UC San Diego UC San Diego Electronic Theses and Dissertations Title Wnt Signaling and Developmental Logic in Human Stem Cell Populations Permalink https://escholarship.org/uc/item/8q9928zm Author Huggins, Ian Joseph Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Wnt Signaling and Developmental Logic in Human Stem Cell Populations A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Ian Joseph Huggins Committee in Charge: Professor Karl Willert, Chair Professor Jerold Chun Professor Terry Gaasterland Professor Larry Goldstein Professor Alysson Muotri 2016 Copyright Ian Joseph Huggins, 2016 All rights reserved. Signature Page The Dissertation of Ian Joseph Huggins is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2016 iii DEDICATION Dedication To Sara You are ever the love of my life and the song of my heart. To my children You are my greatest delight and hope for the future. To my parents, Jennie and Mark For your unfailing love and unshakeable confidence in me. To my Grandma, Marian Budge Who read to a little boy by night and taught him to love raccoons, squirrels, honeybees, hummingbirds and everything under the sun. To my Grandma, Mary Dawn Huggins Whose loving words and deeds are with me always. iv EPIGRAPH Epigraph UNLESS someone like you cares a whole awful lot, nothing is going to get better. It's not. Dr. Seuss v TABLE OF CONTENTS Table of Contents Signature Page .......................................................................................................... iii Dedication .................................................................................................................. iv Epigraph .................................................................................................................... v Table of Contents ....................................................................................................... vi List of Abbreviations ................................................................................................. viii List of Figures ........................................................................................................... xii List of Tables ............................................................................................................ xiv Acknowledgments ..................................................................................................... xv Vita......................................................................................................................... xviii Abstract of the Dissertation ...................................................................................... xix Chapter 1 - Introduction .............................................................................................. 1 1.1 Defining the role of Wnt signaling and SP5 in hPSCs ....................................... 2 1.2 hPSCs and their derivatives .............................................................................. 3 1.3 Wnt Signaling ................................................................................................. 10 1.3.1 Wnt signaling in development and stem cells ........................................... 10 1.3.2 Genetics and Biochemistry of the Wnt Pathway ....................................... 15 1.3.3 Wnt signaling in cancer ............................................................................ 18 1.4 SP5................................................................................................................. 23 1.4.1 Introduction .............................................................................................. 23 1.4.2 SP5 is a developmentally important Wnt target ........................................ 30 1.4.3 SP5 in disease and cancer ....................................................................... 36 1.4.4 SP5 in stem cells ...................................................................................... 40 Chapter 2 - The role of the transcription factor SP5 in human pluripotent stem cells 43 2.1 Abstract .......................................................................................................... 44 2.2 Introduction ..................................................................................................... 45 2.3 Results ........................................................................................................... 47 2.3.1 RNA-Seq identifies differentially expressed genes following Wnt3a treatment ........................................................................................................... 48 2.3.2 Global decrease in SP1 binding following Wnt treatment of hPSCs .......... 52 2.3.3 SP5 drives decreased expression of known Wnt antagonists ................... 55 2.3.4 SP5 and AXIN2 are differentially induced ................................................. 59 2.3.5 SP5 expression impairs endoderm formation through enhanced Wnt signaling ............................................................................................................ 61 2.4 Discussion ...................................................................................................... 64 vi Chapter 3 - Effects of Wnt signaling on gene expression in human pluripotent stem cells .......................................................................................................................... 72 3.1 Abstract .......................................................................................................... 73 3.2 Introduction ..................................................................................................... 74 3.3 Results ........................................................................................................... 76 3.3.1 Morphogen effects of Wnt signaling on hPSC pluripotency and differentiation .................................................................................................... 76 3.3.2 Wnt pathway activation modulates EGR1 expression in hPSCs ............... 83 3.4 Discussion ...................................................................................................... 92 Chapter 4 - Effects of Wnt signaling on gene expression in human neural stem cells ............................................................................................................................... 100 4.1 Abstract ........................................................................................................ 101 4.2 Introduction ................................................................................................... 101 4.3 Results ......................................................................................................... 105 4.3.1 Upregulation of PAX3 and downregulation of PAX6 following Wnt3a Treatment ....................................................................................................... 105 4.3.2 RNA-Seq identifies differentially expressed genes following Wnt3a treatment ......................................................................................................... 107 4.3.3 Effect of Wnt3a treatment on proliferation and multipotency gene expression following FGF withdrawal .............................................................. 109 4.3.4 The role of SP5 in neural differentiation .................................................. 114 4.4 Discussion .................................................................................................... 121 Chapter 5 - Conclusions and future directions ........................................................ 127 Materials and Methods ........................................................................................... 134 References ............................................................................................................. 143 vii LIST OF ABBREVIATIONS List of Abbreviations AAV Adeno-associated Virus APC (Fluorophore) Allophycocyanin APC (Protein) Adenomatous Polyposis Coli bp Base Pairs Btd Buttonhead Buf Wnt Buffer cDNA Complementary Deoxyribonucleic Acid ChIP Chromatin Immunoprecipitation ChIP-Seq Chromatin Immunoprecipitation followed by Sequencing CHIR Small Molecule Glycogen Synthase Kinase Inhibitor CHIR98014 CM Conditioned Medium cm Centimeter CNS Central Nervous System Cre-ER Cre Recombinase Estrogen Receptor Fusion Protein CRISPR Clustered Regularly Interspersed Palindromic Repeats DE Definitive Endoderm Del Deletion dHet Deletion Heterozygote DMEM Dulbecco's Modified Eagle's Medium DMSO Dimethylsulfoxide DNA Deoxyribonucleic Acid dPBS Dulbecco's Phosphate Buffered Saline DVL Dishevelled viii E8 Essential 8 Culture Medium EGFP Enhanced Green Fluorescent Protein EGR Early Growth Response En Embryonic Day n, where n is the number of days F12 Ham's F12 Medium FACS Fluorescence-Activated Cell Sorting FBS Fetal Bovine Serum FGF Fibroblast Growth Factor FLAG FlagTM Peptide FZD Frizzled GEO Gene Expression Omnibus GFP Green Fluorescent Protein GIMP Gnu Image Manipulation Program GSK Glycogen Synthase Kinase GST Glutathione S Transferase Alpha Peptide H1 WA01 (H1) Human Embryonic Stem Cell Line H9 WA09 (H9) Human Embryonic Stem Cell Line hESC Human Embryonic Stem Cell hESC-CM Human Embryonic Stem Cell Conditioned Medium hiPSC Human