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UC San Diego UC San Diego Electronic Theses and Dissertations Title Study of NUP107 in human neurogenetics using zebrafish and induced-pluripotent stem cell models / Permalink https://escholarship.org/uc/item/9t70x07f Author Sotak, Bethany Nicole Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Study of NUP107 in human neurogenetics using zebrafish and induced-pluripotent stem cell models A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Bethany Nicole Sotak Committee in charge: Professor Joseph G. Gleeson, Chair Professor Lawrence S.B. Goldstein Professor Bruce A. Hamilton Professor Martin W. Hetzer Professor Alysson R. Muotri 2012 Copyright Bethany Nicole Sotak, 2012 All rights reserved. The Dissertation of Bethany Nicole Sotak is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2012 iii DEDICATION I dedicate this dissertation to my love, Timothy Allen Ray, and friend Benjamin Horne. These men taught me how to experience life, but were both tragically taken from us too soon. Maintain the light. I cannot hear your laughter. I cannot see your smile. I wish that we could talk again, If only for a while. I know you’re watching over me Seeing everything I do. And though you’ll always be with me, I will always be missing you. You taught me that life is much too short And at any time could end. But know no matter where you are You will always be my best friend. And when it is time for me to go You’ll be there to show me the way. I wish that you could still be here. But I’ll see you again someday -Author Unknown iv TABLE OF CONTENTS Signature Page………………………………………………………………………………………. iii Dedication…………………………………………………………………………………………….. iv Table of Contents……………………………………………………………………………………. v List of Abbreviations…………………………………………………………………………………. viii List of Figures………………………………………………………………………………………… x List of Tables………………………………………………………………………………………..... xi Acknowledgements………………………………………………………………………………….. xii Vita…………………………………………………………………………………………………….. xiv Abstract of the Dissertation…………………………………………………………………………. xvi Chapter 1. Identification of a rare mutation in nucleoporin 107 mapping to a novel 1 autosomal recessive primary microcephaly locus on chromosome 12q15…………………….. Abstract……………………………………………………………………………….……… 1 Background and Significance……………………………………………………………... 2 Results…………………………………………………………………………………….…. 11 Recruitment of family MIC-670…………………………………………………... 11 Homozygous interval mapping identifies a novel locus at 12q15 ……….…... 12 Identification of NUP107 c.G303A transition…………………………………… 14 Conclusion and Discussion…...………………………………………………………….... 15 Materials and Methods……...……………………………………………………………... 17 Acknowledgements…………….………..…………………..……………………………... 18 Chapter 2. A mutation in nucleoporin 107, an integral component of the nuclear pore 19 complex, causes human primary microcephaly…………………………………………………... Abstract………………………………………………………………………………………. 19 Background and Significance……………………………………………………………... 19 Results……………………………………………………………………………………….. 26 c.G303A transition in NUP107 affects the affinity of the splice donor site in 26 v exon 4………………………………………………………………………………. Decreased splicing of exon 4 reduces NUP107 protein with a consequent 29 decrease in nuclear pore density………………………………………………… Morpholino knockdown of Nup107 during zebrafish development results in 30 a microcephalic fish……………………………………………………………….. Conclusion and Discussion…...…………………………………………………………... 33 Materials and Methods……...……………………………………………………………... 35 Acknowledgements…………….………..…………………..……………………………... 39 Chapter 3. Human MCPH modeling in a dish: Generation and characterization of an 41 induced-pluripotent stem cell model of MPCH from MIC-670………………………..……….…. Abstract………………………………………………………………………………….…… 41 Background and Significance………………………………………………………..……. 41 Results……………………………………………………………………………………….. 45 MCPH-iPSCs are pluripotent, have retroviral silencing, and can differentiate 45 into all germ layers………………………………………………………………… c.G303A transition in NUP107 affects the affinity of the splice donor site in exon 4, with a consequent decrease in nuclear pore component proteins 49 and nuclear pore density…………………………………………………………. MCPH-iPSCs can differentiate along the neuronal lineage, but have 54 delayed organization of neuronal rosettes………………………………….…... Decreases in NUP107 do not affect the cell cycle length or Aurora B 56 localization during mitosis………………………………………………………… Lentiviral expression of NUP107 is silenced in MCPH-iPSCs………..………. 58 Conclusion and Discussion…...…………………………………………………………... 60 Materials and Methods……...……………………………………………………………... 62 Acknowledgements…..……...……………………………………………………………... 66 Appendix I. Recipes for cell culture media.………………….………………………………….…. 67 vi Appendix II. Primer sequences for genotyping…………………..……………………………….. 68 Appendix III. Primer sequences for RT-PCR and/or qRT-PCR……………...………………….. 71 75 Appendix IV. Primers for retroviral expression.…………………………..……………………….. 76 Appendix V. Primary and secondary antibodies..………………………..……………………….. 79 Appendix VI. Quantitative RT-PCR for directed neuronal differentiation………………………. References………………………………………....………………………..……………………….. 86 vii LIST OF ABBREVIATIONS AP alkaline phosphatase bFGF basic fibroblast growth factor BRCT domain BRCA1 C-terminal domain BrdU 5-bromo-2’-deoxyuridine CNS central nervous system D/V dorsal/ventral dpf days post fertilization E3 embryo medium FACS fluorescence activated cell sorter FBS fetal bovine serum GT gene trap GWS Galloway-Mowat Syndrome hESC human embryonic stem cell hpf hours post fertilization HRP horseradish peroxidase IACUC Institutional Animal Care and Use Committee ICC immunocytochemistry IKNM interkinetic nuclear migration iPSC induced-pluripotent stem cells IRB Internal Review Board Mb megabase MCPH Autosomal recessive primary microcephaly ml milliliter MRI magnetic resonance imaging nl nanoliter NP neuronal progenitor NPC nuclear pore complex NUP107 nucleoporin 107 NUP133 nucleoporin 133 OFC occipto-frontal head circumference ORF open reading frame P/S Penicillin/Streptomycin PBS phosphate buffered saline PBS-T phosphate-buffered saline containing 0.1% Tween 20 viii PCR polymerase chain reaction pg picogram POL Poly-l-ornithine PTU 1-phenyl-2-thiourea qRT-PCR quantitative reverse-transcriptase PCR R/C rostral/caudal RT room temperature RT-PCR reverse-transcriptase PCR SD standard deviation SDS sodium dodecyl sulfate SEM standard error of the mean SVZ subventricular zone VZ ventricular zone γ-TuRC γ-tubulin ring complex ix LIST OF FIGURES Figure 1.1 Pedigree of family MIC670.………….………………………………………. 11 Figure 1.2 Cranial MRI images from MCPH-affected individuals……..….…………. 13 Figure 1.3 Genome-wide SNP analysis and identification of the 12q15 locus..……. 14 Figure 1.4 Evolutionary conservation of NUP107 c.G303A………..…………………. 15 Figure 1.5 Chromatograms of NUP107 c.G303A in MIC670 family members……... 16 Figure 2.1 Schematic of NUP107 gene and translated protein domain structure….. 26 Figure 2.2 The NUP107 c.G303A transition decreases the affinity of exon splicing, 27 in primary fibroblasts.…………………………………...……………………. Figure 2.3 NUP107 protein is decreased in affected individuals in family 28 MIC670..……….………………………………………………………………. Figure 2.4 Nuclear pore density is decreased in primary fibroblasts from family 29 MIC670….…..…………………………………………………………………. Figure 2.5 Morpholino-mediated knockdown of Nup107 in zebrafish results in 32 microcephaly……………………………….………………………………….. Figure 3.1 Human neuronal rosettes as a model of embryonic corticogenesis…...... 44 Figure 3.2 iPSC colonies have a normal karyotype and express markers of 46 pluripotency……………….…………………..……………………………..… Figure 3.3 Retroviral expression is silenced and endogenous pluripotency genes 48 are expressed in iPSCs………………………………………………….....… Figure 3.4 MCPH-iPSCs have the ability to generate all germ layers in vitro…….… 50 Figure 3.5 NUP107 protein is decreased as well as those in the NUP107-160 51 complex…………………………………………………….…………………... Figure 3.6 NUP107 expression is decreased without changes in expression of 52 other nuclear pore components……………………………………………... Figure 3.7 Nuclear pore density is decreased in MCPH-iPSCs………...…….……… 53 Figure 3.8 Directed differentiation of iPSCs into neurons………………..………..….. 55 Figure 3.9 Directed differentiation of iPSCs into neuronal rosettes is delayed in 56 MCPH-iPSCs ……………………………………………………………..…... Figure 3.10 Cell cycle analysis of MCPH-iPSCs………...……...……………………….. 57 Figure 3.11 Aurora B localization in MCPH-iPSCs………………...……………….…… 58 Figure 3.12 Lentiviral-mediated expression of NUP107 in MCPH-iPSCs from 59 affected individual A2………………………………………………….…...… x LIST OF TABLES Table 1.1 Summary of published loci for autosomal recessive primary 4 microcephaly………………………………………………………………..…. Table 1.2 Clinical features of MIC670 patients with MCPH………………………….. 12 xi ACKNOWLEDGEMENTS I would like to thank Joespeh Gleeson for his guidance and support in my thesis project; his encouragement to push my own boundaries and those of the research, and his assistance in thinking outside the box. I would also like to thank the entire Gleeson lab for their support and guidance in troubleshooting and in teaching me new techniques. Lab members who contributed substatialy to my work include