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 lipids1,2 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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Acknowledgements
We thank C. R. Lee, M. L. Edin and N. Gray for providing reagents; Y. Zhou, A. Dibiase, S. Yang, S. Datta, P. Manos, R. Mathieu and M. Ammerman for technical assistance; H. Huang for providing graphic illustration; R. M. White, T. E. North and C. Mosimann for discussion. Microarray studies were performed by the Molecular Genetics Core Facility at Boston Children's Hospital, supported by NIH-P50-NS40828 and NIH-P30-HD18655. S. Li in Y. Zhang's laboratory at the Longwood HHMI joint core facility helped with RNA-seq. L.I.Z. and G.Q.D. are Howard Hughes Medical Institute (HHMI) investigators. This work was supported by HHMI and National Institutes of Health (NIH) grants R01 HL04880, P015PO1HL32262-32, 5P30 DK49216, 5R01 DK53298, 5U01 HL10001-05, R24 DK092760, and 1R01HL097794-04 (to L.I.Z.). This work was also funded, in part, by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01 ES025034 to D.C.Z.), the National Cancer Institute grant ROCA148633-01A5 (D.P.), and DFG and Care-for-Rare Foundation (V.B.).
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Contributions
P.L. and L.I.Z. designed the study, analysed data and wrote the manuscript, with help from J.L.L. and V.B. P.L. developed the zebrafish competitive transplantation and performed the chemical screen with technical help from E.K.P. P.L. performed the mouse experiments with technical help from T.V.B., S.M. and G.C.H. P.L. performed the zebrafish microarray and embryo chemical/genetic suppressor screens with technical help from E.B.R. J.L.L. performed zebrafish embryo genetic studies and AGM timelapse imaging. V.B. performed RNA-seq and analysis on human cells with technical help from F.G.B. O.J.T. performed CHT time-lapse imaging. T.M.S. provided the chemical library. D.P. and D.C.Z. offered reagents and information related to the EET study. All authors discussed the results and commented on the manuscript.
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Competing interests
L.I.Z. is a founder and stockholder of Fate, Inc. and a founder and stockholder of Scholar Rock. G.Q.D. is a member of the Scientific Advisory Boards of MPM Capital, Inc., Ocata Therapeutics, Raze Therapeutics, Solasia KK and consults to Epizyme, Verastem, and True North.
Extended data figures and tables
Extended Data Figure 1 Zebrafish 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 h 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× PBS plus 5% FBS. Approximately 20,000 treated green WKM and 80,000 untreated red WKM were co-injected retro-orbitally into sublethally irradiated casper zebrafish (n = 10 per chemical). For every independent screening day, negative control (DMSO) and positive control (10 μM dmPGE2) treatments were used for normalization and quality assurance. The engraftment was measured at 4 wpt by fluorescence imaging and ImageJ quantification as described in Fig. 1b. b, EET metabolic pathway: arachidonic acid is released by phospholipase A2 (PLA2) from the membrane lipid bilayer. EETs are synthesized directly from arachidonic acid by the cytochrome P450 family of epoxygenases, especially 2C and 2J in human38, and get degraded by soluble epoxide hydrolase (sEH), generating dihydroxyeicosatrienoic acids (DiHET). Four isomers of EET exist in vivo: 5,6-, 8,9-, 11,12- and 14,15-EET.
Extended Data Figure 2 11,12-EET enhances HSPC specification in the AGM in zebrafish embryos.
Tg(CD41:GFP/flk1:DsRed2) embryos were treated with DMSO or 5 μM 11,12-EET starting at 24 hpf, then mounted for spinning disc confocal timelapse imaging from 30–46 hpf in the presence of the chemicals. Data are 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.
Extended Data Figure 3 11,12-EET treatment between 24 and 48 hpf increases the number of HSPCs in the CHT.
a, Embryos were treated between 24 and 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 h. 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 three independent experiments).
Extended Data Figure 4 EET signalling pathway activates AP-1 family members as primary transcriptional targets, and runx1 as a 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 after 1 h 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 signalling pathway. c, runx1 transcription was induced after 4 h treatment with EET (two columns on the left) and cycloheximide completely blocked EET-induced runx1 expression (two columns on the right). 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 signalling pathway. Representative images from each group (a total of n > 30 from two independent experiments).
Extended Data Figure 5 Knocking down junb and junbl inhibits HSPC specification in the AGM.
a, Wild-type embryos were injected with antisense morpholinos at the one-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). By 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. Representative images from each group (a total of n > 40 from three independent experiments).
Extended Data Figure 6 PI(3)Kγ activation is specifically required for EET-induced gene expression signature.
a, Similar to LY294002 (Fig. 3d–e), another pan-PI(3)K/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 three independent experiments). b, Morpholinos specific to PI(3)Kγ, but not α, β and δ subunits (data not shown), prevented EET-induced runx1 in the AGM and tail. Embryos were injected at 1–2-cell stage with the 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 three experiments, n ≥ 10 embryos for each condition (0, 1 and 2 ng, data are mean and s.e.m.) or one experiment n ≥ 9 for all conditions (4 and 6 ng). c, The PI(3)Kγ-specific inhibitor AS605240 (AS6) recapitulates the morpholino phenotype. Embryos treated from 24 to 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 11,12-EET upregulates genes involved in cell-to-cell signalling and cellular movement in haematopoietic progenitors.
a, Venn diagram showing a common set of 54 genes upregulated (log2(fc) > 0.5) after 2 h of 11,12-EET treatment (5 μM), both in human myeloid U937 cells and human umbilical cord CD34+ HSPCs (see also Supplementary Table 4 for lists of up- and downregulated 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 signalling and cellular movement, were highly enriched, supporting the capability of EETs in enhancing engraftment (see also Supplementary Table 4 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 upregulation of chemokines and cytokines such as CXCL8 and OSM after EET treatment, as well as by upregulation 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 (fragments per kilobase of exon per million reads mapped) values in U937 cells.
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 and 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-PI(3)K inhibitor, suppressed the effect. Representative images from each group (a total of n > 60 from four independent experiments). c, A PI(3)Kγ-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- versus 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 11,12-EET treatment of mouse 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 h. 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 and s.e.m. b, c, In vitro proliferation assay on WBM treated with DMSO or 2 μM 11,12-EET for 4 h, 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 denotes the mean. D, DMSO; E, EET.
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 and 36 hpf. Chemical inhibitors were added 30 min before EET. mRNA or morpholinos (MO) were injected at the one-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 (ref. 30); H89, 5 μM, PKA inhibitor downstream of Gαs5; SQ, SQ22536, 50 μM, adenylate cyclase inhibitor downstream of Gαs5. Representative images from each group (b) (a total of n > 40 from two independent experiments). c–f, 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). f, The results were quantified. Embryos were categorized as having decreased, normal or increased runx1 expression. The bar graph represents the percentage of embryos in each group (n > 30).
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Supplementary Information
This file contains a Supplementary Discussion, Supplementary Tables 1-2 and Supplementary Table 5, full legends for Supplementary Tables 3 and 4 and Supplementary References. (PDF 181 kb)
Supplementary Table 3
This file contains Supplementary Table 3 - see Supplementary Information PDF for full legend. (XLSX 49 kb)
Supplementary Table 4
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Confocal time-lapse imaging of HSPC trafficking to and engrafting CHT (caudal haematopoietic tissue) in zebrafish embryos: DMSO-treated embryo
Tg(Runx1+23:GPF;flk1:DsRed2) embryos were treated with chemicals between 24-46 hpf. Chemicals were washed off at 46 hpf. Embryos were immediately mounted in 1% low-melting-point agarose and imaged between 54-63 hpf. Time-lapse movies were taken on a spinning disk confocal microscope with a 28°C incubation chamber. Images were taken every 2 minutes and focused on the CHT region. (MOV 2513 kb)
Confocal time-lapse imaging of HSPC trafficking to and engrafting CHT (caudal haematopoietic tissue) in zebrafish embryos: 5 μM 11,12-EET-treated embryo
Tg(Runx1+23:GPF;flk1:DsRed2) embryos were treated with chemicals between 24-46 hpf. Chemicals were washed off at 46 hpf. Embryos were immediately mounted in 1% low-melting-point agarose and imaged between 54-63 hpf. Time-lapse movies were taken on a spinning disk confocal microscope with a 28°C incubation chamber. Images were taken every 2 minutes and focused on the CHT region. (MOV 1356 kb)
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Li, P., Lahvic, J., Binder, V. et al. Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. Nature 523, 468–471 (2015). https://doi.org/10.1038/nature14569
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DOI: https://doi.org/10.1038/nature14569
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