The role of the X chromosome in embryonic and postnatal growth Daniel Mark Snell A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy of University College London. Francis Crick Institute/Medical Research Council National Institute for Medical Research University College London January 28, 2018 2 I, Daniel Mark Snell, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the work. Abstract Women born with only a single X chromosome (XO) have Turner syndrome (TS); and they are invariably of short stature. XO female mice are also small: during embryogenesis, female mice with a paternally-inherited X chromosome (XPO) are smaller than XX littermates; whereas during early postnatal life, both XPO and XMO (maternal) mice are smaller than their XX siblings. Here I look to further understand the genetic bases of these phenotypes, and potentially inform areas of future investigation into TS. Mouse pre-implantation embryos preferentially silence the XP via the non-coding RNA Xist.XPO embryos are smaller than XX littermates at embryonic day (E) 10.5, whereas XMO embryos are not. Two possible hypotheses explain this obser- vation. Inappropriate expression of Xist in XPO embryos may cause transcriptional silencing of the single X chromosome and result in embryos nullizygous for X gene products. Alternatively, there could be imprinted genes on the X chromosome that impact on growth and manifest in growth retarded XPO embryos. In contrast, dur- ing the first three weeks of postnatal development, both XPO and XMO mice show a growth deficit when compared with XX littermates. This deficit is not observed in the presence of a second sex chromosome - i.e. in normal XX female mice, or in females with a Y chromosome that lacks Sry - suggesting haploinsufficiency of genes with homologues present on, and expressed from, both sex chromosomes as a cause. Abstract 4 In this thesis I have investigated the role of Xist in XPO embryonic growth retarda- tion; and utilised mouse stem cells to perform an in vitro screen to identify X-linked imprinted genes. To characterise postnatal haploinsufficiency, I identify four can- didate genes and, utilising CRISPR-Cas genome editing, delineate the role of each in the growth deficit phenotype. I further use these X-linked mutants to investigate the functional divergence of the X and Y chromosomes in the context of postnatal survival. Acknowledgements "There is nothing like looking, if you want to find something. You certainly usually find something, if you look, but it is not always quite the something you were after." J.R.R. Tolkien, The Hobbit, 1937 Whilst this thesis bears my name, undoubtedly I couldn’t have finished it without the generous support, advice and encouragement of many colleagues and friends. Firstly, thanks to my supervisor, James, for agreeing to take a punt on me, probably somewhat unexpectedly, after our initial meeting. I am particularly appreciative of your input during the writing process, during which my ability to construct short, logical, umambiguous sentences has improved immeasurably... Thanks to the whole Turner lab, past and present, for collectively making it a plea- sure to come to work: Andrew, Alex, Ben, Bryony, Charlotte, Elias, Shantha, Taka, and Valdone. I pay particular thanks to Fanny, without whose patient teaching and timely words of encouragement I would not be half the molecular biologist (and person) I am today. I am also grateful to Mahesh for analysing the RNA-seq data, and for helpful discussions regarding data interpretation, and likewise to Jasmin. Many thanks to Oana, who is likely the most conscientious and hard working BSc and MSc project student anyone could ever wish for. The writing of this thesis has coincided with the lab migration from an old, char- acterful asylum in Mill Hill to "Sir Paul’s Cathedral" in central London. I’d like to thank Kathy and the Niakan lab for providing me space to work during the migration Acknowledgements 6 and beyond. I’m particularly indebted to Sissy for many fascinating, wide-ranging conversations, both scientific and philosophical. Thanks to the former Developmental Genetics Division for their scientific advise and weekly cake; and in particular to Christophe for motivational words at key times (and for having very slightly less hair than me at all times). None of the work presented here would have been possible without the support of the Biological Research Facility. In chronological order, thanks to Hannah, Sue, Pete, and Pat for providing excellent husbandry over many years. I’m also majorly indebted to the former Procedural Services Section from Mill Hill: almost with- out exception, Sophie expertly carried out all of the microinjection work for my projects, as well as tolerating my occasional moans when things didn’t go to plan. Thanks must also be extended to Sarah, Marta, Nicolle, and Katharine. From UCL, I will be forever grateful to (now) Emeritus Professor Gordon Stewart for giving me the opportunity to pursue the slightly different MBPhD academic track during medicine. I appreciate the input of my Thesis Committee, Alex Gould, Vivian Li, and Robin Lovell-Badge; and thanks to both Rosetrees Trust and Medical Research Council for funding my work. Thanks to my friends - Jack, Lara, Charlie, Ben, and Saba - for showing great understanding when I’ve either been late or not made it to things because I was in the lab. I can’t promise I’ve quite got the balance right just yet, but I’m making progress. This progress is indisputably the result of the influence of Daria. Thank you for helping me to realise the need to live outside of work, and for encouraging me to do things I never imagined I would a year ago. Thank you for being there, unquestioning; and thank you for tolerating me. Saving the final, biggest acknowledgment until last, I thank my parents, Liz and Mark, for their unconditional love, support, and understanding; not only during the past four years, but the past 32 years. Here is, undoubtedly, the most expensive book you’ll never read. This thesis is dedicated to you both. Contents 1 Introduction 22 1.1 Sex . 22 1.1.1 Sex determination and the evolution of the mammalian sex chromosomes . 23 1.2 The mammalian Y chromosome . 25 1.2.1 The Testis Determining Factor: Sry ............. 25 1.2.2 Transposed and ampliconic genes . 26 1.2.3 X degenerate genes . 26 1.3 The mammalian X chromosome . 27 1.3.1 Sexual antagonism drives X chromosome evolution . 28 1.3.2 Pseudoautosomal region (PAR) . 29 1.4 Dosage compensation . 30 1.4.1 X upregulation(XUR) . 30 1.4.2 X chromosome inactivation (XCI) . 32 1.4.3 Escape from X chromosome inactivation . 42 1.5 Aneuploidy . 43 1.5.1 Sex chromosome aneuplodies . 43 1.5.2 Turner syndrome: XO . 46 1.5.3 XO aneuploid mice . 50 1.6 X-Y gene pairs with candidate roles in postnatal growth . 53 1.6.1 Kdm5c ............................ 53 1.6.2 Kdm5d ............................ 55 Contents 8 1.6.3 Kdm6a ............................ 56 1.6.4 Uty .............................. 59 1.6.5 Eif2s3x ............................ 60 1.6.6 Eif2s3y ............................ 61 1.6.7 Ddx3x ............................. 62 1.6.8 Ddx3y ............................. 64 1.6.9 X-Y gene pairs: summary . 65 1.7 Aims . 67 2 Materials and Methods 68 2.1 Mouse work . 68 2.1.1 Mouse strains . 68 2.1.2 Timed matings . 70 2.1.3 Blastocyst isolation . 70 2.1.4 Isolation of post implantation embryos . 71 2.1.5 Weighing of post implantation embryos . 71 2.1.6 Somite counting of post implantation embryos . 71 2.1.7 Generation of CRISPR targeted lines . 71 2.1.8 Weighing postnatal animals . 72 2.1.9 Identification of individuals . 73 2.2 Molecular biology . 73 2.2.1 Genomic DNA (gDNA) extraction . 73 2.2.2 RNA extraction . 74 2.2.3 cDNA synthesis . 74 2.2.4 Polymerase Chain Reaction (PCR) . 74 2.2.5 Quantitative real time PCR . 75 2.2.6 TA cloning . 76 2.2.7 RFLP digest . 76 2.3 Cytology . 77 2.3.1 Metaphase chromosome spreads for karyotyping . 77 2.3.2 DNA FISH . 78 Contents 9 2.4 Protein extraction and western blotting . 80 2.4.1 Tissue harvest and protein extraction . 80 2.4.2 Protein quantification . 80 2.4.3 Western blotting . 80 2.5 Cell culture . 81 2.5.1 Mouse trophoblast stem cells (mTSCs) . 81 2.5.2 Mouse embryonic stem cells (mESCs) . 83 2.5.3 Derivation of MEFs . 84 2.6 Next generation sequencing . 85 2.6.1 Generation of data to screen CRISPR targeted loci . 85 2.6.2 Data analysis . 86 2.6.3 RNA-seq . 86 2.7 Statistical analysis . 88 2.7.1 Weight data analysis . 88 2.7.2 Postnatal survival analysis . 89 3 Examining the role of Xist in the post-implantation growth deficit ob- served in XPO embryos 90 3.1 Introduction . 90 3.2 Results . 92 3.3 Discussion . 96 3.3.1 Xist coating may not directly result in imprinted XCI . 98 3.3.2 The X chromosome may harbour imprinted genes that are primarily expressed in the extraembryonic tissue . 100 3.3.3 Mouse trophoblast stem cells can be utilised to identify im- printed genes in vitro ..................... 102 4 Using in vitro models to identify candidates for X linked imprinted genes in mouse extraembryonic tissue 104 4.1 Introduction . 104 4.1.1 Genomic imprinting . 104 Contents 10 4.1.2 The placenta .