GENETICS AND GENOMICS OF MAMMALIAN PIGMENT PATTERNS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF GENETICS AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Lewis Zuocheng Hong August 2011 © 2011 by Zuocheng Lewis Hong. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/jx191nt1141 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Gregory Barsh, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Andrew Fire I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. David Kingsley I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Arend Sidow Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii iv ABSTRACT Hundreds of coat color loci have been cloned and studied in the laboratory mouse, and most aspects of the mouse pigmentary system are conserved in other mammals. However, comparative zoologic studies suggest that some components of mammalian pigmentation are not represented as coat color mutations in laboratory mice. This dissertation aims to advance our molecular understanding of mammalian pigmentation, and strives to identify novel components of the genetic toolkit involved in generating the diverse array of coat color patterns found in nature. In the Syrian hamster, X-linked inheritance of the Sex-linked yellow (Sly) mutation results in irregular patches of black and yellow hair in heterozygous females. Female- specific variegation of black and yellow coat color is also present in the domestic cat, but curiously absent from other vertebrate species. Using a combination of comparative genomics and linkage mapping, I made key contributions towards defining a candidate interval for the Sly mutation and discovering that the hamster and cat mutations lie in non-homologous regions of the X chromosome. Periodic color patterns on animal skin, such as cheetah spots and zebra stripes, are evident across the mammalian phylogeny, but their molecular basis remains a mystery. Taking advantage of improvements in ultra high-throughput sequencing technologies, I developed a methodology called EDGE, or EcoP15I-tagged Digital Gene Expression, that is well suited for gene expression profiling in non-model organisms. Applying EDGE to black spot and yellow background regions of cheetah skin, I discovered that localized differences in Endothelin-3 expression is likely to be responsible for maintaining black spotting through a pigment-type switching mechanism in hair follicle melanocytes. The use of EDGE and other genomic approaches to generate comparative molecular profiles of mammalian skin patterns is likely to open new avenues of research into mammalian pigment patterning and initiate a molecular renaissance for a set of questions that has mostly resided in the area of theoretical biology for the past few decades. v ACKNOWLEDGEMENTS First and foremost, I would like to thank my thesis advisor, Greg Barsh, for creating a supportive and collaborative environment within the laboratory for me to conduct my thesis research. His visionary leadership and unwavering support during my PhD training was instrumental in the synthesis of this dissertation. Greg’s positive attitude, magnanimity, scientific integrity and superb analytical skills have provided me with an excellent example to follow in my future career. I would like to thank other members of my thesis committee, including Andy Fire, Arend Sidow and David Kingsley, for their guidance and support throughout various stages of graduate school. Past and present members of the Barsh laboratory have also played immensely valuable roles in my scientific training. In particular, I would like to thank Chris Kaelin and Kelly McGowan for their friendship, support and advice throughout graduate school, Hermie Manuel and Stephen Clark for technical assistance, many other members of the Barsh laboratory for their friendship, including Tovi Anderson, Nanibaa’ Garrison and Shiliang Shen, and everyone in the laboratory for contributing towards a collaborative and pleasant working environment. Of course, research rarely happens in isolation, and the work presented in this dissertation would not have been possible without the tireless efforts of many contributors and collaborators. I would like to thank Azita Alizadeh for establishing the Syrian hamster colony that was used to identify the Sex-linked yellow mutation and her heroic efforts in developing molecular markers and a linkage map for the hamster X chromosome. I would also like to thank Chris Kaelin for his advice during the technical development of EDGE. For their help in generating sequencing data, I would like to thank Cheryl Smith, Ziming Weng and Phil Lacroute (under the supervision of Arend Sidow at Stanford University), as well as Flo Pauli, Jason Dilocker, Mike Muratet, Barbara Pusey, K-T Varley and Stephanie Parker (under the supervision of Rick Myers at the HudsonAlpha Institute for Biotechnology). I am indebted to Daniela Witten and Jun Li for their assistance in statistical analysis of transcriptomic and vi epigenomic data. The collection of appropriate skin samples from mammals with pigment patterns was critical towards the success of my research projects. To that end, I am especially grateful to the unselfish efforts of Nancy Nunke and Anne Schmidt- Küntzel for collecting zebra and cheetah skin samples, respectively. I would also like to thank several members of Steve O’Brien’s laboratory—Marilyn Raymond, Victor David, Melody Roelke and Joan Pontius—for providing biological samples and unpublished data during our collaboration on projects on felid coat patterning projects, Wes Warren for sharing unpublished data from the domestic cat transcriptome assembly, and Jim Mullikin for providing access to the unpublished high-coverage domestic cat genome assembly. I would also like to thank faculty members, administrators and students of the Genetics Department for making my experience in graduate school extremely stimulating and enjoyable. Additionally, I am grateful to the Genetics department, the Office of Technology Licensing (Stanford Graduate Fellowship) and the Genentech Foundation for financial support towards my graduate student stipend and tuition fees. Finally, I would like to extend my heartfelt gratitude to my parents, for their tireless support and sacrifice so that I could leave Singapore to pursue an education in the United States, for encouraging me to pursue my interests, and for providing me with timely advice at important junctures of my life. My wife, Yin Nah, has also been an unfailing pillar of support and encouragement throughout graduate school. Her endless optimism and enthusiasm for my research gave me the inspiration and strength to complete this dissertation. vii TABLE OF CONTENTS Abstract ......………………………………………………………………v Acknowledgements ……………………………………………………...vi Table of Contents ………………………………………………………viii List of Tables ………………………………………………………..…xiv List of Figures ……………………………………………………….…xvi Chapter 1: Introduction ........................................................................... 1 Mammalian pigmentary system ........................................................... 2 From laboratory mice to natural populations ...................................... 4 Mammalian pigment patterning ........................................................... 5 Mammalian pigment patterns – a population genetic approach in domestic cats ................................................................................... 7 Digital gene expression in non-model organisms ................................ 8 Mammalian pigment patterns – a functional genomic approach in the cheetah ..................................................................................... 10 References ............................................................................................. 13 viii Chapter 2: Genetics of Sex-linked Yellow in the Syrian hamster ....... 17 Abstract ................................................................................................. 18 Introduction ........................................................................................... 19 Results ................................................................................................... 22 Sex-linked yellow in hamsters: phenotype and genetics .................... 22 Linkage scan of the X chromosome .................................................. 23 Candidate genes for Sly: Agouti and Mc1r ........................................ 24 Relationship to Orange in domestic cats