Vol 447 21 June 2007 doi 10 1038 nature05883 LETTERS Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis Trista E North1 2 Wolfram Goessling1 2 Carl R Walkley1 3 Claudia Lengerke1 Kamden R Kopani1 2 Allegra M Lord1 2 Gerhard J Weber1 2 Teresa V Bowman1 2 Il Ho Jang1 Tilo Grosser4 Garret A FitzGerald4 George Q Daley1 Stuart H Orkin1 2 3 Leonard I Zon1 2 Haematopoietic stem cell HSC homeostasis is tightly controlled by growth factors signalling molecules and transcription factors Definitive HSCs derived during embryogenesis in the aorta gonad mesonephros region subsequently colonize fetal and adult haematopoietic organs1 2 To identify new modulators of HSC formation and homeostasis a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta gonad mesonephros region Here we show that chemicals that enhance prostaglandin PG E2 synthesis increased HSC numbers and those that block prostaglandin synthesis decreased stem cell numbers The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish In murine embryonic stem cell differentiation assays PGE2 caused amplification of multipotent progenitors Furthermore ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes A chemical genetic screen was conducted to identify new pathways modulating definitive HSC formation during zebrafish embryogenesis runx1 and cmyb required for mammalian HSC development are expressed in the ventral wall of the dorsal aorta in a region analogous to the mammalian aorta gonad mesonephros AGM at 36 h post fertilization h p f 3 5 Wild type embryos incubated with individual chemicals were examined for alterations in runx11 cmyb1 HSCs by in situ hybridization expression at 36 h p f A high percentage of compounds 91 7 2 275 of 2 357 failed to alter HSC expression whereas 35 1 4 and 47 1 9 led to increased or decreased numbers of HSCs respectively Among these substances 10 affected the prostaglandin pathway Supplementary Table 1 runx11 cmyb1 HSCs comprise a line of flattened endothelial cells arrow and haematopoietic clusters arrowhead in the aorta Fig 1a c linoleic acid increased HSC numbers 22 altered out of 30 scored whereas celecoxib a cyclooxygenase Cox 2 inhibitor decreased HSCs 26 31 PGE2 is the main effector prostanoid produced in the zebrafish6 and is regulated by both Cox1 also known as Ptgs1 and Cox2 also known as Ptgs2a Treatment of zebrafish embryos with PGE2 increased expression of runx1 cmyb 25 49 whereas Cox inhibition with SC560 Cox1 and NS398 Cox2 Supplementary Fig 1a e decreased HSC numbers in 30 36 and 35 44 cases respectively These findings argue persuasively for a specific role of PGE2 in the formation of AGM HSCs Cox1 is required for the development of the aorta vein endothelial boundary during zebrafish development7 thus alteration in Cox1 activity could have an impact on endothelial derived HSCs By in situ hybridization cox2 was diffusely expressed in the tail region at 36 h p f Supplementary Fig 1f g In FACS isolated blood and endothelial cell populations both cox1 and cox2 were found to be highly expressed during the onset of definitive haematopoiesis cox1 was detected in both Lmo21 endothelial cells and in Cd411 HSCs whereas cox2 was only found in HSCs Supplementary Fig 1h This suggests that Cox1 and Cox2 participate in HSC induction through regulation of the stem cell niche and the HSC itself A long acting derivative of PGE2 16 16 dimethyl PGE2 dmPGE2 caused an increase in runx11 cmyb1 AGM HSCs in 78 of embryos 97 124 Fig 1e h whereas HSCs were inhibited by indomethacin 10 mM treatment in 90 of embryos 92 102 Fig 1k and Supplementary Fig 1j r Mass spectrometry of 36 h p f embryos demonstrated that indomethacin treatment depressed PGE2 formation below detectable levels from 1866 pg per 50 embryos to 2 pg per 50 embryos n 5 3 6 dmPGE2 had minimal effects on the vasculature as shown by flk1 staining Fig 1f i whereas indomethacin slightly altered the intersomitic vessels in 30 15 49 of embryos Fig 1l At 36 h p f live bigenic cmyb gfp lmo2 dsRed green fluorescentprotein labelled HSCs and progenitors red labelled HSCs and endothelium embryos imaged by confocal microscopy exhibited significantly decreased numbers of HSCs yellow following indomethacin treatment and significantly increased HSCs after dmPGE2 exposure Fig 1g j m and Supplementary Fig 1i Quantitative PCR confirmed an enhancement in runx1 and cmyb expression by dmPGE2 whereas indomethacin significantly reduced the expression of each gene Fig 1d To confirm the requirement of PGE2 activity we used morpholino oligonucleotides to knock down expression of Cox1 and Cox2 a low dose 40 mM inhibition of Cox1 minimizes toxicity while mimicking Cox dependent developmental defects6 Morpholino oligonucleotide knockdown of Cox1 Cox2 decreased the levels of prostaglandins and inhibited AGM HSCs Cox1 54 74 Cox2 60 71 Supplementary Fig 1s u The morpholino mediated effects on HSCs were reversed by dmPGE2 Cox1 1 dmPGE2 29 52 rescued Cox2 1 dmPGE2 43 60 Supplementary Fig 1y z a dmPGE2 rescued 25 45 morpholino mediated knockdown of PGE2 synthase 35 50 indicating that PGE2 signalling was sufficient to modulate HSC formation Supplementary Fig 1u b PGE2 signals through receptors Ptger1l Ptger4l ref 8 Morpholino mediated 1 Stem Cell Program and Division of Hematology Oncology Children s Hospital Harvard Stem Cell Institute Harvard Medical School Boston Massachusetts 02115 USA 2Howard Hughes Medical Institute Boston Massachusetts 02115 USA 3Department of Pediatric Oncology Dana Farber Cancer Institute Boston Massachusetts 02115 USA 4Institute for Translational Medicine and Therapeutics University of Pennsylvania School of Medicine Philadelphia Pennsylvania 19104 USA 1007 2007 Nature Publishing Group LETTERS NATURE Vol 447 21 June 2007 knockdown of Ptger2l and Ptger4l diminished runx1 cmyb expression Ptger2l 39 63 Ptger4l 44 67 and was not reversed by dmPGE2 Supplementary Fig 1s t c d