NOVEL BMP ANTAGONISTS AND NEURAL INDUCTION IN THE MOUSE by Lisa Sue De Boer Emmett A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cell and Developmental Biology) in The University of Michigan 2009 Doctoral Committee: Professor Kathy Sue O‘Shea, Chair Associate Professor Catherine Ellen Krull Assistant Professor Scott E. Barolo Assistant Professor Deneen Wellik © Lisa Sue De Boer Emmett All rights reserved 2009 Dedication To my parents, Robert & Sheryl De Boer, my loving husband, John, and my precious son, Aidan. ii Acknowledgements Foremost, I would like to thank my advisor, Dr. Sue O‘Shea, for all her guidance, assistance, and support during my tenure in her lab. Through her tutelage and supervision, I have gained valuable knowledge and skills, and matured as a scientist. I also would like to thank the current and former members of the O‘Shea lab that I have worked with over these years whom acted as my ―second family‖. Without their support, assistance, and friendship, this effort would not have been achievable. Thanks to my former and current undergrads Alyce Whipp, Meg Barry, Amrita George, Derrick Yang, Eric Kilijanczyk, Christine Belzyt, and Michelle Reames for their valuable assistance during my research. Sincere thanks are extended to my fellow graduate students Nicole Slawny, Yao-Chang Tsan, and Dr. Matt Velkey for their collaboration and peer support. My keen appreciation goes to Dr. Theresa Gratsch for her guidance and instruction during my research. I would also like to extend my deepest gratitude to Dr. Maria Morell for her extensive help with new techniques and solutions for my research, and for her unwavering friendship during these years away from my family. Additionally, I would like to thank the faculty and staff at the University of Michigan that have assisted me during this process, especially my committee members Dr. Scott Barolo, Dr. Catherine Krull, and Dr. Deneen Wellik for their invaluable advice and assistance. My thanks go out to members of the Microscopy and Imaging Laboratory: Chris Edwards, Shelley Almburg, Bruce Donohoe, Dotty Sorenson, and Sasha Meshinchi for their instruction and troubleshooting of MIL equipment. My sincere gratitude goes to Thom Saunders and Maggie Van Keuren from the Transgenic Animal Core for their aid and iii advice in making several transgenic animals, and to Stephanie Schmoll and Dr. Marta Dzaman from the Organogenesis Morphology Core for their assistance in cryosectioning and equipment usage. My acknowledgements would be incomplete without recognizing the people that influenced my early scientific career. I am indebted to my former employers at GlaxoSmithKline, Dr. Karen Augustine-Rauch and Dr. Rogely Boyce, whom gave an inexperienced undergraduate her first opportunity to work in a laboratory. Finally, I would like to thank my family, particularly my parents, Robert and Sherry De Boer, and friends for their understanding and support throughout this process. Without their love and friendship, my achievements would not have been possible. I would like to express my heartiest thanks to Susan Johnson and Matt Velkey for taking the time to edit and format this thesis. Sincere thanks go out to my husband John for not only his patience, but his undying love and encouragement over these years. Lastly, I would like to thank my son Aidan. Not only were you a good baby but the joy that you bring to my life has made this entire process worthwhile. iv Table of Contents Dedication .................................................................................................... ii Acknowledgements ...................................................................................... iii List of Figures ............................................................................................... vi List of Tables ................................................................................................ viii List of Appendices ........................................................................................ ix Abstract…………………………………………………………………………….. x Chapter 1. Introduction …..................................................................................... 1 2. Transgenic Misexpression of Noggin in the Early Postimplantation Mouse Embryo …............................................................................... 57 3. RNA Inhibition of BMP-4 Gene Expression in Postimplantation Mouse Embryos …............................................................................. 100 4. Geminin is Required for Gastrulation in Postimplantation Mouse Embryos …......................................................................................... 116 5. Geminin Controls EMT and Neural Crest Specification in the Mouse Embryo .............................................................................................. 173 6. Summary and Future Directions …..................................................... 186 Appendices 1. Transplacental RNAi: Deciphering Gene Function in the Postimplantation-Staged Embryo ...................................................... 198 2. Identification of Novel Genes in the Mouse Embryo Via Laser Capture Microdissection .................................................................... 231 v List of Figures Figure 1-1 Model illustrating how temporally and spatially distinct Nodal activities pattern the anterior-posterior axis …...……………….. 28 1-2 The Node …………………………………..…….………………… 29 1-3 The primitive streak during gastrulation .................................... 30 1-4 Schematic of gastrulation …...................................................... 31 1-5 The Notochord .......................................................................... 33 1-6 The BMP-Smad pathway…....................................................... 34 1-7 The structural and functional domains of Geminin …................ 35 1-8 Geminin‘s roles during development …..................................... 36 2-1 K14-noggin expression constructs ............................................ 79 2-2 Keratin 14 expression ............................................................... 81 2-3 EGFP expression in transgenic embryos ….............................. 83 2-4 Morphologies of transgenic embryos ….................................... 85 2-5 Scanning electron microscopy ….............................................. 86 2-6 Histological analysis of E7.5 embryos …................................... 88 2-7 Means areas of wild-type and K14-noggin transgenic tissues .. 89 2-8 In Situ hybridization analysis .................................................... 90 3-1 Predicted stem loop .................................................................. 108 3-2 Expression of Bmp-4i or DsRed control .................................... 109 3-3 Immunohistochemical localization of Bmp-4 protein in embryos exposed to pBmp-4i (A-D), and DsRed control embryos (E,F) ........................................................................... 111 3-4 RT-PCR analysis of gene expression in pCS2/Red control and pBmp-4i-exposed embryos….................................................... 112 4-1 Geminin expression in the early postimplantation embryo ....... 144 4-2 Geminin is required for gastrulation .......................................... 146 4-3 Alterations at the Node ….......................................................... 148 4-4 Organization of the primitive streak …....................................... 150 4-5 Geminin is required for anterior patterning …............................ 152 4-6 Geminin is required for patterning the cephalic region ….......... 154 S4-1 Geminin targeting constructs .................................................... 157 S4-2 Geminin is required for axis elongation, closure of the anterior neural folds and branchial arch morphogenesis ....................... 159 S4-3 Geminin expression is significantly reduced in Geminin shRNA embryos ........................................................................ 161 S4-4 Geminin alters neural crest restricted gene expression ............ 162 S4-5 Embryos that survive to E8.75-E9.0 are mispatterned ............. 164 vi List of Figures (continued) Figure 5-1 In situ hybridization of Geminin ................................................ 179 5-2 Geminin controls proliferation and closure of the neural folds .. 180 5-3 Geminin controls expression of Snail1 involved in EMT and neural crest specification …....................................................... 181 5-4 Geminin controls expression of Msx2 involved in neural crest specification and body wall closure……………………………… 182 5-5 Wnt signaling is strongly affected by Geminin dosage………… 183 6-1 Schematic illustrating the overlapping expression patterns at gastrulation…………………………………………………………. 193 A1-1 Tail vein injections …................................................................. 213 A1-2 Effects of geminin shRNA ......................................................... 214 A1-3 Geminin knockdown ….............................................................. 215 A1-4 Nanog knockdown …................................................................. 216 A1-5 Oas1 PCR ................................................................................. 217 A1-6 Effects of Bmp shRNA…........................................................... 219 A1-7 Bmp4 knockdown ….................................................................. 220 A1-8 Longevity of the RNAi ..............................................................