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. 2015 Jul 23;523(7561):468-71.
doi: 10.1038/nature14569.

Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment

Pulin Li  1 , Jamie L Lahvic  2 , Vera Binder  3 , Emily K Pugach  2 , Elizabeth B Riley  2 , Owen J Tamplin  2 , Dipak Panigrahy  4 , Teresa V Bowman  2 , Francesca G Barrett  2 , Garrett C Heffner  2 , Shannon McKinney-Freeman  5 , Thorsten M Schlaeger  2 , George Q Daley  2 , Darryl C Zeldin  6 , Leonard I Zon  1
Affiliations

Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment

Pulin Li et al. Nature. .

Erratum in

Abstract

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

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Figures

Extended Data Figure 1
Extended Data Figure 1. Zebrafish whole kidney marrow (WKM) competitive transplantation-based chemical screen identifies EETs as enhancers of marrow engraftment
a, WKM from Tg(β-actin:GFP) donors were dissected, dissociated as single cell suspension, and incubated with chemicals at room temperature for 4 hrs in a round-bottom 96-well plate. Meanwhile, WKM were dissected from RedGlo® zebrafish, counted and kept on ice. After the drug treatment, chemicals were washed off and cells were resuspended in 0.9 X DPBS + 5% FBS. 20,000 treated green WKM and 80,000 untreated red WKM were co-injected retro-orbitally into sublethally irradiated capser zebrafish (n=10 per chemical). For every independent screening day, negative control (DMSO) and positive control (10 μM dmPGE2) were used for normalization and quality assurance. The engraftment was measured at 4 wpt (weeks post transplant) by fluorescence imaging and ImageJ quantification as described in Fig. 1b. b, EET metabolic pathway: arachidonic acid is released by PLA2 (phospholipase A2) from the membrane lipid bilayer. EETs (epoxyeicosatrienoic acids) are synthesized directly from arachidonic acid by the cytochrome P450 family of epoxygenases, especially 2C and 2J in human, and get degraded by soluble epoxide hydrolase (sEH), generating DiHET (dihydroxyeicosatrienoic acids). Four isomers of EET exist in vivo: 5,6-, 8,9-, 11,12- and 14,15-EET.
Extended Data Figure 2
Extended Data Figure 2. 11,12-EET enhances HSPC specification in the AGM in zebrafish embryos
Tg(CD41:GFP/flk1:NRAS-mCherry) embryos were treated with DMSO or 5 μM 11,12-EET starting at 24 hpf (hours post fertilization), then mounted for spinning disc confocal timelapse imaging from 30–46 hpf in the presence of the chemicals. Bars show mean and s.e.m., unpaired two-tailed t-tests, n=10 for DMSO, n=7 for EET. a, More HSPCs are directly specified in EET-treated AGM. Graph shows HSPCs born by direct specification/budding only, excluding cells born by division of an already-budding cell. b–c, 11,12-EET does not influence the rate of HSPC division in the AGM, shown by per movie, percentage of budding HSPCs that divide at least once (b) and divide twice or more (c) before leaving the AGM or before the end of timelapse recording. n.s., not statistically significant.
Extended Data Figure 3
Extended Data Figure 3. 11,12-EET treatment between 24–48 hpf increases the number of HSPCs in the CHT
a, Embryos were treated between 24–48 hpf with either DMSO or 5 μM 11,12-EET. Chemicals were washed off at 48 hpf, and embryos grew in drug-free environment for another 24 hrs. b, 11,12-EET treatment increased the number of mCherry+ HSPCs in the CHT in Tg(Runx1+23:mCherry) embryos (see also Fig. 2e). Representative images of the CHT from the two groups. c, The same chemical treatment increased the staining of cmyb, a HSPC marker, by whole-mount RNA in situ hybridization. Representative images from each group (a total of n>60 from 3 independent experiments).
Extended Data Figure 4
Extended Data Figure 4. EET signaling pathway activates AP-1 family members as primary transcriptional targets, and runx1 as secondary transcriptional target
a, Wild-type embryos were incubated with 300 μM cycloheximide, a translation blocker, for 30 min before the addition of 5 μM 11,12-EET at 24 hpf. Embryos were fixed for in situ hybridization at 25 hpf or 28 hpf. b. AP-1 transcription was induced upon 1 hr treatment with 11,12-EET, insensitive to cycloheximide inhibition. This means AP-1 induction does not depend on de novo protein synthesis, indicating AP-1 members are primary transcriptional targets of the EET signaling pathway. c. runx1 transcription was induced upon 4 hr treatment with EET (left two columns) and cycloheximide completely blocked EET-induced runx1 expression (right two columns). This suggests runx1 transcription depends on de novo protein synthesis of an upstream factor(s) upon EET stimulation, indicating that runx1 is a secondary transcriptional target of the EET signaling pathway. Representative images from each group (a total of n>30 from 2 independent experiments).
Extended Data Figure 5
Extended Data Figure 5. Knocking down junb/junbl inhibits HSPC specification in the AGM
a, Wild-type (WT) embryos were injected with antisense morpholinos at 1-cell stage, and treated with DMSO or 5 μM 11,12-EET starting from 24 hpf. Embryos were fixed at 36 hpf for in situ hybridization of runx1. b, Knocking down junb completely blocked runx1 expression at 36 hpf both in the AGM and the tail non-haematopoietic tissue (middle row). In contrast, knocking down c-jun did not block the increase of runx1 (bottom row), consistent with the lack of c-jun upregulation in EET-treated embryos (data not shown). c, junb morphants still developed normal vascular structure in the AGM at 28 hpf, as shown by endothelial marker flk1 (c). Representative images from each group (a total of n>40 from 3 independent experiments).
Extended Data Figure 6
Extended Data Figure 6. PI3Kγ activation is specifically required for EET-induced gene expression signature
a, Similar to LY294002 (Fig. 3e), another pan-PI3K/AKT inhibitor, wortmannin (1 μM) blocked EET-induced runx1 expression both in the AGM and tail. Representative images from each group (a total of n>60 from 3 independent experiments). b, Morpholinos specific to PI3Kγ, but not α, β, and δ subunits (data not shown), prevented EET-induced runx1 in the AGM and tail. Embryos injected at 1–2 cell stage with indicated amount of morpholino and treated with DMSO or 5 μM 11,12-EET from 24–36 hpf. In situ hybridization for runx1 performed at 36 hpf and percentages of embryos having high, medium, or low expression in the AGM and present or absent expression in the tail are shown. Graph summarizes 3 experiments, n≥10 embryos for each condition (0, 1, and 2 ng, bars show mean and s.e.m.) or one experiment n≥9 for all conditions (4 and 6 ng). c, The PI3Kγ specific inhibitor AS605240 (AS6) recapitulates the morpholino phenotype. Embryos treated from 24–36 hpf with DMSO or 5 μM 11,12-EET, with or without 0.3–1.0 μM AS6, then fixed and stained for runx1 at 36 hpf. DMSO, n=23; EET, n=33; EET+0.3 μM AS6, n=35; EET+1.0 μM AS6, n= 38. * p<0.05, *** p<0.001, two-tailed Fisher’s Exact Test.
Extended Data Figure 7
Extended Data Figure 7. 11,12-EET up-regulates genes involved in cell-to-cell signaling and cellular movement in haematopoietic progenitors
a, Venn diagram showing a common set of 54 genes up-regulated (log2fc>0.5) after 2 hrs of 11,12-EET treatment (5 μM), both in human myeloid U937 cells and human umbilical cord CD34+ HSPCs (see also Supplementary Table S4 for lists of up- and down-regulated genes). b–c, Ingenuity Pathway Analysis (IPA) of the overlapping gene set between the two cell types for enrichment of bio-functions. b, Biological processes, such as cell-to-cell signaling and cellular movement, were highly enriched, supporting EETs’ capability of enhancing engraftment (see also Supplementary Table S4 for a comprehensive list of all biological functions predicted to be activated or suppressed based on the same gene set). c, Activation of recruitment of blood cells is caused by up-regulation of chemokines and cytokines such as CXCL8 and OSM after EET treatment, as well as by up-regulation of transcription factors, such as AP-1 genes (FOS). Orange dashed arrows depict activation. Shades of red represent the level of activation. Numbers underneath factors show RNAseq FPKM values in U937 cells.
Extended Data Figure 8
Extended Data Figure 8. 11,12-EET treatment after HSPC specification still enhances the number of HSPCs in the CHT
a, Embryos were treated with DMSO or 5 μM 11,12-EET between 48–72 hpf to bypass the HSPC specification process in the AGM. 72 hpf embryos were fixed and tested on the following assays. b, In situ hybridization for cmyb, a marker for HSPCs. EET treatment significantly increased the staining, while LY294002, a pan-PI3K inhibitor, suppressed the effect. Representative images from each group (a total of n>60 from 4 independent experiments). c, A PI3Kγ-specific inhibitor AS605240 (AS6) also blocked the EET-induced increase of cmyb staining. Percentage of embryos having high, medium, or low expression in the CHT is shown. n≥11 for all conditions. Chi-square analysis. d, The increase of HSPCs in the CHT is not due to effects on proliferation. Immunofluorescence staining for phospho-Histone H3 (pH3) as a marker for proliferating cells. The number of pH3 positive cells was manually counted. Two-tailed t-test showed no significant difference between DMSO vs EET treated embryos. n=9 for DMSO, n=10 for EET. e, TUNEL staining as an assay for apoptotic cells. Apoptosis was minimal in the CHT at 72 hpf. As a staining control, obvious apoptosis was detected in the same embryos in the brain region, and was comparable between DMSO and EET treated embryos (data not shown).
Extended Data Figure 9
Extended Data Figure 9. 11,12-EET treatment of mouse whole bone marrow (WBM) does not lead to immediate changes in cell proliferation or apoptosis
a, in vitro apoptosis assay on WBM treated with DMSO or 2 μM 11,12-EET for 4 hrs. The 7-AAD negative and AnnexinV positive population are the cells undergoing apoptosis. No significant differences between the two groups were observed either in Lin-Sca-Kit+ or Lin-Sca+Kit+ progenitor populations (n=4 each), mean with s.e.m.. b–c, in vitro proliferation assay on WBM treated with DMSO or 2 μM 11,12-EET for 4 hrs, in the presence of 10 μM BrdU. No significant differences between the two groups were observed either in Lin-Sca-Kit+ (b) or Lin-Sca+Kit+ populations (c) for any cell-cycle stage. Unpaired two-tailed t-test, n=4 each, bar showing mean. D, DMSO; E, EET; n.s., not significant.
Extended Data Figure 10
Extended Data Figure 10. Gα12/13 is specifically required for EET-induced phenotypes in zebrafish embryos
All embryos were treated with DMSO or 5 μM 11,12-EET between 24–36 hpf. Chemical inhibitors were added 30 min before EET. mRNA or morpholinos (MO) were injected at 1-cell stage. a–b, Inhibiting Gαs or Gαi had no effect on EET-induced runx1 expression. Embryos were categorized into two groups with either normal or increased runx1 expression level (n>20 each). PtxA, pertussis toxin A, 3 pg, inhibiting Gαi; H89, 5 μM, PKA inhibitor downstream of Gαs; SQ, SQ22536, 50 μM, adenylate cyclase inhibitor downstream of Gαs. Representative images from each group (b) (a total of n>40 from 2 independent experiments). c–e, Synergistic effects of gna12/13a/13b knockdown on suppressing runx1 expression. Knocking down gna13a/b or gna12 alone partially inhibited EET-induced runx1 expression in the AGM and tail (c). gna12 MO: 2 ng; gna13a/13b MOs: 1 ng each. Triple morpholinos against gna12, gna13a and gna13b (0.67 ng each) completely blocked EET-induced multiple gene expression, including runx1, genes in regeneration (fosl2) and cholesterol metabolism (hmgcs1) (d), while other major tissue development processes were not significantly affected, such as notochord (shh), muscle (myoD), and blood vessels (flk, ephrinB2) (e). The results were quantified in (f). Embryos were categorized as having decreased, normal or increased runx1 expression. The bar graph represents the percentage of embryos in each group (n>30).
Figure 1
Figure 1. Zebrafish whole kidney marrow (WKM) competitive transplantation-based chemical screen identifies EETs as enhancers of marrow engraftment
a, Schematic of zebrafish WKM competitive transplantation. b, Calculation of relative engraftment capability (G/R). White dashed line: kidney; Gkid/Rkid, kidney fluorescence intensity; Gbkg/Rbkg, background fluorescence intensity. c, The G/R ratios from imaging linearly correlated with flow cytometry analysis of the same recipients (linear regression). wpt, weeks post transplant. d, Serial dilution competitive transplantation with varying donor GFP:DsRed2 ratios. e, 4 hr transient chemical treatment increased WKM engraftment. 11,12- and 14,15-EET, 0.5 μM. Unpaired two-tailed t-test, mean with s.e.m. (d–e).
Figure 2
Figure 2. 11,12-EET enhances HSPC specification in the zebrafish embryo AGM
a, Schematic of HSPC development in zebrafish embryos. hpf, hours post fertilization; AGM, aorta-gonad-mesonephros; CHT, caudal haematopoietic tissue. b, Representative images of whole-mount in situ hybridization showing 11,12-EET (24–36 hpf treatment) induced HSPC marker runx1 in the AGM and a tail non-haematopoietic tissue (>8 independent experiments, n>100). c–d, 11,12-EET (24–46 hpf) enhanced CD41:GFP/flk1:NRAS-mCherry double positive HSPCs (white arrowheads) emerging in the AGM. Arrows indicate blood flow. e–f, Same treatment increased the number of HSPCs in the CHT. mCherry+ HSPCs quantified in the Tg(Runx1+23:mCherry) CHT (e). Representative montage images of Runx1+23:GFP HSPCs (white arrowheads) engrafting CHT. flk1:DsRed2, endothelial cells (f). Unpaired two-tailed t-test, mean with s.e.m. (d–e).
Figure 3
Figure 3. 11,12-EET induces a PI3K-dependent AP-1/runx1 transcriptional program to increase HSPC specification
a–b, Stable flk1:dnJUNB-2A-GFP expression blocking AP-1 function suppressed 11,12-EET-enhanced HSPCs in the AGM. Representative images of runx1/cmyb in situ hybridization (a) and quantification (b) after 11,12-EET treatment (24–36 hpf). Embryos scored as high, medium, or low runx1/cmyb, summed across 4 experiments. *, p=0.01; ***, p<0.0001 by Chi-square. WT, wild-type. c, Schematic of chemical screen for EET signaling pathway suppressors. d–e,11,12-EET induced AP-1 family transcription factors (fosl2, junb/junbl) (d) and runx1 (e), suppressed by cotreatment with LY294002, a PI3K inhibitor, in the AGM and tail (d–e) (3 independent experiments, n>40). Same images from Fig. 2b as staining controls (e).
Figure 4
Figure 4. 11,12-EET enhances HSPC engraftment and homing in mammals
a, Schematic of mouse WBM (whole-bone marrow) competitive transplantation. RT, room temperature. b–c, 4 hrs of 11,12-EET treatment promoted short-term WBM engraftment at 4 wpt (b) and long-term multilineage engraftment at 24 wpt (c). WBC, white blood cells; M, myeloid cells; B, B cells; T, T cells. 2 independent experiments combined, n=20 total. d, Schematic of WBM competitive homing assay. hpt, hours post transplant. e, 11,12-EET increased homing efficiency of Lin- cells and Lin-Kit+ HSPCs (n=5). f, PI3K activation is required for EET-enhanced mouse WBM engraftment (n=10). LY, 10 μM LY294002. Recipients characterized as engrafted or non-engrafted based on peripheral blood WBC chimerism, two-tailed Fisher’s Exact test (b,f); unpaired two-tailed t-test (c,e), mean with s.e.m..
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