The Role of the X-Chromosomal Porcupine homolog Gene in Mouse Development by Steffen Biechele A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Steffen Biechele 2013 The Role of the X-Chromosomal Porcupine homolog Gene in Mouse Embryonic Development Steffen Biechele Doctor of Philosophy Department of Molecular Genetics University of Toronto 2013 Abstract WNT ligands are secreted proteins that act as signals between cells. WNTs activate several interconnected signaling pathways that are required for embryonic development as well as tissue homeostasis in adults. The X-chromosomal Porcn gene encodes a membrane-bound O-acyl transferase that is required for the acylation of all 19 WNT ligands encoded in the mammalian genome. Non-acylated WNTs fail to be secreted from the producing cell and thus do not activate downstream signaling targets. In my thesis research, I have investigated the function of Porcn in mouse embryonic development. In vitro, I have shown that Porcn is required for canonical WNT signaling in ES cells and further, for their differentiation into endodermal and mesodermal derivatives. Taking advantage of a mouse line carrying a conditional (floxed) Porcn allele that I have generated, I have focused my studies on the early embryonic roles of Porcn using Cre recombinase-mediated and X chromosome inactivation-based ablation of Porcn function in vivo. I have found that the earliest requirement for Porcn in mouse development is the induction of gastrulation. In contrast to findings from in vitro studies, I have provided evidence that Porcn is not required for pre-implantation development in vivo. Dissecting embryonic and extra- embryonic roles of Porcn, I have been able to show that Porcn is required in the extra-embryonic chorion in order to mediate chorio-allantoic fusion, whereas ablation in the extra-embryonic ii visceral endoderm had no apparent effects. The extra-embryonic requirement for Porcn results in a parent-of-origin effect in Porcn heterozygous females due to X chromosome inactivation. In contrast to the placentation defect causing embryonic lethality of maternal allele mutants, deletion of the paternal allele caused variable fetal defects resulting in perinatal lethality with only rare survivors to adulthood. Both fetuses and adults represent a mouse model for Focal Dermal Hypoplasia (FDH), the syndrome caused by mutations in the human PORCN gene. My studies highlight the importance of PORCN-mediated WNT signaling for gastrulation, placentation, and fetal development, but suggest that endogenous WNT secretion does not play an essential role in either implantation or blastocyst lineage specification. iii Acknowledgments I would like to thank my supervisor, Dr. Janet Rossant for her guidance over the course of my Ph.D, in particular, throughout the ups and downs of the Porcn project. I would also like to thank my supervisory committee Dr. Helen McNeill, Dr. Ian Scott, and Dr. Gordon Keller for seeing me through this endeavour. I am incredibly indebted to Dr. Brian Cox, who has been a brilliant mentor, friend, and organizer of coffee breaks. Another huge thank-you to Jorge Cabezas, who always went above and beyond to help with mouse work. I want to thank Valerie Prideaux for protecting me from bureaucracy and keeping us organized, Andres Nieto for putting up with all my unfiltered thoughts, Dr. Amy Wong for teaching me how to run, Dr. Fredrik Lanner for great advice and for dissociating blastocysts, Dr. Oliver Tam for having an answer for everything, Angela McDonald for being my flow-cytometry princess, Katie Cockburn for being the queen of both blastocysts and reality TV, and Jodi Garner for countless hours of fun in- and outside of tissue culture. Thank you, also, to several unforgettable former lab members: Dr. Amy Ralston, Dr. Cheryle Seguin, Dr. Yojiro Yamanaka, Dr. Peter Rugg-Gunn and Dr. Jon Draper. I cannot express my gratitude enough for everything you all have done. The vast majority of my project involved mice and all imaginable aspects of mouse generation and breeding, which would have been impossible without the expert help of Sue MacMaster, Sandra Tondat, Linda Wei, Dr. Monica Perreira and Marina Gertsenstein from the Transgenic Core. I would like to acknowledge the Toronto Centre for Phenogenomics, specifically, Emi Yano, Betty-Jo Edgell, and Shella Paje. As well, I would like to thank the members of the Centre for Modeling Human Disease; Dr. Ann Flenniken, Igor Vukobradovic, Lois Kelsey, Zorana Berberovic, Celeste Owen, Lily Morikawa, and Dr. Susan Newbigging. A special thank-you to Dr. Hibret Adissu who was invaluable in analyzing, interpreting and discussing phenotypes. Last, but not least, I want to thank my friends and family. Thank you for sharing joy and pain – even when you did not know what I was talking about. Finally, thank you to my family for their endless encouragement, support and faith in me. Danke. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi List of Abbreviations ................................................................................................................... xiv 1 Introduction ................................................................................................................................ 1 1.1 The WNT family of secreted signaling molecules ............................................................. 1 1.2 WNT ligand biogenesis ....................................................................................................... 2 1.2.1 Post-translational lipid-modification ...................................................................... 4 1.2.2 Post-translational glycosylation .............................................................................. 5 1.3 The importance of PORCN for WNT ligand biogenesis .................................................... 5 1.4 Specialized mechanisms for WNT secretion ...................................................................... 7 1.5 Receptors and pathways activated by WNT ligands ........................................................... 8 1.5.1 The FZD receptor family ...................................................................................... 10 1.5.1.1 Heterodimeric FZD/LRP complexes activate the canonical, Beta- Catenin-mediated WNT signaling pathway ........................................... 10 1.5.1.2 Non-canonical use of FZD receptors ...................................................... 11 1.5.1.3 FZD receptor regulation by R-spondin signaling ................................... 13 1.5.2 ROR receptor family ............................................................................................. 13 1.5.3 RYK receptor ........................................................................................................ 14 1.6 WNT ligands - conserved secretion & complex response ................................................ 14 1.7 WNT signaling in mouse embryogenesis ......................................................................... 16 1.7.1 WNT signaling in pre-implantation development ................................................ 19 1.7.2 WNT signaling in post-implantation development ............................................... 21 1.8 WNT and PORCN mutations in human disease ................................................................ 24 v 1.9 Thesis Research ................................................................................................................ 24 2 Porcupine homolog is required for canonical WNT signaling and gastrulation in mouse embryos .................................................................................................................................... 26 2.1 Contributions ..................................................................................................................... 26 2.2 Abstract ............................................................................................................................. 26 2.3 Introduction ....................................................................................................................... 26 2.4 Results ............................................................................................................................... 27 2.4.1 Porcn expression analysis in the peri-gastrulation mouse embryo ....................... 27 2.4.2 Porcn genetrap ES cells exhibit defects in canonical WNT signaling ................. 29 2.4.3 Normal NODAL secretion and signaling in Porcn genetrap ES cells .................. 32 2.4.4 Porcn null epiblasts fail to differentiate and establish anterior-posterior identity .................................................................................................................. 33 2.4.5 Porcn null ES cells fail to differentiate into mesoderm and endoderm derivatives in vitro ...............................................................................................