〈 0.05). There was no significant increase in donor cell engraftment in the FL at earlier time points associated with PGE2 treatment. PGE2 treatment was also associated with increased survival of donor cells compared to vehicle treated cells as indicated by increased donor cell expression of survivin (19.8±5.6% vs. 4.6±1.3%; p

Description: During vertebrate embryogenesis, definitive hematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM). Based on the functional conservation of AGM hematopoiesis from fish to man, an evolutionary advantage for the production of stem cells within the aorta must exist. The identification of the signals that induce HSCs at this developmental stage is of significant interest. Through a chemical genetic screen in zebrafish, a diverse group of compounds that regulate blood flow were found to affect the production of runx1/cmyb+ HSCs. These compounds represented modulators of the adrenergic and renin/angiotensin pathways, and Ca+, Na+ and nitric oxide (NO) signaling. In general, we determined that compounds which increased blood flow enhanced HSC number, whereas chemicals that decreased blood flow diminished runx1/cmyb expression. The conserved physiological mechanism of action of each compound on the vasculature was confirmed in vivo by confocal microscopy of transgenic fli1:GFP reporter fish. In the zebrafish, the step-wise initiation of heartbeat, establishment of vigorous circulation and onset of definitive hematopoiesis in the aorta-gonad-mesonephros region (AGM) suggests that blood flow may trigger HSC formation. silent heart (sih) embryos that lack a heartbeat and fail to establish blood circulation exhibit severely reduced numbers of runx1+ HSCs in the AGM. Blood flow modifying agents primarily exerted their effects after the onset of the heartbeat (〉24 hpf), however, only compounds that increase NO production (L-Arginine, S-nitroso-N-acetyl-penicillamine (SNAP)) could induce HSC formation prior to the initiation of circulation (5 somites to 22 hpf). Furthermore, SNAP rescued HSC production in sih mutant zebrafish, whereas other drugs that increased blood flow could not. Treatment with the NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), and morpholino-oligonucleotide (MO)-knockdown of nos1 (nnos/enos) blocked HSC development. Additionally, modulation of downstream components of the NO pathway affected HSC production in the zebrafish embryo. Together these data indicate that NO signaling is the downstream effector of blood flow on AGM HSC induction. To document that NO-mediated regulation of HSC formation was conserved across vertebrate species, we examined definitive HSC production in the murine AGM. Nos3 (eNos) was found to be expressed in the AGM endothelium and aortic hematopoietic clusters. Additionally, Nos3 expression specifically marks the population of HSCs with long-term adult bone marrow repopulating activity. Intrauterine NOS inhibition with L-NAME resulted in a lack of hematopoietic clusters in the AGM and a failure to generate transplantable hematopoietic progenitors. Our work provides a direct link between the initiation of circulation and the onset of AGM hematopoiesis, and identifies NO signaling as a conserved downstream regulator of HSC development. ^TEN and WG contributed equally to this work

Description: Hematopoietic stem and progenitor cells (HSPCs) are exposed to a variety of intrinsic and extrinsic factors regulating all processes needed during development, and for successful engraftment after transplantation. In order to decipher the molecular pathways that may promote engraftment of HSPCs after marrow transplantation, we performed a competitive transplantation screen using chemical genetics in zebrafish. Green fluorescent protein-labeled kidney marrow cells (equivalent to mammalian bone marrow cells) were treated ex vivo with single compounds of a chemical library of known biologically active compounds, and administered by retro-orbital venous injection to lethally irradiated recipient zebrafish. About 500 chemicals were screened. Untreated kidney marrow cells labeled with a red fluorescent protein were used as competitors. Imaging-based assessment of short-term engraftment demonstrated that 1,2-Didecanoylglycerol, a membrane permeable but non-physiologic analogue of diacylglycerol (DAG), significantly improved engraftment compared to competitor cells. Follow-up by FACS analysis showed a 3.5 fold increase of long-term repopulating units after DAG treatment. To interrogate whether DAG treatment not only affects HSPCs under transplant conditions, but also during normal embryonic development, we treated zebrafish embryos within the time window of HSC formation in the dorsal aorta. DAG treatment increased expression of the HSPC markers Runx1 and c-myb in the AGM (Aorto-Gonad-Mesonephros). Treatment after HSC specification also led to an upregulation of HSPC markers in the caudal hematopoietic tissue (equivalent to fetal liver in mammals). These data suggest that DAG affects not only HSC formation, but also migration and engraftment of HSPCs as hematopoiesis transitions from the AGM to the CHT during development. To determine whether HSPCs respond to DAG in a cell autonomous manner, and to identify the underlying molecular mechanism, we treated human CD34+ cells from umbilical cord blood with DAG and performed RNA-seq analysis. Ingenuity Pathway Analysis of the 395 differentially expressed genes (q-value 〈 0.05) implicated the MAP kinase pathway as an upstream regulator. Human Phosphokinase array analysis of treated CD34+ showed ERK 1/2 activation. DAG is known to activate Protein Kinase C (PKC) with subsequent Raf kinase phosphorylation, which has the potential to activate ERK. Co-treatment of CD34+ cells with DAG and the ERK inhibitor PD98059 blocked upregulation of downstream ERK-targets (e.g. AREG, CSF2, EGR1, HMOX, SERPINE1, DUSP4, DUSP6), whereas the PI3K family inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190 did not alter the effect of DAG on expression of these genes. This demonstrates that DAG activates ERK and its downstream targets. Our competitive marrow transplantation-based chemical screen has led to the discovery of 1,2-Didecanoylglycerol as a novel modulator of HSPC development and engraftment after transplantation. This discovery may be of clinical relevance to marrow or cord blood hematopoietic stem cell transplantation. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Description: Abstract 506 Hematopoietic stem and progenitor cells (HSPC) self-renew and give rise to all blood cell types throughout adulthood. Definitive HSPC arise from the hemogenic endothelium of the dorsal aorta, are released into circulation, and then seed an intermediate hematopoietic tissue before colonizing the adult marrow. In mammals this intermediate tissue is the fetal liver, and in the zebrafish it is the caudal hematopoietic tissue (CHT), a vascular plexus in the ventral tail of the embryo. We have generated the first highly specific zebrafish transgenic reporter of HSPC, using the previously described mouse Runx1 +23 kb intronic enhancer driving GFP (or mCherry) fluorescent protein. We have demonstrated that these Runx1+23 positive cells are capable of long-term engraftment and multi-lineage contribution. Using time-lapse live imaging in the embryo, we followed HSPC as they migrate to the CHT niche. Together with endothelial (kdrl(flk1):DsRed2) or stromal (cxcl12a(sdf-1a):DsRed2) reporter lines, we could visualize stem cell behavior directly in the endogenous niche. Upon arrival, HSPC underwent a number of distinct steps to engraftment, including: 1) adherence to the vessel wall; 2) extravasation; 3) migration to the abluminal space; 4) triggering of niche formation—endothelial cells actually remodel around a single HSPC to create a niche; 5) cell division decisions. To determine if endothelial niche formation is conserved in mammals during ontogeny, we performed live imaging of mouse fetal liver explants at embryonic day 11.5, the earliest stage of seeding by HSPC. We observed rare c-kit+/Ly6a(Sca1):GFP+ HSPC become centered in a rosette of CD31+/Lyve1+ sinusoidal endothelial cells. This dynamic remodeling of endothelial cells around an HSPC in the niche was strikingly similar to the cellular behaviors we observed in zebrafish. We hypothesized that chemical genetics could reveal the molecular mechanisms and signaling pathways that are associated with the distinct steps of HSPC engraftment. As proof-of-concept, we tested the CXCR4 antagonist AMD3100 because the CXCR4-CXCL12 receptors and ligands are expressed in the CHT, and found that it suppressed CHT hematopoiesis. Next, we performed a chemical genetic screen by applying ∼2400 individual compounds of known action to zebrafish embryos during colonization of the CHT. We found 40 compounds that increased and 107 compounds that decreased CHT hematopoiesis. Applying selected chemical hits in our live imaging assay we found that certain compounds actually modulated distinct steps during engraftment. We identified a role for sphingosine-1-phosphate signaling during extravasation. We observed that regulators of the transcription factor hypoxia inducible factor (HIF)-1α modulated migration into abluminal spaces. The HIF-1α stabilizer dimethyloxalylglycine (DMOG) promoted migration into hypoxic abluminal spaces, while conversely the HIF-1α inhibitor YC-1 promoted migration into normoxic luminal spaces. We found the plant alkaloid Lycorine promoted endothelial niche formation, creating more locations for HSPC and allowing longer residence times in the CHT. The transforming growth factor (TGF)-β receptor inhibitor SB-431542 increased the rate of HSPC division after they had arrived in the niche. Our studies provide the first genetic approach to understanding engraftment, and the chemicals found could be used therapeutically for patients receiving marrow transplantation. Disclosures: Tamplin: Boston Children's Hospital: Employment, Patents & Royalties. Zon:Fate Therapeutics: Founder Other.

Description: Small molecule treatment of hematopoietic stem cells ex vivo has the potential to expand these cells or increase their engraftability. Previously, we discovered that ex vivo treatment of marrow with 11,12-epoxyeicosatrienoic acid (EET) enhances the engraftment of hematopoietic stem and progenitor cells in both zebrafish and mammals. Additionally, EET treatment promotes specification of HSPC from the hemogenic endothelium, suggesting a broad pro-hematopoietic role of this molecule. Indeed, bioactive lipids play an important role as signaling molecules both during embryo development and adult tissue homeostasis. However, due to their small-molecule nature, identifying their receptors biochemically has been a long-standing challenge which impedes the understanding of the biological processes they regulate. The identity of the EET receptor remains unknown despite more than a decade of research. Here, we utilized a novel bioinformatic approach to identify candidate EET receptors and identified a candidate functional in cell culture, zebrafish and mouse assays. EET signaling is known to be G-protein dependent, suggesting its receptor is a G-protein coupled receptor (GPCR). We performed RNAseq on U937 monocytes, EaHy endothelial cells, and PC3M-LN4 prostate cancer cells, three human cell lines with clear EET-responsive phenotypes. These three cell lines expressed 37 GPCR in common at a basal level of greater than or equal to 0.3 fragments per kilobase per million reads (FPKM). 27 of these GPCR were also expressed in a non-EET-responsive cell line, HEK293, leaving only 10 candidate EET receptors. We screened 7 of these candidates for EET-responsiveness using a cell-culture based β-arrestin recruitment assay. Of these, only GPR132 exhibited EET-dependent recruitment of β-arrestin to the cell membrane, indicating GPCR activation. GPR132 was previously identified as a receptor for a variety of small oxygenated fatty acids, and we confirmed that these related molecules induce GPR132-dependent β-arrestin recruitment. We additionally treated developing zebrafish embryos with these molecules. Like EET, these GPR132 ligands increased HSPC numbers in the zebrafish aorta-gonad-mesonephros (AGM) and caused ectopic expression of the HSPC marker runx1 in the zebrafish tail, a phenotype that was previously seen only with EET treatment. To test the requirement of GPR132 for EET signaling, we knocked down the zebrafish ortholog of GPR132 by morpholino injection, which prevented the EET-induced increase of runx1in both the AGM and tail. Finally, we performed competitive whole bone marrow transplant using wildtype and GPR132-/- mice as donors and found that while treatment with EET increases engraftment of WT donor cells, no such improvement is seen in GPR132-/- cells. GPR132 is thus required in both zebrafish and mice for EET phenotypes. Combining bioinformatic, biochemical, and genetic approaches, we identified GPR132 as a receptor for EET involved in regulating hematopoiesis and marrow transplant. GPR132 thus represents a therapeutic target for the enhancement of hematopoietic stem cell transplant, and genetic manipulation of GPR132 could help illuminate the endogenous roles of its fatty acid ligands. Disclosures Zon: Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Description: Transplanted hematopoietic stem cells (HSCs) home to the marrow space, engraft and self-renew. The process of engraftment is difficult to study in most vertebrates because it is impossible to visualize the engraftment directly. Here we have developed competitive transplantation of fluorescent donor marrow samples into a completely transparent adult zebrafish called casper. This allows the direct visualization of engraftment and the process of HSC competition. Transplantation of zebrafish whole kidney marrow (WKM) cells, containing HSCs, progenitors and mature blood cells, can rescue lethally irradiated recipient fish and repopulate all the blood lineages. We used different GFP-/DsRed-labeled transgenic fish as donors in WKM transplantation, such as the myeloid specific Tg(mpo:GFP), or the ubiquitous expression of Tg(β-actin:GFP), and Red GloFish® to study the kinetics of engraftment. Engraftment can be visualized with a simple fluorescent microscope, and was evident in the recipient kidney as early as 2 weeks post transplant (wpt). By 4 wpt, mature hematopoietic cells differentiated from donor HSCs or progenitors can be observed in the recipient circulation as well as in epidermis as tissue residential macrophages. At 8 wpt, donor-derived lymphoid cells also repopulate the recipient immune system. The fluorescence intensity of engrafted cells was quantified by image analysis software and positively correlated with the engraftment efficiency quantified by FACS. Using Red GloFish® as competitor donor and Tg(β-actin:GFP) as test donor, we were able to see the competition between the two donors both by fluorescence intensity analysis and FACS analysis. To test the sensitivity of this competitive transplant system to the alteration of the ratios between the two colors, we increased the ratio of the test vs. competitor donor from 1:3 to 1:2 to 1:1. At 4wpt, in the 1:3 ratio group, only 14% of the recipients had stronger GFP intensity than the red (n=7), while the rest had weaker or equivalent GFP intensity than the red; however, this percentage increased to 43% and 50% in the 1:2 (n=7) and 1:1(n=8) groups respectively. Therefore, the relative engraftment ratio can be fast detected in vivo by direct fluorescent visualization with the transparent zebrafish recipients. Next, we treated the test donor cells with 16,16-methyl prostaglandin E2 (dmPGE2) or DMSO for 2 hours in vitro at room temperature before transplant. The engraftment efficiency of the dmPGE2 group dramatically increased by at least 2 folds compared with the DMSO group. The colors of competitor and test donors were switched, and the increase of engraftment efficiency by dmPGE2 treatment of the test donor was still evident. In conclusion, direct visualization of competitive marrow repopulation greatly facilitates the developmental, physiological or pathological study of HSCs and progenitors and should provide a new platform for the assay of specific genes or chemicals that could alter homing, engraftment or self-renewal.

Description: Abstract 3870 The BMP and WNT signaling pathways are two highly conserved signaling pathways that cooperate in many developmental processes, ultimately through alteration of transcription via SMAD and TCF transcription factors. These pathways elicit pleiotropic outcomes across cell types, yet only a few cell-specific direct target genes are known for the signaling transcription factors that mitigate these effects. We took a genome-wide approach to define the binding sites of BMP and WNT-directed transcription factors in different hematopoietic lineages. Using heat-shock inducible transgenic fish lines that overexpress BMP2 or WNT8, we demonstrated accelerated marrow recovery following irradiation. Irradiation recovery was blunted by heat shock induced overexpression of the respective inhibitors Chordin and DKK1. Similar to the zebrafish regeneration results, competitive transplants with mouse bone marrow treated with the WNT agonist BIO led to enhanced chimerism. Inhibition of BMP diminished peripheral blood contribution even in the presence of WNT stimulation, suggesting a conserved and cell intrinsic interaction for these signaling pathways in adult stress hematopoiesis. To examine potential target genes that could account for the synergy, we performed chromatin immunoprecipitation with WNT- and BMP-activated transcription factors followed by sequencing (ChIP-seq) in K562 cells. ChIP-seq was performed with TCF7L2/TCF4, a mediator of the WNT pathway, and SMAD1, a mediator of the BMP signaling pathway, and 〉2000 binding sites were identified for each factor. Motif discovery revealed that the DNA sequences bound by TCF7L2 and SMAD1 were not only enriched for TCF and SMAD binding elements, respectively, but were also enriched for a GATA motif. Comparison of the TCF7L2 and SMAD1 bound genes with published ChIP-Seq data for GATA1 and GATA2 in K562 cells revealed that both signaling factors bind more than 40% of GATA1 bound genes and greater than 70% of GATA2 bound genes. Ingenuity and GSEA analysis revealed that genes important for erythropoiesis were among the genes co-bound by these factors. To evaluate the effect of cell lineage on signaling factor binding, ChIP-seq of TCF7L2 and SMAD1 in U937, a monocytic leukemia cell line, was performed. Motif discovery of sequences bound in U937 found enrichment for an ETS motif, which is bound by the key myeloid transcription factor Pu.1. In addition, TCF7L2 and SMAD1 bound genes in U937 overlapped genes bound by C/EBPalpha in U937 by greater than 70%. These genes are implicated in monocytic development. The overlap of binding between TCF7L2 in K562 and U937 was less than 15% and the overlap of SMAD1 binding sites between the cell lines was less than 10%, indicating a substantial influence of cell lineage on transcription factor binding. Confirmation of cell type selective binding of TCF7L2 and SMAD1 in vivo was accomplished by ChIP of the transcription factors in zebrafish nucleated erythrocytes. Binding of TCF7L2 and SMAD1 in these cells showed that these factors co-bind with GATA1 in many genes with established roles in erythropoiesis. Together our data suggest the co-binding of WNT- and BMP-specific transcription factors with master regulators of each hematopoietic cell type results in regulation of distinct blood genes based on lineage. (First two authors contributed equally to this work) Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Hematopoietic stem cells (HSC) reside in the bone marrow niche and sustain the production of blood throughout life. The entire pool of these rare and important cells is generated during a brief window of embryonic development. HSC are produced by the hemogenic endothelium of the dorsal aorta, migrate to and expand in the fetal liver, and then migrate again to seed the bone marrow. The zebrafish is a highly conserved and well-established model for HSC development. Similar to mammals, HSC emerge from the dorsal aorta, but then colonize a vascular plexus in the tail of the embryo—the caudal hematopoietic tissue (CHT). It is difficult to directly observe the interactions between an endogenous HSC and its niche, so we have developed the CHT as a model for HSC-niche interactions. To track HSC in vivo we have generated a transgenic reporter using the previously described mouse Runx1 +23 kb intronic enhancer. The purity of the stem cell pool marked by this reporter was determined. Using adult-to-adult limiting dilution transplantation with as few as one Runx1+23 positive cell, we have estimated the HSC purity to be approximately 1/35 (without immune matching), or similar to Kit+Sca1+Lin- (KSL) in mouse. This is the most pure stem cell population defined in the zebrafish system. Using embryo-to-embryo transplantation, a technique that is unique to zebrafish, we sorted Runx1+23 positive cells from one group of embryos and transplanted them to another by injection directly into circulation. Embryos are then grown to adulthood and marrow is tested for long-term engraftment between 3 and 5 months. This transplantation technique precedes formation of the thymus, thereby removing any chance of immune rejection. Highly stringent dilution of HSC in our embryo-to-embryo transplants has estimated a stem cell purity of one in two cells. Next, we applied our highly specific reporter to visualize HSC migration to the CHT niche. After arrival of the HSC, we have described 5 distinct steps during colonization: 1) adherence; 2) extravasation; 3) abluminal migration; 4) endothelial niche formation (“cuddling”); and 5) cell fate decisions. Live imaging analysis of HSC together with endothelial and stromal transgenic reporters has allowed us to quantify the relationship between different cell types within the CHT. For example, we observe preferential localization of HSC in close proximity to cxcl12a positive stromal cells. Lastly, we have sought to identify the molecular mechanisms involved in interactions between HSC and their niche. A chemical genetic screen identified the natural product lycorine as a small molecule that increases hematopoiesis in the CHT and promotes HSC-endothelial cell interactions. Combined chemical treatment and live imaging revealed that lycorine significantly increased the residence time of HSC in the niche. To test if treatment during the window of CHT colonization (2-3 days post fertilization) had long-term effects on HSC and the stem cell pool, the compound was washed off at 3 days and the Runx1+23 positive population was quantified by FACS. At 7 days post fertilization, after colonization of the marrow, there was a sustained and significant increase in Runx1+23 positive HSC. Strikingly, after 3 months, when treated embryos were raised to adulthood, we discovered that the increased HSC-endothelial cell interactions we observed in the CHT niche had in fact had an impact on the number of HSC in the adult. Our studies establish that the Runx1+23 transgenic is a highly specific reporter of HSC both in the embryo and adult, and that we can use this reporter for in vivo observation of an endogenous HSC niche. Furthermore, we show that the size of the adult stem cell pool can be altered by a transient signal during development. Disclosures: Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.