Role of CHD7 in neural development and maintenance by Wanda Sherrie Layman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Human Genetics) in The University of Michigan 2011 Doctoral Committee: Associate Professor Donna M. Martin, Chair Professor Sally A. Camper Professor Jeffrey W. Innis Professor Miriam H. Meisler Associate Professor Jeffrey R. Martens 1 © Wanda Sherrie Layman 2011 2 Dedication To my Bear and cub, with your love and support all things are possible… ii Acknowledgements First, I would like to thank Donna Martin for her mentorship and support. I have benefitted greatly from having you as my mentor. I would also like to thank the members of the Martin lab. Liz, you were definitely the best half of the CHD7 dynamic duo. I’m not sure I would have survived the last several years in the lab without you. Jennifer, Mindy, Jillian, and Joe – thank you for all of your help and support. I also need to thank Jeff Martens and some former members of the Martens lab, Dyke and Paul. When I first started this project, I knew absolutely nothing about olfaction or olfactory tissues. My thesis project was greatly influenced by your help and support. Many individuals contributed to making this work possible. I am grateful to the members of my thesis committee Sally Camper, Jeff Innis, Miriam Meisler, and Jeff Martens for their support and guidance. I could not have found better scientists and mentors to guide me through this project. Yehoash Raphael and the Raphael lab were also incredibly beneficial to this work. Thanks also to the Program in Biomedical Sciences (PIBS), the Human Genetics department, and the University of Michigan for taking on a graduate student that did not have the best grades, test scores, or experience. Finally, I would like to thank my family. Brian and Xavier, I don’t know how you put up with me all of these years, but I would never have continued on to this point in my life without both of you. Thank you Mom, words cannot express how much I respect iii and love you. Dad, I guess that I will finally be Dr. Wanda; I wish you could have lived to see it happen. iv Table of Contents ontents Dedication .......................................................................................................................... ii Acknowledgements .......................................................................................................... iii List of Figures ................................................................................................................. viii List of Tables ..................................................................................................................... x Abstract ............................................................................................................................. xi Chapter 1 Introduction....................................................................................................... 1 Abstract ........................................................................................................................... 1 Introduction ..................................................................................................................... 2 CHD7 mutations cause CHARGE syndrome ................................................................. 3 Mouse models of CHARGE and tissue specific defects ................................................. 4 Inner ear abnormalities in humans and mice with CHD7 deficiency ............................. 5 Impaired olfaction in humans and mice with CHD7 deficiency..................................... 6 CHD7 and other CHD proteins: classification and characteristics ................................. 9 CHD7 binding sites and interacting proteins ................................................................ 10 Summary and future studies.......................................................................................... 11 v Chapter 1 Notes............................................................................................................. 12 References ..................................................................................................................... 20 Chapter 2 Defects in neural stem cell proliferation and olfaction in Chd7 deficient mice indicate a mechanism for hyposmia in human CHARGE syndrome ............................... 35 Abstract ......................................................................................................................... 35 Introduction ................................................................................................................... 36 Results ........................................................................................................................... 38 Discussion ..................................................................................................................... 49 Materials & Methods .................................................................................................... 55 Chapter 2 Notes............................................................................................................. 63 References ..................................................................................................................... 75 Chapter 3 Reproductive dysfunction and decreased GnRH neurogenesis in a mouse model of CHARGE syndrome .......................................................................................... 88 Abstract ......................................................................................................................... 88 Introduction ................................................................................................................... 89 Results ........................................................................................................................... 90 Discussion ................................................................................................................... 100 Materials and Methods ................................................................................................ 105 Chapter 3 Notes........................................................................................................... 112 References ................................................................................................................... 119 vi Chapter 4 CHD7 deficient mice have defects in neural stem cell maintenance and cell fate................................................................................................................................... 137 Introduction ................................................................................................................. 137 Results ......................................................................................................................... 139 Discussion ................................................................................................................... 143 Materials and Methods ................................................................................................ 145 References ................................................................................................................... 155 Chapter 5 Conclusion .................................................................................................... 164 CHD7 in olfactory tissues ........................................................................................... 164 CHD7 and reproductive dysfunction .......................................................................... 169 vii List of Figures Figure 1.1 Chd7 is expressed in CHARGE related tissues during development ............ 13 Figure 1.2 Model of inner ear development in wild type and Chd7 heterozygous mutant mice. .................................................................................................................................. 14 Figure 1.3 Model of olfactory function in wild type and Chd7Gt/+ mice ........................ 15 Figure 1.4 The chromodomain family of proteins contains 9 members that are subdivided into 3 classes on the basis of shared protein motifs. ...................................... 16 Figure 1.5 Model for CHD7 transcriptional regulation .................................................. 17 Figure 2.1 Chd7Gt/+ mice have severely impaired olfaction. ......................................... 64 Figure 2.2 Chd7 is expressed in developing and mature olfactory tissues. ................... 65 Figure 2. 3 Chd7 is expressed in basal stem cells in the mature olfactory epithelium. .. 66 Figure 2.4 Chd7 mutant mice have olfactory bulb hypoplasia ....................................... 67 Figure 2.5 Olfactory sensory neurons and sustentacular cells are physiologically intact in early postnatal Chd7Gt/+ mice ....................................................................................... 68 Figure 2.6 Components of the olfactory cilia are intact in Chd7Gt/+ mice ...................... 69 Figure 2.7 Olfactory sensory neurons in Chd7Gt/+ mutant mice are disorganized and reduced in number............................................................................................................. 70 Figure 2.8 Olfactory epithelial basal cell proliferation is reduced in Chd7Gt/+ mice. ..... 71 viii Figure 2.9 Chd7Gt/+ mice exhibit altered regeneration of olfactory sensory neurons in the olfactory epithelium after chemical ablation. ..................................................................