Oxygenated Fatty Acids Enhance Hematopoiesis via the Receptor GPR132 The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Lahvic, Jamie L. 2017. Oxygenated Fatty Acids Enhance Hematopoiesis via the Receptor GPR132. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42061504 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Oxygenated Fatty Acids Enhance Hematopoiesis via the Receptor GPR132 A dissertation presented by Jamie L. Lahvic to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Developmental and Regenerative Biology Harvard University Cambridge, Massachusetts May 2017 © 2017 Jamie L. Lahvic All rights reserved. Dissertation Advisor: Leonard I. Zon Jamie L. Lahvic Oxygenated Fatty Acids Enhance Hematopoiesis via the Receptor GPR132 Abstract After their specification in early development, hematopoietic stem cells (HSCs) maintain the entire blood system throughout adulthood as well as upon transplantation. The processes of HSC specification, renewal, and homing to the niche are regulated by protein, as well as lipid signaling molecules. A screen for chemical enhancers of marrow transplant in the zebrafish identified the endogenous lipid signaling molecule 11,12-epoxyeicosatrienoic acid (11,12-EET). EET has vasodilatory properties, but had no previously described function on HSCs. EET treatment enhanced mouse marrow transplant, and time-lapse imaging showed that this lipid increased HSC specification in zebrafish embryos. These phenotypes were dependent on PI3Kγ signaling as well as AP-1 signaling, which in the zebrafish was specifically required in endothelial cells of the AGM or CHT. EET is known to signal via a G-protein coupled receptor (GPCR), but the identity of this receptor remains unknown, impeding the progress of EET to the clinic and preventing genetic studies of EET signaling. A novel bioinformatic approach identified 10 candidate EET receptors that are expressed in three EET-binding human cell lines, but missing from an EET-non-binding line. Of these, only GPR132 showed EET-responsiveness in vitro. Knockdown of zebrafish gpr132b prevented EET- induced hematopoiesis in the embryo, and marrow from GPR132 KO mice showed a decreased ability to transplant long-term. Others have shown that GPR132 has affinity for a variety of oxygenated fatty acids in vitro. Treatment of zebrafish embryos with these putative GPR132 ligands produced EET-like phenotypes in vivo. Structure-activity-relationship analyses using both in vitro and in vivo assays revealed that a carboxylic acid moiety is required for activity, and oxygenated, unsaturated fatty acids are stronger activators. I have identified GPR132 as an oxygenated fatty acid receptor that mediates both embryonic and adult hematopoiesis. This receptor is a promising target for therapeutic modulation of hematopoiesis and for genetic interrogation of fatty acid signaling. Together these studies reveal the biological and potentially therapeutic importance of lipid signaling molecules and their target GPCRs in regulating stem cell behavior. iii Contents Abstract...................................................................................................................................... iii Acknowledgements ..................................................................................................................... v Chapter 1. Introduction ........................................................................................................... 1 Chapter 2. Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. ..................................................................................................................20 Chapter 3. Oxygenated Fatty Acids Enhance Hematopoiesis via the Receptor GPR132 ......54 Chapter 4. GATA Factor-G-Protein-Coupled-Receptor Circuit Suppresses Hematopoiesis ...81 Chapter 5. Discussion ......................................................................................................... 109 References ............................................................................................................................. 134 Appendix 1: Zebrafish Microarray............................................................................................ 165 Appendix 2: GPCR expression levels ...................................................................................... 167 Appendix 3: Chapter 4 Supplemental Experimental Procedures ............................................. 172 Appendix 4: Digital Supplemental Files ................................................................................... 176 iv Acknowledgements This thesis has been many years in the making and all along I’ve been supported by so many. At Harvard, the BBS office smoothes the many logistical and administrative hurdles, always with a smile. My DAC was full of helpful members- Stu Orkin for gave insightful comments on hematopoiesis, Patricia D’Amore was always ready with tough questions for me and for Len. Charles Serhan turned out to be essential for his expert perspective on lipid biochemistry, and I’m grateful to have learned many new techniques in his lab as well. Most of all, Caroline Burns encouraged me at every step of my graduate career; her advice on my science and professional development has always been spot-on. In the Zon lab, Len has recruited a talented group of positive, collaborative scientists. Ilya Shestapolov provided an upbeat welcome and mentored my rotation project. Pulin Li gave me a whirlwind introduction to her project, and dazzled me with her scientific precision and sheer efficiency. Vera Binder helped me through my first real experience of publishing a paper. Ellen Durand and Alison Taylor served as wise older grad students. Michelle Lin taught me how to do Western blots and Jon Henninger taught me how to properly CRISPR a zebrafish gene, and they, along with John Gansner, served as cheerful baymates and nearby sources of advice. Julie Perlin and Eva Fast became good friends in the lab who I could turn to for discussions about science or life in general. I’ve surely learned from every single Zon lab member, and I’ve been motivated and inspired by the quality work they present. The data I present here relies directly on the work of many people. Kara Maloney and Christian Lawrence kept my fish healthy and thriving. Ron Bernier supported my FACS analysis. Bruce Barut, Dorothy Giarla, and Hannah DiCicco made sure that reagents arrived and meetings got booked. Two summer students, Michael Chase and Olivia Weiss, produced outstanding work. And I’ve had the honor to work with talented technicians who’ve arrived early, stayed late, and followed my project wherever it leads: Michelle Ammerman, and Emma Stillman. I’m so grateful to them, and to Megan Blair, especially for performing the mouse procedures that I couldn’t handle. Lastly, Dr. Len Zon has been an outstanding mentor throughout these years. His advice has been invaluable in designing and executing these projects, and especially in bringing them to the scientific world in the form of papers, presentations, and grants. Len has a deep understanding of stem cell biology that gives him an impressive intuition for explaining results and linking ideas. Len also cares deeply about the v success of his mentees. He spent dozens of hours with me discussing my future, my goals, and how best to reach them. His encouragement, his confidence in my abilities, and his optimistic, can-do attitude about science helped me through the slow months and the dead ends that are inevitable in research. Of course, a lot of life happens during grad school, as well, and many people become dear. Elaine biked the whole Cape with me, through sunshine and rain and flat tires. Marc drove halfway down the coast and back with me in a long weekend, just so we could each see our families for Thanksgiving. Deepali and Evan never hesitated to share their joys or their sorrows. So many other members of my BBS class have been great company- Huixin, Charles, Jennifer, John, Hao, Linda, and many more. I’m grateful too to my fellow DRBers, Adrianna, Olivia, and Tyler. Jamie and Devereux made fabulous roommates and always reminded me of the good old times. My extended circle of friends in the Boston dance and music community, in turn, made sure to remind me that there is life beyond science. I’m grateful to Sharon and Ari for baking, cooking, and plotting the resistance with me. And I’m grateful to Will, who brought me so much light and love, so many hugs and so many new perspectives in this past year. But Harvard, BBS, and science, none of this would have happened without my family, a constant source of support and encouragement. Mom and Dad raised me with a confidence that I could do anything, and with an outgoing, collaborative spirit that has served me well. They sustained me during my graduate career with phone calls, visits, and shipments of baked goods- they once even flew to Boston with frozen fruit cobbler in their suitcase. Grandpa Segler’s Skype calls were always brief, but frequent- he would light up