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GDF11 in Ocular Development and MOTA Mapping by Robertino Ralph Karlo Peralta Mateo

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GDF11 in Ocular Development and MOTA Mapping by Robertino Ralph Karlo Peralta Mateo University of Alberta GDF11 in Ocular Development and MOTA Mapping by Robertino Ralph Karlo Peralta Mateo A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science In Medical Sciences – Medical Genetics ©Robertino Ralph Karlo Peralta Mateo Fall 2012 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Abstract Vision relies on the ability of the eye to receive, process, and send signals to the brain for interpretation. To perform these functions, the eye must properly form during embryogenesis which requires the interaction of genes encoding proteins with various functions during development such as cellular differentiation, migration, and proliferation. In this thesis, I investigate ocular formation and disease. One project assesses the role of gdf11 in a zebrafish animal model to study the eye formation. I also explore the effect of human GDF11 sequence variants in ocular disorders. The second project involves mapping a genomic interval responsible for an autosomal recessive disorder known as Manitoba Oculotrichoanal syndrome. The interval detected is in the vicinity of FREM1, whose paralogs cause Fraser Syndrome, a disease with phenotypic overlaps. Understanding the molecular basis of ocular diseases can aid the development of new methods to potentially better manage, treat, and reduce their occurrences. Acknowledgements The work presented in this thesis would not have been possible without the aid and guidance of many individuals. First of all I would like to thank my mentor Dr. Ordan J. Lehmann for accepting me as a trainee. I have been privileged to study in the Department of Medical Genetics and Dr. Lehmann‟s position as a clinical-scientist allowed for the pursuit of projects regarding ocular development and disease. His high expectations, patience, and understanding made me a better scientist in terms of my ability to plan experiments, analyze data, and develop the skills required to write a manuscript. I thank my committee members Dr. Andrew J. Waskiewicz and Dr. Michael A. Walter for their input and guidance in my training at committee meeting presentations or at various poster presentations. I would also like to thank Dr. Nipavan Chiamvimonvat from the University of California Davis for recommending the University of Alberta as an institution where I can continue my studies. I wish to express my gratitude to the following individuals who have trained me in numerous molecular assays and analytical techniques. To Dr. Mika Asai- Coakwell and Ming Ye I thank you both for offering assistants and guidance in trouble shooting GC rich PCRs, molecular cloning, cell transfection and western analysis training. To Dr. Xiao Hua thanks you for creating template plasmids for subsequent cloning assays. I thank past and present members of the Waskiewicz especially Karyn Berry who guided me in performing in situ hybridization, morpholino injections, and demonstrating the patience and dedication required to perform zebrafish for experiments as well as Curtis French, Timothy Erickson, Patricia Gongal, and Laura Pillay for their encouragement and the occasional Friday afternoon beers. To Dr. Ted Allison and his trainee Michele DuVal, thank you for your assistance and supply of photoreceptor antibodies. A special thanks goes to Azam Khorshidi for training me in the use of PLINK and GenomeStudio software suite for my mapping project. To Dr. Hardeep P. Singh and Dr. Sameer D. Pant I thank for their opinions and input on my mapping project and paper. To Dr. Sarah Hughes, thank you for letting me use your confocal microscope facility. I would also like to thank technicians May Yu for plating and upkeep of cells used for western analysis, Aleah McCoury for maintaining a healthy zebrafish facility, and Erin Strachan for helping to keep the Lehmann lab running smoothly. I would like to thank the various funding sources I have been fortunate to receive in my time as a graduate research assistant. First off, I would like to thank Dr. Lehmann for providing and supplementing my stipend from some of the lab‟s funding sources, notably from the Canadian Institute of Health Research and Alberta Innovates Health Solutions. Secondly I would like to thank the Department of Medical Genetics for paying half of my annual tuition, which as an international is quite a substantial sum. I would like to also recognize the Faculty of Medicine & Dentistry and Alberta Health Services for the Graduate Student Recruitment Studentship and a Graduate Research Assistantship awards and the Women and Children Health Research Institute (WCHRI) for financing a Graduate Studentship. The work presented in this thesis made it possible to attend conferences at the provincial, national, and international level. I also enjoyed presenting at several research days organized in Edmonton by WCHRI, Team to Prevent Blindness, or the Medical Genetics Students‟ Association. I lastly thank my friends and family members for their support whether they are in Edmonton, California, or the Philippines. I am grateful for the experiences I have had and I am proud call Canada my home. Table of Contents Chapter 1 General Introduction .......................................................................................1 The visual system in brief ............................................................................................2 Ocular morphogenesis .................................................................................................2 Anterior segment and eyelid morphogenesis ................................................................3 Retinal Development ...................................................................................................4 Zebrafish as a Visual System Model Organism ............................................................5 Morpholino utility, strengths, and weaknesses .............................................................6 Ocular Disease ............................................................................................................8 TGF-β family and BMP signaling................................................................................9 Role of BMPs in eye and retinal development ...........................................................11 Retinal dystrophies ....................................................................................................12 Gene mapping strategies and SNP arrays ...................................................................13 References ................................................................................................................22 Chapter 2 Investigating the role of GDF11 in ocular development and disease...............33 Introduction ..............................................................................................................34 Methods ....................................................................................................................37 Zebrafish Usage and Growth .................................................................................37 Zebrafish gdf11 DIG labeled probe synthesis .........................................................37 Cloning of gdf11 PCR product ...............................................................................38 DIG labeled Probe Synthesis .................................................................................39 Whole embryo In Situ Hybridization .....................................................................39 Whole Embryo Immunohistochemistry ..................................................................41 Microscopy and automated cell counting ...............................................................42 Morpholino use and injection ................................................................................43 Reverse transcription PCR for morpholino efficacy studies ....................................44 Screening GDF11 in human DNA samples ............................................................45 Size Fragment Genotyping ....................................................................................48 Constructions of expression vectors containing sequence variants for western analysis .................................................................................................................49 Construction of GDF11 polyalanine vectors ..........................................................51 Transient transfection and maintenance of COS cells for v-5 tagged westerns ........52 Protein Quantification............................................................................................53 Western Assay.......................................................................................................54 Results ......................................................................................................................56
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