THE GENETIC ARCHITECTURE OF THE DDK SYNDROME: AN EARLY EMBRYONIC LETHAL PHENOTYPE IN THE MOUSE Folami Yetunde Ideraabdullah A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology. Chapel Hill 2007 Approved by: Fernando Pardo-Manuel de Villena, PhD Scott Bultman, PhD Beverly Koller, PhD Karen Mohlke, PhD David Threadgill, PhD ABSTRACT Folami Yetunde Ideraabdullah: The Genetic Architecture of the DDK Syndrome: An Early Embryonic Lethal Phenotype in the Mouse (Under the direction of Fernando Pardo-Manuel de Villena) The DDK syndrome is a polar early embryonic lethal phenotype that occurs when DDK females are mated to males of other inbred mouse strains. Lethality is parent of origin dependent and results from an incompatibility between an ooplasmic DDK factor and a non- DDK paternal gene, both of which map to the Ovum mutant (Om) locus on chromosome 11. Here, I utilize naturally occurring genetic variation in classical and wild-derived inbred strains to characterize the genetic architecture of the DDK syndrome. I show that genetic variation among wild-derived strains is uniformly distributed and significantly higher than previously reported for other mammalian species. The high levels of diversity present among laboratory strains suggests that the effective population size of the Mus lineage has been relatively large and constant over a long period of time. Overall, these findings demonstrate that wild- derived inbred strains are a valuable resource for genetic studies. By utilizing this resource in recombination mapping and association mapping experiments, we have reduced the candidate interval for the paternal gene of the DDK syndrome to a 23 kb region encompassing a single gene. We have also defined a candidate interval for the gene encoding the maternal factor, and demonstrated that the maternal and paternal components of the DDK syndrome are non-allelic. I have identified three Mus musculus domesticus wild- derived strains carrying modifiers that completely rescue the DDK syndrome lethality. In at ii least two of these strains, the major modifier loci are unlinked to Om and rescue lethality in a parent of origin dependent manner that is independent of allelic exclusion at Om. Taken together, these data reveal that the DDK syndrome requires a specific combination of alleles at multiple loci. The fact that all of these alleles, with the exception of the allele encoding the maternal DDK factor, segregate in natural populations of mice suggests that they may be part of an important molecular pathway. In conclusion, further characterization of the genes responsible for this rescue phenotype will not only provide significant insight into the DDK syndrome, it should also increase our understanding of the molecular framework underlying early mammalian development. iii DEDICATION To my parents for overcoming the odds and living their lives as an inspiration to others; and to my sister and three brothers for making me tough and believing in me. iv ACKNOWLEDGEMENTS First and foremost, I would like to acknowledge my faculty advisor, Fernando Pardo- Manuel de Villena for his dedication to teaching, as well as his support, guidance, patience, and active involvement in the studies described herein. I would like to thank all those in and around the lab who not only made this work possible but also made my time spent in the lab quite enjoyable, especially Timothy Bell, Kuikwon Kim, and Clemencio Salvador. Furthermore, I would like to express my deepest gratitude to Kirk Wilhelmsen, Daniel Pomp, Ethan Lange and Leslie Lange who were gracious enough to lend their know-how to these projects, as well as Elena de la Casa-Esperón and Carmen Sapienza for their enthusiasm and willingness to collaborate and offer advice over the years. Finally, I would like to thank my committee members, Beverly Koller, Scott Bultman, Karen Molke and David Threadgill for their guidance, and Sylvia Frazier-Bowers for all of her support and encouragement throughout the course of my dissertation. v TABLE OF CONTENTS LIST OF TABLES .................................................................................................................. x LIST OF FIGURES ............................................................................................................... xi LIST OF ABBREVIATIONS..................................................................................................xiii Chapter: 1. BACKGROUND AND INTRODUCTION I. Specific aims........................................................................................................1 II. Early embryonic mouse development ...................................................................2 a) Mammalian gametes ........................................................................................2 b) Fertilization ......................................................................................................3 c) Preimplantation development ...........................................................................4 d) Genetic and epigenetic interactions in the preimplantation embryo ...................6 III. The DDK syndrome..............................................................................................7 a) Timing of lethality .............................................................................................9 b) Physiological cause of lethality ....................................................................... 10 c) Genetic basis of lethality… ............................................................................. 11 d) Mapping genetic components of the DDK syndrome....................................... 13 e) Modifiers of the DDK syndrome ...................................................................... 19 f) Relevance of the DDK syndrome..................................................................... 20 IV. Laboratory inbred mouse strains as tools for genetic studies............................... 21 a) Classical inbred strains................................................................................... 22 vi b) Wild-derived inbred strains ............................................................................. 23 c) Haplotype structure of inbred strains............................................................... 25 2. CHARACTERIZING GENETIC DIVERSITY AMONG WILD-DERIVED INBRED STRAINS................................................................................................... 27 I. Frequency and distribution of sequence variants ................................................ 27 II. Analysis of insertion/deletions............................................................................. 33 III. Strain distribution patterns.................................................................................. 35 IV. Phylogenetic history of genetic variation found in Mus musculus......................... 37 V. Discussion ......................................................................................................... 39 3. HIGH RESOLUTION MAPPING OF THE PATERNAL GENE OF THE DDK SYNDROME............................................................................................ 47 I. Mapping strategies............................................................................................. 47 II. Recombination mapping..................................................................................... 48 III. Association mapping using recombinant males................................................... 48 IV. Characterization of the paternal gene compatibility of various inbred strains ..................................................................................................... 53 V. Association mapping using inbred strains ........................................................... 54 VI. Reducing the candidate interval for the gene encoding the maternal factor ......... 58 VII. Discussion ......................................................................................................... 58 a) Mapping the DDK syndrome paternal gene candidate region.......................... 59 b) Evolutionary history of the Om region and the DDK syndrome ........................ 62 4. MODIFIERS OF THE DDK SYNDROME .................................................................. 67 I. A sensitized screen reveals modifiers that completely rescue the DDK syndrome lethality............................................................................................ 67 II. A major modifier locus maps to proximal chromosome 13................................... 69 a) Rmod1........................................................................................................... 71 b) Rmod2........................................................................................................... 73 vii III. Rescue is independent of allelic exclusion at Om................................................ 73 IV. Parent of origin dependent rescue of lethality by PERA or PERC alleles at Rmod1 and Rmod2 ............................................................................. 73 V. Homozygosity for C57BL/6 alleles at Rmod2 is associated with an increase in DDK syndrome lethality ............................................................... 77 VI. Discussion ........................................................................................................