MORPHOLOGY, BEHAVIOR, AND THE SONIC HEDGEHOG PATHWAY IN MOUSE MODELS OF DOWN SYNDROME by Tara Dutka A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July, 2014 © 2014 Tara Dutka All Rights Reserved Abstract Down Syndrome (DS) is caused by a triplication of human chromosome 21 (Hsa21). Ts65Dn, a mouse model of DS, contains a freely segregating extra chromosome consisting of the distal portion of mouse chromosome 16 (Mmu16), a region orthologous to part of Hsa21, and a non-Hsa21 orthologous region of mouse chromosome 17. All individuals with DS display some level of craniofacial dysmorphology, brain structural and functional changes, and cognitive impairment. Ts65Dn recapitulates these features of DS and aspects of each of these traits have been linked in Ts65Dn to a reduced response to Sonic Hedgehog (SHH) in trisomic cells. Dp(16)1Yey is a new mouse model of DS which has a direct duplication of the entire Hsa21 orthologous region of Mmu16. Dp(16)1Yey’s creators found similar behavioral deficits to those seen in Ts65Dn. We performed a quantitative investigation of the skull and brain of Dp(16)1Yey as compared to Ts65Dn and found that DS-like changes to brain and craniofacial morphology were similar in both models. Our results validate examination of the genetic basis for these phenotypes in Dp(16)1Yey mice and the genetic links for these phenotypes previously found in Ts65Dn , i.e., reduced response to SHH. Further, we hypothesized that if all trisomic cells show a reduced response to SHH, then up-regulation of the SHH pathway might ameliorate multiple phenotypes. We crossed Ts65Dn mice with Ptch1tm1Mps/+ mice, which up-regulate the canonical SHH pathway through the loss of function of one Ptch1 allele. The resulting four mouse genotypes were examined for craniofacial, behavioral and brain phenotypes. We found that Ptch1 heterozygotes displayed craniofacial and behavioral phenotypes that were mostly distinct from the effects of trisomy rather than complementary. However, as predicted some brain structural deficits were ameliorated in the Ts65Dn Ptch1 heterozygotes. ii Our studies comprise the first evaluation of Dp(16)1Yey morphology to confirm it as a DS model and our additional DS model investigations indicate a more complex pathogenicity for SHH related phenotypes in DS than constitutive uniform pathway repression. Readers: Dr. Roger Reeves, Advisor Dr. Mikhail Pletnikov, Thesis Committee Member iii Acknowledgements I am extremely grateful to the many people who have provided their guidance and support over the last six years while I have toiled toward completing this doctoral research. First I would like to thank my advisor Dr. Roger Reeves. Though I have been a member of his lab for a relatively short period, just about three and a half years, I have learned so much in that time. Roger taught me not only to execute experiments, but to think critically about what question I was asking. He also encouraged me to learn as much as I could and come at a problem from every angle, as least until the grants run out. I will also always be grateful to Roger for turning what could have been a huge upset in my academic career into a great opportunity. I would like to thank the members of my thesis committee, Dr. Jonathan Pevsner, Dr. William Pavan, and Dr. Mikhai Pletnikov for their tireless efforts to make my research the best it could be. Specifically I would like to thank: Dr. Pevsner for encouraging me to really learn and understand statistics to a far greater depth than I might have done on my own; Dr. Pavan for his insights into the SHH pathway and the genetics underlying my experiments; and Dr. Pletnikov for helping in the design and analysis of all of my behavioral tests. Many others also contributed to the completion of this body of work. I am thankful for our wonderful collaborators at Penn State University, Dr. Joan Richtsmeier, her post-doc Dr. Nandini Singh, and her former student Dr. John Starbuck who completed all of the craniofacial analyses described in this work. The microCT images utilized in these craniofacial studies were completed with the assistance of the Johns Hopkins Small Animal Imaging Resource Program. Additionally, my behavioral test would not have been possible without the help of Joshua Crawford and all the members of the Johns Hopkins School of Medicine Rodent Behavior Core who aided in designing my behavior tests, trained me to use the core, and kept all equipment running. iv I want to thank all the members of the Reeves’ lab for making our lab such a great environment to work in. I always had someone willing to help with my behavioral tests, which made them significantly shorter and kept me from loosing mice and my sanity. Without Benjamin Devenney, who taught me to handle all the mice without fear and handled them for me when they were too fast for me to catch I do not think I would have ever finished one behavior test, let alone six sets. I would also like to thank Dr. Donna Klinedinst who answered every question I had about previous lab experiments, helped run several weeks of rotarod, and started the Ptch mouse colony for me. My fellow students, Dr. Sarah Edie, Dr. Renita Polk, Jennifer Poitras, Dr. Annan Yang, and Duane Currier, made the lab a great place to be, with lots of good times and good beer, and all helped run at least one test for me. I would also like to thank our post-docs Dr. Huiqing Li and Dr. Fabian Fernandez for their valuable scientific insight. I would also like to thank the former lab members, Dr. Randall Roper, Dr. Lisa Olson and Dr. Laura Baxter for aiding me in deciphering their experiments and notes from their days in the lab. Finally I would like to thank my undergrad intern Tabetha Ratliff who completed many days of tedious genotyping and imaging for this project and was a joy to work with. I could not have completed this work without the Human Genetics graduate program which has been my home for the last 6 years. Dr. Dave Valle, Dr. Kirby Smith and Dr. Andy McCallion have been wonderful mentors and worked very hard to make our program as rigorous and fulfilling as possible. Sandy Muscelli, our program administrator, kept the whole thing running and always had candy. My fellow students and all the friends I have made in the program made Hopkins a great place to be. I would like to thank all my mentors in science without whom I could never have reached this goal. I had many wonderful instructors, first in the Montgomery County Public Schools, then at University of Maryland, and finally here at Hopkins. I also had the privilege of working with some truly excellent mentors during my three internships each of which taught me new exciting aspects of research: Dr. Minoru Ko, Dr. Mark Carter, Dr. Sarah Via, Dr. Roger Woodgate, and v Dr. John McDonald. Finally I would like to thank my advisors from my three rotations at Hopkins, Dr. Steve Leach, Dr. Susan Michaelis, and Dr. Josh Mendel for their time and effort in training me to be a better geneticist. I would not have made it to Hopkins and through this thesis without the love and support of my friends and my huge family. My dear friends outside of science, especially Carmel, have listened to me complain and forced me to relax. The close friends I have made at Hopkins, especially Sarah, Renita and Sam, have helped me trouble shoot my projects and commiserated when I couldn’t get something to work. My siblings, Caitie, Tim and Meg, have cheered me on and cheered me up. My paternal grandparents, Grandmommy and Granddaddy, Maxwell and Lee Howard, were so proud to have me attend Granddaddy’s alma mater. I wish they could have seen this dream completed. My maternal grandparents, Grandma and Grandfather, Mary and James Flaherty, have always listened to everything I had to say no matter if they understood me or not and I will be forever grateful for that safe space. My mom, Mary Howard, has been my inspiration my whole life. She showed me what smart, strong women could do and remains to this day one of the smartest people I have ever met. My dad, Regan Howard, helped inspire my love of science and showed me how to sustain joy in science and discovery. I hope to still love it as much as he does when I reach his age. Last but not least I would like to dedicate this thesis to my husband Mike Dutka. He has been my anchor in the graduate school sea of insanity. He has stuck by me through this entire grueling process while he worked on his own thesis. He made sure I ate when I would have forgotten and cheered me up when everything exploded. He was the best and easiest decision I made in the last six years and I don’t know what I would have done without him. vi Table of Contents Page Title…………………………………………………………………………………………i Abstract…………………………………………………………………………………......ii-iii Acknowledgements………………………………………………………………………....iv-vi Table of Contents…………………………………………………………………………...vii-x List of Tables……………………………………………………………………………….xi List of Figures………………………………………………………………………………xii-xiii Chapter 1: Introduction……………………………………………………………………..1-15 Down Syndrome…………………………………………………………………...1-2 Mouse Models……………………………………………………………………..2-4 The Cerebellum in DS……………………………………………………………..5-9 The SHH Pathway…………………………………………………………………9-12 Connection of Central DS Phenotypes to SHH …………………………………...13-14 Treatment of the Cerebellum with SAG…………………………………………...14 Thesis ……………………………………………………………………...............15 Chapter 2: Overlapping Trisomies for Human Chromosome