UNIVERSITY of CALIFORNIA, SAN DIEGO Study of NUP107 in Human Neurogenetics Using Zebrafish and Induced-Pluripotent Stem Cell

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

Biomedical Sciences

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

Bethany Nicole Sotak, 2012

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 Jennifer Silhavy, who drove the genetic alaysis of this project;

Stephanie Bielas, who provided intellectual feedback and experimental assistance; Ji-Eun Lee, who provided zebrafish expertise and training; and Jana Schroth, Gaia Novarino and Ali Crawford for their patient guidance in research and life.

I would like to thank the members of my committee: Lawrence Goldstein, Alysson Muotri,

Martin Hetzer, and Bruce Hamilton for their invaluable insights and guidance throughout my graduate school carreer. I would also like to thank the labs of Martin Hetzer, Douglass Forbes, and Larry Gerace for use of their equipment and valuable scientific discusssions.

I would like to thank the lab of Neil Chi, particularly Shu Tu and Hongbo Yang, who provided technical assistance and trainging as well as equipment and crtical scientific discusssions. As well as the lab of Alysson Muotri, especially Alysson Muotri and Cassiano

Carromeu, for valuable training and troubleshooting necessary to bring stem cell techniques to the Gleeson Lab.

I would like to thank my students, Amanda Phillips Yazguirre, Sofia Infante, Whitney

Thuong, and Andrew Segina for their patience and hard work while teching me how to become a good mentor.

I would like to thank the following agencies that have provided my funding: California

Institute of Regenerative Medicice (CIRM) and the Ruth L. Kirschstein National Research Service

Award (NRSA) from the National Institute of Neurological Disorders and Stroke (NINDS).

Lastly, I would like to thank my friends and family, whose support and encouragement gave me the necessary strength. Special thanks to my family, Kerin Sotak, James Sotak, Erica

Sotak Bolvin, Sofia and Audrey, Vicki Reynolds, Greg Reynolds, and especially Timothy Ray,

xii who were always are there to give their invaluable support. Thank you to my friends: Diane

Bushman, Margaret Butko, Ariana Lorenzana-Mortovesky, Erilynn Heinrichson, Emily Witham,

Emily Loui, Michael Libutti, and Reina Juarez; the Friday Night Swim Club and members Mark

Stuckelman, Sandi Smith, Bryan Larish, Thomas Johnson, Greg Barry, and Greg McDonald; and the community of Tri Club San Diego.

Chapters 1, 2 and 3 contains work submitted, in part, for publication by authors Bethany

N. Sotak, Stephanie L. Bielas, Jennifer L. Silhavy, Shu Tu, Ozgur Rosti, Hulya Kayserilli, Amanda

D. Yazguirre, Sofia Infante, Whitney Thuong, Neil Chi, Joseph G. Gleeson. The dissertation author was the primary investigator and author of this material.

xiii VITA

Education

2003 Bachelor of Science, University of Washington

2012 Doctor of Philosophy, University of California, San Diego

Publications

B. N. Sotak, S. A. Bielas, J. L. Silhavy, S. Tu, O. Rosti, H. Kayserilli, A. D. Yazguirre, S. Infante, W. Thuong, N. Chi, J. G. Gleeson. “Splice mutation in NUP107, encoding an integral component of the nuclear pore complex, leads to primary microcephaly.” (in preparation)

B. N. Sotak, J. G. Gleeson. “Can’t get there from here: cilia and hydrocephalus.” Nat Med. 2012 Dec;18(12):1742-3.

J. Yuan, M. Darvas, B. Sotak, G. Hatzidimitriou, U. D. McCann, R. D. Palmiter, G. A. Ricaurte. “Dopamine is not essential for the development of methamphetamine-induced neurotoxicity.” J Neurochem. 2010 Aug;114(4):1135-42

C. Nerii, C. Ghelardini, B. Sotak, R. D. Palmiter, M. Guarna, G. Stefano, E. Bianchi. “Dopamine is necessary to endogenous morphine formation in mammalian brain in vivo.” J Neurochem. 2008 Spet;106(6):2337-44.

T. S. Hnasko*, B. N. Sotak*, R. D. Palmiter. “Cocaine-conditioned place preference by dopamine-deficient mice is mediated by serotonin.” J Neurosci. 2007 Nov 14;27(46):12484-8.

T. S. Hnasko, R. M. Hnasko, B. N. Sotak, R. P. Kapur, R. D. Palmiter. “Genetic disruption of dopamine production results in pituitary adenomas and severe prolactinemia.” Neuroendocrinology. 2007;86(1):48-57.

S. Robinson, B. N. Sotak, M. J. During, R. D. Palmiter. “Local production in the dorsal striatum restored goal-directed behavior in dopamine-deficient mice". Behav Neurosci. 2006 Feb;120(1):196-200

T. S. Hnasko, B. N. Sotak, R. D. Palmiter. "Morphine reward in dopamine-deficient mice." Nature. 2005 Dec 8;438(7069):854-7.

B. N. Sotak, T. S. Hnasko, S. Robinson, E. J. Kremer, R.D. Palmiter. “Dysregulation of dopamine signaling in the dorsal striatum inhibits feeding”. Brain Res. 2005 Nov 9;1061(2):88-96.

Awards

Best poster at retreat, Biomedical Sciences PhD Program, 2011

Predoctoral fellow, California Institute of Regenerative Medicine, 2008-2010

xiv Ruth L. Kirschstein National Research Service Award (NRSA), National Institute of Neurological

Disorders and Stroke (NINDS), 2010-2012

xv ABSTRACT OF THE DISSERTATION

Study of NUP107 in human neurogenetics using zebrafish and induced-pluripotent stem cell models

Bethany Nicole Sotak

Doctor of Philosophy in Biomedical Sciences

University of California, San Diego, 2012

Professor Joseph G. Gleeson, Chair

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a great reduction of brain growth in an otherwise architecturally normal brain.

The incidence of MCPH is 1:1,000,000 in Caucasian populations but can be as high as one in

10,000 in some Arab and Asian populations. Using linkage analysis and direct sequencing, we identify a mutation in a highly conserved region of nucleoporin 107 (NUP107), a critical component of the nuclear pore complex. Using primary patient fibroblasts, we verified a reduction of NUP107 transcript that results in decreased levels of NUP107 protein and a functional decrease in the density of nuclear pores within the nuclear envelope.

One difficulty in studying neurodevelopmental diseases is the ability to access specific cell types, such as human neurons, from affected individuals. In order to study the role of

NUP107 in human neurogenesis, we generated induced-pluripotent stem cells from an affected

MCPH patient. These iPSCs can generate neuronal progenitors and neurons that contain the genetic information of the MCPH donor. All iPSCs expressed markers of pluripotency, show retroviral silencing, and can differentiate into all three germ layers. When differentiated via dual-

xvi SMAD inhibition, control cells start to form ZO-1+ neuronal rosettes by day 7; affected patient iPSCs form rosettes by day 30. With a delay in expansion of the neuronal progenitor pool, this could account for fewer cells being generated during neurogenesis. Thus, we have developed the first in vitro iPSC model of a human neurodevelopmental disease.

We further validate NUP107 as a causative gene for MCPH in a zebrafish model of development. Using a translation blocking antisense morpholino, we show a reduction in nup107 protein and a 15% reduction in dorsal brain area without significant alterations to body size. This phenotype can be reversed with co-injection of the human NUP107 ortholog, indicating a critical role of NUP107 in developmental brain size regulation.

Overall, the identification of MCPH genes can increase our knowledge about the control of neuronal development and also how cortical expansion has changed throughout human evolution. These results are important because they contribute to our overall understanding of human intelligence, which can potentially help us to develop treatments for neurodevelopmental diseases leading to intellectual disability and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or stroke.

xvii CHAPTER 1.

Identification of a rare mutation in nucleoporin 107 mapping to a novel autosomal

recessive primary microcephaly locus on chromosome

ABSTRACT

During primate evolution, the cerebral cortex has undergone a dramatic increase in surface area and brain size varies considerably between species. This evolutionary expansion is thought to be important for the high-order cognitive functions such as language2. In proportion to body size, the brain of humans is three times larger than that of any non-human primate. One strategy to identify genetic modifications that have occurred during brain during evolution is to identify genes involved in the regulation of human brain size.

Autosomal recessive primary microcephaly (MCPH) is characterized by the reduction of brain size and reduced cortical surface area, without alterations to the cortical thickness3, which suggests that causative MCPH genes could be good candidates for a role in the evolutionary expansion of the human brain. Mutations in MCPH genes can reduce the volume of the brain more to a size roughly comparable to our hominid ancestors4,5. There is also evidence that four genes have evolved rapidly and have shown a strong selective pressure during evolution. Here, we report the identification of a novel MCPH locus in a consanguineous Turkish family. Identity- by-descent modeling of SNP array data identified a single homozygous interval of 1.8 Mb on chromosome 12q15 containing 15 known genes. Using Sanger sequencing, a rare homozygous variant was identified in a highly conserved region of NUP107 (NM_020401) at c.G303A.

Overall, MCPH genes can teach us about the control of neuronal development and how neuronal development has changed through evolution. These results are important because they contribute to our understanding of human neuronal development, which can potentially help us to develop treatments for brain diseases such as Alzheimer’s disease, Parkinson’s disease, or stroke.

1 2

BACKGROUND AND SIGNIFICANCE

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder of brain growth characterized by a reduction in occipito-frontal head circumference

(OFC) of at least 3 standard deviations below (-3SD) the mean for equivalent ethnicity, age and sex. In the past, some studies have used -2SD as a cutoff for microcephaly6 but using this definition will include ~2% of normal individuals. Using a -3SD cutoff ensures there are no false negatives using a -4SD cutoff ensure no false positives7. Within a family, the OFC usually does not vary by more than 2SD between affected individuals8.

MCPH correlates closely with reduced cranial volume and mental retardation without further neurological findings. Based on magnetic resonance imaging (MRI) studies, affected individuals show an overall smaller brain with normal cytoarchitecture. The size reduction is most prevalent in the cerebral cortex where the cortex has simplified gyri and sulci with slight reductions of white matter consistent with the small size of the brain6,9. Patients experience varying degrees of intellectual disability; the smaller the head circumference, the higher the chances of associated intellectual disability. It is also possible that patients can have delayed development of motor functions. As the child grows, the reduced size of the skull becomes more apparent; in some cases, the body can also be smaller or lighter.

Although brain size closely correlates with occipito-frontal head circumference (OFC), it may not in rare situations like craniosynostosis. In craniosynostosis, the sutures between the bones prematurely fuse and the skull expands upwards, rather than front to back. Due to the altered skull shape, the children may have a small OFC but normal brain size.

Microcephaly can be divided into primary or secondary based on the onset of the decreased head circumference:

Primary microcephaly, recessive microcephaly, or autosomal recessive primary microcephaly (MCPH)10 is characterized by having an autosomal recessive mode of inheritance.

MCPH also been called “true microcephaly” or “microcephaly vera” due to the lack of any pathological abnormalities other than the reduced size of the brain. Primary microcephaly is

3 evident by the 32nd week of gestation11 and is typically associated with non-progressive mild mental retardation.

Secondary microcephaly is also called acquired microcephaly. At birth, the head size and brain function are normal but microcephaly develops during infancy or early childhood, usually between 6-18 months of age12,13. Causes of secondary microcephaly have been linked to environmental and maternal factors, such as exposure to chemicals, maternal syphilis, poor prenatal care, or maternal diabetes. Secondary microcephaly is not always linked to environmental factors; it has also been identified in genetic disorders, such as Rett or Angelman syndromes or a central nervous system (CNS) degenerative disease.

The incidence of MCPH varies greatly within the world: 1/30,000 in Japan14, 1/250,000 in

Holland15, 1/2,000,000 in Scotland16, and 1/1,000,000 in the Yorkshire region of Britain17, and as high as 1/10,000 in northern Pakistan10. Presumably, the higher incidence of MCPH is seen in populations, such as the Middle East and parts of South Asia, where consanguineous marriages are customary18. Indeed, it has been estimated that 70% of marriages that happen in Pakistan are consanguineous19.

MCPH is autosomal recessive and follows Mendelian genetics. Recessive diseases are more common in individuals born from consanguineous marriages, because they have inherited identical ancestral genomic segments from both parents. When two parents each carry the disease-causing mutation, the risk of having an affected child would be 1:4. To date, ten causative genes and eleven loci have been reported for MCPH: Microcephalin20 (MCPH121),

WDR623 (MCPH222), CDK5RAP223 (MCPH324), CEP15225 (MCPH426), ASPM27 (MCPH526),

CENPJ23 (MCPH628), STIL (MCPH729), CEP153 (MCPH830), CEP6331, CASC532 and an additional locus at 10q33 (Table 1.1).

It has been hypothesized that MCPH may be caused by a decrease in the number of neurons generated during neurogenesis, most likely by controlling the size of the neuronal progenitor pool at the onset of neurogenesis. During corticogenesis, each RG gives rise to a single column on neurons34, with the number of neurons in each column roughly constant from

Table 1.1 Summary of published loci for autosomal recessive primary microcephaly.

mouse, cat, macaque and humans35. The net result of a bigger progenitor pool is an increase in the cortical surface area with little effect on cortical thickness36. Indeed, larger brains can result by increasing the number of neuronal progenitor symmetrical divisions37,38.

The size of the NPC pool can be regulated by the following ways: 1) Controlling of symmetrical/asymmetrical divisions through mitotic spindle alterations or microtubule dynamics 2)

Apoptosis 3) Increase in cell cycle length. Overall, the end result is a smaller brain and microcephaly.

One way that MCPH genes may control the NPC pool is by control of symmetric/asymmetric divisions. During development, each cell type must be generated at the correct time and place. In brain development, the neuroepithelial (NE) progenitors are located along the ventricle in an area called the ventricular zone (VZ), which is formed when the neural tube polarizes NE cells are connected by tight and adherens junctions39. Radial glia (RG) cells, a more neural fate-restricted progenitor than NE cells40,41, also have highly polarized apicobasal cell polarity accompanies by the disappearance of the tight junctions while the adherens junctions remain. The RG cell bodies are located at the apical surface or the ventricle, but the RG send long processes that attach to the basal (pial) surface in the developing mammalian brain41,42.

Only 1-3% of the RG plasma membrane is exposed to the ventricle9; this area is highly polarized

5 and contains a high concentration of the apical determinants such as adherens junctions43-45,

PAR complex proteins (Par6, Par3, aPKC)43,46,47, and Crumbs complex proteins (Crumbs, Pals1,

PatJ)47. When a RG cell divides, this apical domain is partitioned between the resulting cells and can be distributed evenly, resulting in symmetric division, or unevenly, resulting in asymmetric division39,48,49. It is important to note the developmental differences between symmetric and asymmetric divisions. During corticogenesis, the RG go through a period of exponential expansion, which correlates, to symmetrical divisions. After this period of RG expansion, the cells shift towards asymmetric divisions that directly correlate to neurogenesis9. Although asymmetric divisions were once though to occur only when the cleavage plane was parallel to the ventricular surface in Drosophila, asymmetrical divisions can occur in vertebrates when the cleavage plane is perpendicular to the ventricular surface if the apical domain is unevenly distributed9,15,50. Mutations that disrupt the polarity of vertebrate neuroepithelial cells can result in changes in cell division plane, interkinetic nuclear migration (IKNM), proliferation, and neurogenesis51-55

The centrioles that form during mitosis are not equal in structure or function56.

Centrosomes are involved in the generation of and organization of the microtubule cytoskeleton, mitotic spindle and cilia. During neurogenesis, the RG preferentially inherit the mother centriole, and this inheritance is required for maintenance of the RG progenitor pool57. Typically, the mother centrosome is tethered to the apical side of the mitotic event58,59 while the nucleus undergoes specialize movements called interkinetic nuclear migration (IKNM). Mutations in complex proteins of the microtubules or microtubule attachments60 could affect centrosome inheritance leading to a premature depletion of the RG pool.

During mitosis, the nuclei of RG cells undergo a process in which nuclei oscillate between apical and basal positioning that correlates to the progression of the cell cycle61,62 called

IKNM. M-phase nuclei position themselves at the apical VZ surface while moving to more basal locations during G1/S phase. During G2 phase, nuclei again move towards the apical surface where it enters mitosis and undergoes cytokinesis63,64. The mechanisms of IKNM are not well

6 understood in RG cells65. In order to undergo IKNM, the nucleus must elongate in shape to migrate up and return to a ball shape when migration stops and is suggested to be dependent on microtubules and actin66-71.

During IKNM, the progenitor nuclei are exposed a gradient of Notch signaling 46,72,73. If changes occur to increase the length of G1/S phase, cell nuclei will spend more time in the region of high Notch signaling, biasing the cell toward neurogenic divisions in the next mitosis74,75. In fact, the farther nuclei move from the apical surface, the more likely the progenitor cell produces neurons in the next division76 and the length of G1/S phase increases as cell become progressively more restricted in cell fate77.

Although length of G1 is associated with more restrictive cell fates, activation of cell cycle checkpoints can increase the length of the cell cycle leading to slower proliferation rates or cell cycle arrest and apoptosis. Inactivation of cell cycle checkpoints can also lead to apoptosis through DNA damage response pathways. Lastly, apoptosis can occur when progenitor cells spend too much time in an area of high notch signaling78.

When changes to cell cycle, IKNM, and apoptosis are considered, it suggests a unifying cellular mechanism of the MCPH genes in the modulation of proliferation and differentiation of neuronal progenitor cells. Each gene, known functions, animal models, and predicted role in the etiology of MCPH is discussed below.

The gene product of MCPH121, microcephalin20, is predicted to contain three BRCA1 C- terminal (BRCT) domains. It is known that BRCT domains specifically bind to phosphorylated proteins involved in DNA damage response pathways. Microcephalin is one protein in a DNA damage response complex that participates in DNA repair, cell cycle checkpoint control, and maintenance of genomic integrity. Mutations in MCPH1 have been shown to cause premature chromosome condensation in early G2 phase and delayed decondensation post mitosis79.

Presumably, MCPH1 has a role in maintaining inhibitory cyclin-dependent kinase 1 phosphorylation, which prevents entry into mitosis80. Mutant drosophila showed premature chromosome condensation, genomic instability, premature separation, and centrosome

7 detachment; although adult brain size is normal81,82. The Mcph1 gene trap (GT) mouse

(Mcph1GT/GT), in which Mcph1 function is defective, confirms the misregulated chromosome condensation in vivo, although mice do not have any alterations to brain or body size83. Similar negative results occur when the DNA damage response is fine in both MCPH1 patient cells and cell cultures generated from Mcph1GT/GT mice. Mutations in MCPH1 have also been linked to

Premature Chromosome Condensation Syndrome79 and Craniosynostosis-Microcephaly with

Chromosomal Breakage84. Mcph1 is expressed ubiquitously, but with higher levels in the brain, testes, pancreas, and liver. In the fetal brain, Mcph1 expression is highest in the developing forebrain and on the lateral walls of the ventricle in the NPC population20.

The MCPH222 locus encodes WDR623 and contains 15 WD repeats; WD repeats have been implicated in signal transduction, transcriptional regulation, cell cycle control, autophagy, and apoptosis. In humans, WDR62 has been shown to play a role in neuronal migration and proliferation; in addition to microcephaly, some patients exhibit lissencephaly, schizencephaly, and polymicrogyria3,85,86. During mitosis, WDR62 accumulates at the spindle poles, the centrosome, and is expressed exclusively apical precursors of the neuroepithelium during embryonic neurogenesis3,86. Overall, WDR62 is the second most common form of MCPH.

The MCPH324 locus encodes for cyclin dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2)23. The CDK5RAP2 protein contains two putative structural maintenance of chromosome (SRC) domains that play a role in the cohesion and condensation of chromosomes during mitosis87. CDK5RAP2 has been shown to localize to the centrosome throughout the cell

23,88 cycle as well as to the γ-tubulin ring complex (γ-TuRC). Inhibition of CDK5RAP2 results in chromosome miss-segregation due to its requirement for spindle checkpoint function89. In drosophila, the CDK5RAP2 ortholog, centrosomin (cnn) knockout flies show an uncoupling on the centrosome to the pericentriolar matrix, although the adult brains had a normal size90. In the developing brain or human and mice, CDK5RAP2 is widely expressed in embryonic tissue23,91, but has the highest expression in the brain and spinal cord, particularly in the neuroepithelial cells that line the ventricle89.

Centrosomal protein of 152 kDa (CEP152)25 is encoded by the MCPH426 locus. CEP152 is required for the initiation of centriole duplication and functions as a molecular scaffold for PLK4 and CENPJ. CEP152 is also involved in regulating genomic integrity and cellular response to

DNA damage; mutations are associated with Seckel syndrome and caused by accumulation of genomic defects92. CEP152 is also required for ciliogenesis93. The Drosophila ortholog is asterless (asl) and mutations in fly or morpholino knockdown in zebrafish result in mitotic and ciliary defects due to the lack of centrioles93,94 Although CEP152 can be detected in the brain, the location has yet to be determined.

The MCPH526 locus encodes abnormal spindle-like, microcephaly associated (APSM)27, the most common cause of MCPH with over 80 mutations identified95. The human ASPM gene contains 81 isoleucine-glutamine (IQ) motifs, which is thought to be directly involved in the increased cortical sized during evolution96,97. Several isoforms with different numbers of IQ repeats have been identified, suggesting that isoforms may have different functions96. ASPM mutations result in a truncated protein ranging from 116 to 3357 amino acids 8,27,98,99. Drosophila homolog, abnormal spindle (asp), is essential for normal mitotic spindle function in embryonic neuroblasts100 and fly mutants were referred to as ‘mini brain’. ASPM organizes microtubules at the spindle pole during mitosis and at the central spindle during cytokinesis100,101 and expression has been shown to maintain symmetrical cell divisions. In the developing mice and zebrafish,

ASPM has the highest expression in regions of active neurogenesis and both mouse mutants and zebrafish morpholino knockdown results in smaller brains with early progenitors undergoing mitotic arrest102,103. Expression is downregulated when neurogenesis is complete 27, although there is some expression in adult tissues8,95.

Centromere associated protein J (CENPJ)23 or CPAP is encoded by the MCPH628 locus.

CPAP protein requires for control of centriole length and inhibits microtubule assembly104. CENPJ is localized within the centrosome during mitosis and is concentrated at the spindle poles during prometaphase and metaphase23,104,105; additionally, it has been suggested the CENPJ can inhibit microtubule nucleation and depolymerization106. Like CEP152, CENPJ is also associated with

Seckel syndrome107. Cells lacking CENPJ arrest in mitosis with multipolar spindles108 while knockout of the drosophila ortholog dsas-4 resulted in flies without centrioles109. CENPJ is widely expressed in the developing embryo with the highest expression in the brain and spinal cord; the most highly express in the VZ of the developing brain23.

The MCPH7 locus encodes the SCL/TAL1 interrupting locus (STIL)29 or SIL; STIL localizes to the mitotic spindle during metaphase is required, along with CENPJ 110, for centriole duplication in human cells. Homozygous loss of function mutations in the zebrafish homolog, cassiopeia (csp), result in neuronal death, metaphase arrest, and mitotic spindle defects lacking one or more centrosome(s)111. Mouse mutants are embryonic lethal and have abnormal left/right development, increased apoptosis and smaller size112. STIL is expressed in the proliferating cells of the brain during early neurogenesis23.

MCPH8 encodes the centrosomal protein of 135 kDa (CEP135).30 CEP135 localizes to the centrosome throughout the cell cycle and also has a role in microtubule organization113 and centriole biogenesis114. Affected patient fibroblasts show fragmented centrosomes, disorganized microtubules, and reduced growth rate.

Centrosomal protein of 63 kDa (CEP63)31 forms a complex with CEP152; together these genes are essential for maintenance of centrosome numbers by regulating centrosome duplication115. In human lymphocytes, there are no defects of centrosome number, but there is a small increase in the centriole distances during mitosis. CEP63 associates with the mother centriole during mitosis in the VZ neuroepithelial cells of the developing mouse cortex31.

Recently, an MCPH mutation was identified in the cancer susceptibility candidate 5

(CASC5)116. CASC5 is part of the kinetochore network and is required for microtubule attachment to the centrosome and for spindle assembly checkpoint activation during mitosis, as shown by siRNA knockdown in HeLa cells117. But, in CASC5 patient lymphoblasts, no defects in cell cycle, spindle morphology or chromosome alignment was seen. However, when compared to expression in the cortical plate, CASC5 is highly expressed in the ventricular zone in the developing human brain116.

A new MCPH locus was identified at 10q11.23-21.3 in a Turkish consanguineous family; the disease-causing variant was not identified. Analysis of affected MCPH patient fibroblasts identified an elevated rate of chromosome nondisjunction and altered anaphase disruption of

Aurora B and INCENP, which remain abnormally associated with chromosomes during anaphase instead of transferring to the spindle midzone33. This did not affect cell cycle progression or spindle checkpoint activity and had no effect on DNA damage response.

As of this date, all of the identified MCPH mutations participate in cytoskeletal control of the mitotic machinery, such as mitotic spindle assembly or centriole biogenesis. CDK5RAP2,

CENPJ, STIL, WDR62, CEP152, CEP135 and CEP62 all play a role in centriole duplication.

Abnormal centriole numbers can lead to improper spindle alignment in dividing RG cell. Although the MCPH proteins may have a common localization, the genes most likely have unique functions and may have evolved compensatory mechanisms in many cell types.

Of the identified MCPH genes, five genes have been shown to have accelerated evolution under positive selection in human: ASPM96,118,119, MCPH1120,121, CDK5RAP2, CENPJ122 and CASC532. Although, there is debate as to whether CENPJ and MCPH1 have been linked to brain size evolution; CDK5RAP2 and ASPM have been linked to an increased neonatal brain mass with a debate as to the evidence of increased brain size122,. Interestingly, it has been hypothesized that a new allele of ASPM appeared between 14,000 and 500 years ago, with a mean estimated age of 5,800 years ago123. Roughly, this correlates with the appearance of the first true written language (3600 BCE), the development of civilizations (3500 BCE), and the development of agriculture (10,000 BCE – 3000 BCE) 124.

Here, we test the hypothesis that we can identify a new MCPH locus in a consanguineous Turkish family, MIC-670. We identify a novel locus of 1.8 Mb on chromosome

12q15 using single nucleotide polymorphism (SNP) mapping. We further use Sanger sequencing to identify a single, rare homozygous variant in NUP107 within this interval.

RESULTS

Recruitment of family MIC670

This study focuses on one family, MIC670, recruited by doctors Dr. Huyla Kayserilli and

Dr. Ozgur Rosti. MIC670 is a large two-branch consanguineous family of Turkish decent with four affected family members from a common founder; three in branch I from a first-cousin marriage and one in branch II resulting from a marriage of third-cousins once removed. Saliva or peripheral blood was collected from all individuals in generations V and VI, denoted by * (Figure 1.1). Upon physical examination, the age of affected individuals (A1, A2, A3 and A4) ranged from 0–8 years.

Figure 1.1 Pedigree of family MIC-670. A) Pedigree of a two-branch multiplex consanguineous family of Turkish descent, displaying autosomal recessive primary microcephaly (MCPH). Squares indicated male, circles indicate females, black symbols indicate affected individuals, and double connecting lines indicate a documented consanguineous marriage. Asteriks denote family members enrolled in the study B) Images showing the facial features of affected individual A3 at age 8 months.

MRI image analysis shows normal brain cytoarchitecture with simplified gyri and sulci (Figure 1.2)

Based on OFC measurements, all affected individuals have head circumference 5-7 standard deviation (SD) below the ethnically matched age and sex mean. All affected individuals exhibit no major motor deficits; however, developmental milestones were reached with a mild-moderate delay. Additionally, patients A1 and A2 have been diagnosed with minimal change proteinuria that has progressed to focal segmental glomerulsclerosis (FSGS) in A2; she is currently awaiting a

Table 1.2 Clinical features of MIC-670 patients with MCPH. N/A, not available; OFC, occipital-frontal head circumference; PSMR, profoundly and mentally retarded; FSGS, focal segmental glomerulosclerosis.

kidney transplant. A detailed summary of individual phenotypes is presented in Table 1.2.

Homozygous interval mapping identifies a novel locus at 12q15

Children from consanguineous unions have segments of their genome that are homozygous as a result of inheriting identical genomic fragments from both parents. Analysis of the pedigree suggests an autosomal recessive mode of inheritance. If so, all affected family members must share the same region from a common individual (in generation I) harboring the disease-causing mutation.

In order to identify homozygous genomic intervals associated with MCPH, DNA from family members in generation VI were subjected to an Affymetrix 250K NspI SNP mapping array. Analysis of identity-by-descent identified a single homozygous interval of 1.8 Mb on chromosome 12q15 between rs775645 and rs11177550 (Figure 1.3A) that contains 15 known genes (Figure 1.3B). An

Figure 1.2 Cranial MRI imaging of affected patients. A) Comparison of MRI images from control (left) and microcephaly affected (A2, right) individuals. Note the simplified gyral pattern and severe microcephaly while the overall brain architecture is retained. B) First column: patient A1; second column, patient A2. Top: midline sagittal MR images. Middle: Axial views at the level of the cerebrum Bottom: axial views at the level of the medulla. additional copy-number scan, based on the SNP signal, did not show any large (>100kb) deletion or duplication in the affected individuals.

Identification of NUP107 c.G303A transition

To detect potentially disease-causing variants, we performed direct sequencing analysis on 11/15 of the candidate genes. As part of our exclusion criteria, mutations that are present in dbSNP, the 1000 Genomes Project, or within our internal database of over 1300 ethnically matched control were excluded. Additionally, potential variants must segregate by phenotype within the family. Within the interval, a single rare homozygous c.G303A variant was identified in nucleoporin 107 (NUP107). NUP107 has 28 exons and the G to A transition occurs in the last base pair of exon 4, encoding a consensus sequence for a splice donor site. This base pair is

Figure 1.3 Genome wide SNP analysis and identification of the 12q15 locus. A) Ancestral identity by descent (IBD) mapping analysis representing areas of shared homozygous homology between affected individuals, not present in unaffected family members. IBD block size (cM) is plotted against genomic position. A single candidate interval is present on 12q15. B) The 12q15 interval between rs775645 and rs1117550 contains 15 known genes. Exons and surrounding intronic regions were sequenced for 11/15 genes and the NUP107 c.G303A transition was the only rare homozygous variant that was identified and was not present in ethnically matched control samples.

15 conserved through zebrafish (danio rerio) as determined by manual alignment of base pairs reported in the Human Genome Browser (http://www.genome.ucsc.edu) (Figure 1.4). Additional sequencing verified the homozygous segregation of c.G303A with the affected individuals (A/A).

As predicted, the parents were confirmed as carrier heterozygotes (G/A) and individual U1 as an unaffected non-carrier (G/G) (Figure 1.5).

CONCLUSION AND DISCUSSION

The current study describes identification of a nonsense mutation in the NUP107 gene in a consanguineous family of Turkish descent. The affected individuals presented with a deceased

OFC between -5 and -7SD and simplified gyri and sulci with an otherwise normal brain cytoarchitecture. They have hallmarks indicative of MCPH, such as micrognathia, bitemporal hollowing and smooth philtrum along with a mild-moderate delay in developmental milestones. In branch I, two individual A1 and A2 have been diagnosed with minimal change proteinuria, which

Figure 1.4 Evolutionary conservation of NUP107 exon 4 and splicing into exon 5. Comparative sequences for NUP107 splice donor site at the end of exon 4 and splice acceptor site at the beginning of exon 5. Conservation includes a range of evolution from human down to reptile (lizard) and fish (zebrafish), has progressed to FSGS in individual A2 at an age of 11 years. No changes in kidney function have been observed in individuals A3 or A4, who are currently 3 and 6 years old, respectively.

Figure 1.5 Sequencing chromatograms. Chromatograms of the NUP107 c.G303A transition at the end of exon 4 in MIC670 family members. The unaffected individual is homozygous wildtype (U1, G/G), heterozygous G/A in the mother (U2; G/A) and affected individuals are homozygous for the mutation (A2, A/A). U1: unaffected non-carrier (G/G); U2: heterozygous unaffected carrier (G/A); A2: homozygous affected (A/A).

Microcephaly in presence of FSGS is very rare and could be indicative of Galloway-Mowat syndrome (GWS).

GWS is a rare autosomal disorder consisting of a microcephaly and early onset of nephrotic syndrome125, with only 25 patients diagnosed between 1965 and 1999. Often, a variety of other features, such as hiatal hernia, infantile spasm are present but are not consistently present126. Various renal pathologies have been reported with the most common being FSGS127-

129, minimal change disease125, and diffuse mesangial sclerosis130; it is unclear whether these findings represent different stages of the disease or variations that occur within GWS131. Although nephrotic syndrome usually begins before the age of 2 years132, there has been a report of a child first experiencing nephrotic changes at age 7 years133. There is a wide variation in the clinical manifestation of GWS134. It is possible that family MIC-670 may represent a new variant of late- onset nephrotic syndrome in GMS.

Also, individuals A3 and A4 were born respectively with clinodactyly and bifid thumb. A bifid thumb is a rare form of polydactyly where there is a complete of partial duplication of the thumb. This typically occurs as an isolated deformity that is unassociated with other malformation syndromes and it is considered to occur sporadically, rather than having genetic causes135.

Experimental evidence suggests that a bifid thumb is cause by a temporary growth disturbance between the mesoderm and ectoderm of the preaxial limb136. Clinodactyly is when there is a bend or curvature of the pinky towards the other fingers. It is fairly common and can occur in combination of many genetic syndromes like Down Syndrome.

Homozygosity mapping revealed a region of 1.8 Mb at 12q15, which does not map to any known MCPH loci. We identified smaller regions of homozygosity, but were all smaller than the threshold of 1 cM that was set as a significant length consistent with autozygosity. Only 11/15 genes were sequenced, so it is possible that there are deleterious mutations within the intronic region or within these 4 genes. The c.G303A NUP107 transition segregates with the phenotype.

It is also possible that branches I and II consist of two different diseases. Although, the

OFC deviation between affected family members is within 2SD8, suggesting that the microcephaly seen in branches I and II is related.

This chapter demonstrates the identification of a new MPCH locus. In this locus, we identified a mutation in NUP107, which has never before been linked to neurogenesis.

Identification of novel genes involved in the regulation of human neurogenesis and brain size will aid in our understanding of human development and evolution.

MATERIALS AND METHODS

Patient recruitment. Consanguineous families with affected individuals having an OFC less than 3 standard deviations below the age and sex mean were recruited throughout the

Middle East. All individuals were enrolled in the study under the Institutional Review Board (IRB)- approved protocol of informed consent at University of California, San Diego.

DNA isolation. Genomic DNA was isolated one of two ways: saliva or blood leukocytes.

For saliva, samples were collected and processed with OrageneDISCOVER DNA collection kits

(DNA Genotek) according to manufacturer instructions. For blood, samples were collected in blood tubes containing a standard anticoagulant and stored at -70°C. Genomic DNA was isolated with the Blood & Cell Culture Maxi Kit (Qiagen) according to manufacturer instructions.

Linkage analysis and mapping. Fine mapping was performed on family MIC-670-V with the Affymetrix 250K NspI SNP array. Commonly shared homozygous intervals from all affected family members were calculated using a custom script implemented in Mathematica (Barry

Merriman, UCLA, unpublished). This script identifies all homozygous intervals longer than 2 Mb

18 for which there are no more than 1% heterozygous calls (to permit potential genotyping errors).

Segregation of the identified mutations was investigated in all available family members.

Sequencing. To screen for mutations, we performed direct sequencing of 11/15 genes within the homozygous interval. PCR primers were designed for each gene using Primer3

(Whitehead Institute for Biomedical Research) to flank every coding exon and adjacent intronic junctions (primers listed in Appendix II). The PCR products were treated with Exonuclease I

(Fermentas) and shrimp alkaline phosphatase (Promega); both strands were sequenced using a

BigDye Terminator v3.1 sequencing kit with an ABI3100 automated sequencer (Applied

Biosystems) and chromatograms analyzed with Sequencher software (GeneCodes). All available family members were tested for segregation of any potential disease-causing mutation. Exclusion criteria for a mutation excludes mutations found in dbSNP, the 100 Genomes Project, or within our internal whole exome database of over 1300 ethnically matched controls.

ACKNOWLEDGEMENTS

Chapter 1 contains work submitted for publication by authors Bethany N. Sotak,

Stephanie A. Bielas, Jennifer L. Silhavy, Shu Tu, Ozgur Rosti, Hulya Kayserilli, Amanda D.

Yazguirre, Sofia Infante, Whitney Thuong, Neil Chi, Joseph G. Gleeson. The dissertation author was the primary investigator and author of this material.

CHAPTER 2.

A mutation in nucleoporin 107, an integral component of the nuclear pore complex, causes

human primary microcephaly

ABSTRACT

Autosomal recessive primary microcephaly (MCPH) is a disorder characterized by the reduction of brain size in the absence of structural abnormalities. It has been hypothesized that suggests causative MCPH genes could be good candidates for a role in the evolutionary expansion of the human brain. We have previously identified a MCPH locus on chromosome 12 in a Turkish consanguineous family and identified a potentially deleterious c.G303A in nucleoporin 107 (NUP107).

Here, we provide evidence to link NUP107 to human brain size during development. In family MIC-670, a c.G303A transition in the last base pair of exon 4 decreases the affinity of the splice donor site. This induces skipping of exon 4 and generated a product that undergoes nonsense-mediated decay. Overall, less NUP107 protein is generated. Without NUP107, the

NUP107-160 complex of the nuclear pore is unstable and results functionally in lower nuclear pore density. Using zebrafish, morpholino-mediated knockdown of Nup107 results in decreased skull size by 5 days post fertilization. These results indicate that NUP107 has a role in developmental brain size.

BACKGROUND AND SIGNIFICANCE

In eukaryotic cells, the nucleus and cytoplasm are separated by a double membrane called the nuclear envelope (NE). The bidirectional movement of molecules between the nucleus and cytoplasm is mediated by nuclear pore complexes (NPCs). In vertebrates, the nuclear pore complex (NPC) contains many protein complexes and has an estimated mass of 60Mda137.

19 20

NPCs are embedded within the nuclear envelope and provide a size-selective gate where 138-140 molecules up to 20-40 kDa can freely diffuse; larger molecules (<40 kDa) are excluded unless actively transported through the NPC[139]. Molecules in the cytoplasm, destined for the nucleus, are labeled by a nuclear localization symbols for nuclear import141,142, while molecules in the nucleus, destined for the cytoplasm, are labeled by the nuclear export signal143,144.

The NPC has an eightfold rotational symmetry and is made up of approximately 30 distinct proteins, the nucleoporins (Nups), present in multiples of eight copies each145 137. These

Nups can be partitioned into three structural groups146: transmembrane, scaffold, and transport.

147,148 The transmembrane Nups, which consist of transmembrane α-helices and cadherin folds , span the nuclear pore and attach into the membrane. The scaffold Nups consist of α-solenoid and β-propeller folds and make up the central framework of the NPC, connecting the cytoplasm to the nucleus. The transport Nups are mainly composed of FG-Nups149, which contain FG, GLFG or FxFG repeats (x = any amino acid) separated by regions of hydrophilic amino acids. These

FG-Nups represent ~1/3 of all Nups137 and most are anchored on the inner surface of the NPC central pore and have large, highly disordered regions. These flexible regions effectively fill the

NPC central core145,150,151 to establish a barrier that can selectively interact with carriers to selectively transport cargo across the NE152. The antibody, MAB414, recognizes the FG-repeat

Nups NUP62, NUP153, NUP214 and NUP358, and is used as a general marker for nuclear pores153,154.

In proliferating human cells, there are 2000-5000 NPCs/nucleus, depending on the cell type155,156. The core of the nuclear pore constitutes ~50% of the entire nuclear pore and is made up of only 2 subunits, the NUP107-160 and NUP205 complexes157. The NUP107-160 complex is composed of nine nucleoporins (NUP107, NUP160, NUP133, NUP96, NUP43, NUP85, NUP37,

SEC13, and SEH1)158,159 and is a biochemically stable “Y”-shaped structure160. NUP107 interacts with NUP133 on the N-terminus and NUP96 at the C-terminus161 in the bottom section of the “Y”.

During mitosis, the nuclear volume and number of nuclear pore almost double during interphase of dividing cells162; this increase in the number of NPCs is controlled by cyclin-

21 dependent protein kinase, CDK1, mechanism163; the nuclear growth is regulated by inhibition of

MEK1 (9). Inactivation or Ras/Erk signal keeps NPC density low, whereas activation increases

NPC density164.

One difference between mitosis in yeast and fungi versus metazoa is that metazoa must perform “open mitosis” which requires breakdown the nuclear envelope to allow the spindle access to the chromosomes165. Yeast and fungi can undergo “closed mitosis” where the spindle microtubules can penetrate the nuclear membrane166,167. Cells that undergo “open” mitosis must re-establish the nuclear compartment and rebuild the NE168-170.

One of the initial events during NE breakdown is the release of nucleoporin 98 (NUP98) into the cytoplasm171. Then, the rest of the Nups are dispersed synchronously into the cytoplasm within their subcomplexes 172-174, allowing the NPC complex to reform rapidly at the end of mitosis.

At the end of mitosis, the intact NPC subunits are reused to form new NPCs within NEs of the two daughter cells165,175.

There are two periods of NPC formation, during interphase and during post-mitotic assembly139,175-178; each is differentially regulated179. During post-mitotic assembly, siRNA depletion of NUP107-160 complex in vitro and in vitro demonstrates a crucial role of the NUP107-

160 complex at a very early stage of NPC assembly 153,173,174. Indeed, the NUP107-160 complex is the earliest subunit recruited to the surface of the chromatin in anaphase 180 to form a scaffold complex; this process is mediated by Elys/Mel28181{Rasala, 2006 #674,182,183}. Then, the NUP107-160 complex then recruits Pom121 and other pore components.

During interphase assembly, pores must be inserted into the NE. Pom121 slowly accumulates and then recruits NUP107-160184, which is opposite from that of NPC formation in post-mitotic cells. This difference may be due to the fact that a component of the NUP107-160 complex, nucleoporin 133 (NUP133) has a membrane curvature-sensing domain that is necessary for interphase, but not post-mitotic, NPC formation 181,185. Pom121 first accumulates at the site of nuclear pore assembly, and recruits the protein gp210186, which will contribute to the

22 formation of a hole through the intact nuclear membrane. Once a hole is formed, the NUP107-

160 complex is recruited to form the nuclear pore from both sides of the NE187,188.

Also during mitosis, NUP160, NUP107, NUP96, and NUP133, components of the

NUP107-160 complex, are phosphorylated189. It is thought that phosphorylation of these components regulates breakdown of the nuclear envelope190-192.

Throughout mitosis, the constituents of the NUP107-160 complex remain associated193 and the entire complex has been shown to localize to the spindle poles, proximal spindle fibers, and kinetochores in prophase mammalian cells and throughout spindle in Xenopus egg extracts194. But only ~5-10% of the entire NUP107-160 subcomplex associates with the kinetochores180, which link spindles to chromosomes for separation of DNA during mitosis195.

Localization of the NUP107-160 complex to kinetochores is dependent on NDC80 and CENP-F196.

Kinetochores depleted of the NUP107-160 complex fail to establish proper MT attachments by recruiting an altered set of kinetochore constituents, inducing a checkpoint-dependent mitotic delay158,196-198. Depletion of the N-terminal domain is dispensable for NPC formation, but causes a delay or failure to complete cytokinesis199. The N-terminal domain of NUP133 recruits CENP-F to kinetochores and depletion of CENP-F decreases tension between the kinetochores and decreased stability of kinetochore microtubules200-204. At the G2/M transition, NUP133 tethers the centrosomes to the NE to contribute to early stages of bipolar assembly205

Depletion of NUP107 or NUP133 results in protein co-depletion and a NE devoid of nuclear pores173,174. Presumably, this is due to the instability of the NUP107-160 complex as depletion of NUP107 or NUP133 depletes other NUP107-160 complex proteins without altering transcription153. Cell missing the NUP107/NUP133 components prevent the assembly of

NUP214 and NUP358 on the cytoplasmic side, and NUP153 to the nucleoplasmic side.

Presumably, NUP107 and NUP133 stabilize each other through interactions; expression of the C- terminal NUP133 can rescue pore formation in cells devoid of NUP133205.

Levels of NUP107-160 complex transcripts are regulated by the cell cycle. NUP96 is regulated at the protein level during mitosis by the ubiquitin-proteasome pathway206. At the

23 transcript level, all Nups are expressed in proliferating cells while scaffold Nups are downregulated when cells exit the cell cycle207. Without NE breakdown, the scaffold Nups do not turn over in post-mitotic cells and an age-dependent NPC deterioration occurs in cells, affecting the nuclear pore permeability208.

NPCs regulate gene function and gene expression by controlling the access of transcription factors or the export of specific messages207. Mutations in multiples Nups have been linked to tissue-specific cellular and developmental defects. A mutation in a scaffold Nup,

NUP155, is linked to sudden cardiac death due to atrial fibrillation209. Homozygous mice are lethal, but heterozygous mice have the atrial fibrillation phenotype indicating that disease can be caused by a tissue-specific reduction of Nup155. Interestingly, the NUP155 protein homolog in drosophila,

NUP154, is crucial for gametogenesis210. Translocations and fusions of NUP98 and NUP214 to transcriptional and signaling receptors have been shown to lead to acute myelogenous leukemia in humans211-213.

Loss of function mutations in mice have been reported for seven Nups, all of which have been embryonic lethal. Nup214, ELYS, and Rae1/Gle2 null mutations led to developmental arrest at implantation, consistent with the depletion of the maternal stores of protein214-216. Loss of

Nup98 or Nup50 led to embryonic death at early and late gastrulation, respectively217,218.

Perhaps the most informative mouse model for NUP107 is the mermaid (merm) mouse, a functional-null allele of the NUP107 binding partner, Nup133. Merm was identified during an N- ethyl-N-nitrosourea (ENU) mutagenesis screen for gastrulation219. Nup133 expression is first detected ubiquitously within the epiblast. But, after gastrulation expression varied between developmental stage and tissue with the highest expression in the neuroepithelium and paraxial mesoderm, just lateral to the neural tube. Presumably, merm embryos were lethal at midgestation due to circulatory defects. In the absence of functional Nup133 protein, nuclear pores could still incorporate Nup107 and form a stable mESCs line. When differentiated, in vitro, the merm mESCs differentiate into neuronal progenitors but abnormally maintain markers of the epiblast and inefficiently differentiate into post-mitotic cells. In vitro, merm mESCs inappropriately

24 maintained markers of pluripotency and preferentially differentiated into mesoderm and endoderm220. Interestingly, knockdown studies of NUP133 HeLa cells resulted in an NE devoid of nuclear pores. Yet, in vivo studies in many whole organisms find that Nup133 is not necessary for cell viability or essential for any cell function of the NUP107-160 complex221-225.

Anther example of cell-type specificity of NPC complex proteins is the elys zebrafish mutant, flotte lotte (flo)226. ELYS associates with the NUP107-160 complex and is expressed throughout the developing embryo as well as in many adult tissues in humans199 and its homolog,

Mel-28, has been associated with nuclear integrity in C. Elegans 227,228. During zebrafish embryogenesis, strong maternal elys expression may assist during development. However, at later stages, NPC formation was disrupted in rapidly proliferating cells, where the maternal Elys protein is depleted. Therefore, the most affected organs in the flo mutants are the brain, eye, and intestines229-232. In these organs, cells undergo apoptosis consistent with G1 arrest233.

There are two in vitro NUP107 mutant models; one in C. elegans234 one in zebrafish235. In

C. elegans, genetic disruption of the NUP107 homolog, npp-5 is dispensable for incorporation of most nuclear pore components, nuclear barrier permeability, and importin α/β-mediated nuclear import but worms do not complete development. NPP-5 is necessary for NUP133/NPP-15 and

NUP43/NPP-23 localization but is dispensable for ELYS/MEL-28 and NUP96/NPP-10C localization at the kinetochores during mitosis. In npp-5 mutants, absence of NPP-5 reduced nuclear growth and delayed entry into mitosis when the spindle assembly checkpoint (SAC), which ensures that chromosome segregation is correct. Decreases in NPP-5 decrease localization of the chromosomal passenger complex (CPC) protein Aurora B/AIR-2 and the kinetochore protein NUF2/HIM-10 at the kinetochores. NPP-5 directly interacts with the SAC protein MAD1/MDF-1 and assists in MAD1 accumulation at the NE. Overall, the npp-5 mutant suggests a role of NPP-5 in chromosome segregation.

In nup107 mutant zebrafish, nuclear pore number is greatly reduced in highly proliferative regions during development, leading to the impaired mRNA, but not protein, export. Zebrafish are missing pharyngeal cartilages, intestinal degeneration, non-existent swim bladder and smaller

25 eyes due to widespread apoptosis, but not altered proliferation, in the affected tissues.

Additionally, cell cycle was not globally disturbed in these tissues. Pharyngeal arches were missing due to decreased chondrocyte maturation. The authors did not comment on the obvious change to head size with increased apoptosis in the head235.

NUP96, the other NUP107 binding partner, affects G1/S progression through regulating

206 of expression of cell cycle regulators, like IκBα specifically in T and B cells within the immune system. NUP96 levels affect the nuclear/cytoplasmic ratio of certain mRNAs that encode G1 cell cycle regulators. These suggest that the nucleocytoplasmic transport of the NPC varies in a cell- cycle dependent manner. Distribution of Cyclin D3, CDK6 and IκBα became more cytoplasmic in

G1206. Cyclin D3 and CDK6 are drivers of cell cycle progression and stimulate G1 progression236.

Although NUP107 has a role in cell cycle, two knockdown studies did not show cell cycle defects in human neuronal cell lines207,237. Instead, these lines showed increased apoptosis, consistent with NUP107 RNAi knockdown data suggesting metaphase alignment problems and apoptosis in human cells238. These data further support the in vivo NUP107 zebrafish mutant exhibiting cell-type specific apoptosis during development235.

Other studies link the NPC to direct regulation of gene expression139,239,240. NUP98 and

NUP153, which can bind RNA export factors C241 and RNA242, are components of the nuclear basket. Their movement depends on active transcription243. Not only have the Nups been implicated in the transport-dependent movement of transcription factors and mRNA, but also in transport-independent gene regulation. The nuclear pore physically interacts with segments of the genome, presumably affecting chromatin organization and therefore transcriptional regulation of those genes 244,245. Therefore, when examining the cell-type dependent changes to nuclear pores, the transport-dependent and independent roles of Nups must be considered.

In this chapter, we test the hypothesis that the NUP107 c.G303A transition is the cause of MCPH in family MIC-670. We do this by showing that this mutation alters expression and decreases protein levels; the overall result is a decrease in nuclear pore density. Furthermore, we

26 use MO injection in zebrafish embryos to study the role of NUP107 during early embryogenesis in vivo.

RESULTS

c.G303A transition in NUP107 affects the affinity of the splice donor site in exon 4

In chapter 1, we identified a single rare homozygous c.G303A variant in nucleoporin 107

(NUP107). The G to A transition occurs in the last base pair of exon 4, encoding a consensus sequence for a splice donor site. In order to study the expression and biochemical properties of the underlying affects of c.G303A transition, we obtained skin fibroblast samples and generated primary fibroblast lines from 2 unaffected (U1, U2) and 3 affected (A1, A2, A4) individuals for this study.

Primary fibroblasts are fast growing, can undergo a large number of population doublings before undergoing senescence, and are derived from minimally invasive skin biopsies. They also maintain the euploid karyotype of the donor, making them suitable for genomic studies. This also

Figure 2.1 Schematic representations of the NUP107 gene and protein domains. A) NUP107 contains 28 exons. The start codon begins in the middle of exon 1 (ben arrow) and the stop codon in the beginning of exon 28 (line). The c.G303A transition is located at the end of exon 4 (arrow). B) The NUP107 protein contains an α-solenoid fold at the C-terminus with a highly disordered region at the N-terminus. There are three phosphorylation sites within the disordered region, thought to be important for disassembly of the nuclear envelope during mitosis. The p.M101I mutation does not disrupt any known domains (X).

27 offers an advantage over transformed cell line because the abnormalities underlying the disease should be present in the cells, only if the gene in question is expressed in this somatic cell type.

To investigate the affect of the c.G303A transition on splicing, we extracted total RNA from patient fibroblasts and generated cDNA using reverse transcriptase with oligo-dt primers, which prime at the poly-A tails of capped mRNAs. Mature mRNA will only contain the spliced exons. Primers were designed to amplify the cDNA fragment between NUP107 exons 3 and 5

(Figure 2.2A); the PCR product was then inserted into the pCR2.1-TOPO vector and Sanger

Figure 2.2 Exon 4 splicing in affected patient fibroblasts is skipped due to the c.G303A transition. A) Schematic of exon splicing from exons 345 and 35. Primer locations are located within exons 3 and 5 (arrows) B) RT-PCR from unaffected (U1), unaffected carrier (U2), and affected MCPH patients (A1, A2, A4). Splicing from exons 345 produces a 182 bp product. Splicing from exons 35 produced a product of 66bp (arrow) C) Quantification of band intensity compared to GAPDH shows decreased 345 splice product in the affected patient cells, not an increase in the amount of 35 splice product.

28 sequenced to identify the splicing of the coding exons. The resulting sequences verified splicing of exons 3à4à5 in fibroblasts from individual U1. In an affected individual, A2, there was also the presence of a transcript directly splicing exons 3à5 (Figure 2.2B; arrow).

Using the same primers to amplify between exons 3 and 5, we performed semiquantitative RT-PCR to determine the ratio of transcripts containing splicing of exons

3à4à5 to 3à5 (Figure 2.2B). When the products were visualized on a 1% agarose gel, an expected band of 182 bp from splicing of exons 3à4à5 was present in the unaffected homozygous (U1) and unaffected heterozygous carrier (U2) cells. Low levels of 3à4à5 spicing can also bee seen in the cells from all affected individuals tested (A1, A2, A4). An additional band of 66bp, corresponding to splicing of exons 3à5, is seen in both the heterozygous carrier (U2) and the affected individuals (A1, A2, A4) samples (Figure 2.2C). A reaction using primers for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was run as a loading control.

The intensity of each band was calculated using densitometry software (ImageJ) and normalized to GADPH intensity. This revealed similar levels of the 3à4à5 transcript in U1 and

U2 while A1, A2 and A4 express this transcript at ~40% the level of U1. This suggests a decrease in affinity, not the abolishment, of the exon 4 splice donor site. Furthermore, the 3à5 transcript was not visualized in individual U1, but similar levels were identified in individuals U2, A1, A2 and

A4. As a heterozygote, individual U2 should have similar levels of the 3à4à5 and the 3à5

Figure 2.3 NUP107 protein levels in patient fibroblasts. A) Schematic representation of protein translation of 345 and 35 mRNA splice products. The c.G303A transition results in a truncated protein with a stop codon at the beginning of exon 5. Non-coding regions are in exon 1 (white) and exon 28 (red) B) Western blots for NUP107 and NUP133 expression. NUP107 and NUP133 protein levels are decreased in affected patient cells (A1, A2, A4) compared to unaffected patients (U1, U2). α-tubulin was used as a loading control.

29 transcript, but this was not seen suggesting nonsense mediated decay of the 3à5 transcript.

Nonsense mediated decay also explains why equivalent levels of 3à5 transcript were seen in individuals U2, A1, A2 and A4.

Decreased splicing of exon 4 reduces NUP107 protein with a consequent decrease in nuclear pore density

With a decrease in the amount of NUP107 transcript, we would expect a decrease in

NUP107 protein expression in the fibroblasts from the affected individuals. When splicing occurs from 3à4à5, a protein is translated starting toward the end of exon 1 and ending toward the beginning of exon 28. But, when the c.G303A transition occurs, then direct splicing exons 3à5 encodes a premature stop codon at the beginning of exon 5 (Figure2.2A). Western blot analysis of proteins expressed in non-senescent fibroblasts show that protein expression of NUP107 and

NUP133 are greatly reduced in fibroblasts from affected individuals (A1, A2, A4) (Figure 2.2B). All commercial antibodies recognize the C-terminal end of NUP107, so the smaller protein produced from the 3à5 transcript would not be seen. We predict that this protein is never made due to the nonsense-mediated decay of the transcript.

NUP107 is a critical component of the NUP107-160 complex. Cellular knockdown of

Figure 2.4 Affected patient fibroblasts have fewer nuclear pores. A) Representative fluorescent images of MAB414 (green, top) expression on fibroblast nuclei. A 2µm by 2µm square from the middle of the nuclei was used for nuclear pore quantification and was counted 2 manually (white, bottom). B) Quantification of nuclear pore number per 1µm for an unaffected (U1), unaffected carrier (U2) and affected (A2) patient fibroblasts. Affected patient nuclei have ~40% fewer nuclear pores. **P<0.01

30 either NUP107 or its binding partner, NUP133, results in a nucleus devoid of nuclear pores. Using an antibody against MAB414, which recognizes a subset of nucleoporins containing an FG repeat motif, we performed immunocytochemistry to visualize the number of nuclear pores on the nuclear envelope. Fluorescent nuclei were visualized via a confocal microscope. First, Z-stack images from the top of the nucleus through the bottom were collected. Then, a maximum intensity projection, that projects 3-D objects in the visual plane, was generated with software (Figure

2.4A). A 2µm x 2µm square section was taken from the middle of the nuclei for quantification, which represents the number of nuclear pores on the top and bottom of the nucleus throughout

2 that area (Figure 2.4B). There was no difference in the number of nuclear pores per µm between the unaffected individuals (U1, U2). But, there was ~40% decrease in the number of nuclear pores on nuclei from affected individuals (A1, A2, A4) compared to the unaffected. This is consistent with the decrease in NUP107 protein expression.

Morpholino knockdown of Nup107 during zebrafish development results in a microcephalic fish

A discrepancy exists between the in vitro knockdown and in vivo knockout studies. In vitro, knockdown of NUP107 or NUP133 results in a nuclei devoid or nuclear pores. Yet, in the

NUP133 null mouse and in npp-5 mutant C. Elegans, nuclear pores still form and are functional.

We already confirmed that a reduction in NUP107 in primary human cells result in fewer nuclear pores. In order to confirm that a decrease in NUP107 expression leaves result in a reduction of brain size in vivo, we use a nup107 translation blocking morpholino (MO) to reduce nup107 expression in zebrafish embryos. NUP107 knockdown efficiency was assessed by western blot

60 hours post fertilization (hpf), and we see a significant reduction in protein levels after MO treatment. This reduction can be attenuated via co-injection of the human NUP107 RNA (Figure

2.5B).

By 5 days post fertilization (dpf), there was an obvious small head phenotype in over 75% of the MO-injected embryos. This is compared to ~1% in the AB control embryos, and attenuated to ~40% in MO+RNA rescued embryos (Figure 2.5A). Occasionally, there were fish that

31 developed abnormally and did not live to 5 dpf. This was increased from ~2% in AB embryos to

~7% in MO-treated embryos and attenuated to ~4% in MO+RNA rescued embryos.

Quantification of body measurements, lateral images were collected at 5 dpf. When comparing rostral/caudal (R/C) body width and dorsal/ventral (D/V) body length, there were no differences between AB, MO, or MO+RNA groups (Figure 2.5I and 2.5J). Quantification of head and eye size were calculated from lateral and dorsal images. The MO-treated embryos had an increase distance between the eyes (D/V eye) compared to AB (Figure 2.5G). Co-injection with

RNA rescued this effect. The measurement across the widest part of the head (D/V head) was significantly reduced with MO injection and could not be rescued with RNA co-injection (Figure

2.5F).

The area of the eye (lateral eye) and the area of the skull (dorsal skull) were significantly reduced with MO injection (~35% and 15%, respectively) and partially rescued via RNA co- injection (~15% and ~9%, respectively decreased when compared to the AB control) (Figure 2.5D and 2.5E). The 15% decrease corresponds to a single plane through the skull. If we assume the skull is a sphere in shape, this corresponds to an ~20% in the volume of the skull.

Figure 2.5 Nup107 morpholino knockdown and rescue with NUP107 in zebrafish. (A) Injection of Nup107 translation blocking morpholino (MO) altered neurodevelopment. Range of phenotypes include normal, small head/eyes, and abnormal. Abnormal represents severely deformed and lethal phenotypes. Injection of the human NUP107 RNA rescues the small head phenotype at 100 pg. (B) Western blot showing reduction of NUP107 after MO injection. Levels are restored with injection of the human ortholog. (C) Lateral, Dorsal/Ventral (D/V) and Dorsal head images of AB, MO and MO+RNA zebrafish and measurements used for quantification. (D) Quantification of dorsal skull area (E) Lateral eye area (F) D/V head or (G) D/V between the eye measurements compared to the AB control. MO injection significantly decreased dorsal skull and lateral eye areas, compared to AB while RNA rescue partially rescued this decrease. (H) Lateral images of AB, MO, and MO+RNA zebrafish and measurements used for quantification of body size (I) Rostral/Caudal and D/V body measurements compares to AB control. There are no significant differences in body size between the groups. Data is expressed as mean SEM. ** p>0.01, *** p>0.001

CONCLUSION AND DISCUSSION

In this study, we investigate the role that the c.G303A NUP107 transition plays in the expression of NUP107 within MCPH affected patient fibroblasts. This transition does not affect any known domain or phosphorylation sites within the NUP107 protein. Changing the last base pair of exon 4 from a G to an A results in the decreased affinity of the splice site and a transcript lacking exon 4, as shown from RT-PCR. Skipping of exon 4 results in a premature stop codon near the beginning of exon 5; this, along with the fact that small amounts of the 3à5 transcript are seen in the affected patient cell suggests nonsense mediated decay of the 3à5 transcript.

The most important observation is that some normal 3à4à5 transcript was seen in MCPH affected patient cells. This suggests that the c.G303A transition does not represent a null allele of

NUP107, but a hypomorphic allele that acts to decrease the amount of wildtype transcript expressed. This result also suggests that some normal protein product is generated.

Indeed, western blot of NUP107 reveals a large decrease in the amount of protein generated in fibroblasts from affected, compared to unaffected individuals. In accordance to the literature, NUP133 is also depleted with NUP107 protein, further supporting the role of NUP107 in the stability of NUP133 incorporation.

Numerous knockdown studies of NUP107 have yield conflicting results on the necessity for NUP107 in nuclear pore formation. In C. Elegans, NUP107 is not required for nuclear pore formation; siRNA knockdown in human-derived HeLa cells and mutant zebrafish suggest that

NUP107 is essential for nuclear pore formation. One reason for this discrepancy may be that the authors of the C. Elegans paper may not have looked at the appropriate cell type for NUP107 affected on the nuclear pore. Indeed, the nup107 mutant zebrafish show decreased pore formation in the most proliferative area such as the head and digestive system but no change in pore density within the skeletal muscle.

Using an antibody against MAB414 to visualize nuclear pore density, we observe a ~40% decrease in the density. This suggests that the decreases in NUP107 destabilize the NUP107-

160 complexes not incorporated into the nuclear pore, and agrees with the previous human knockdown studies.

Furthermore, to confirm that a reduction of NUP107 in vivo results in brain size reduction, we used a MO to knockdown nup107 in zebrafish. MO-treated fish experience a ~15% reduction in skull area as seen from the dorsal view at 5 dpf, which is partially reverse with co-injection of the human NUP107 open reading frame. We would expect this small, but highly significant decrease in skull size to be more significant as the fish develops, but fish are lethal by 5 dpf.

Increasing the concentration of the MO results in a greater reduction of head size, but fish are ill before 5 dpf. In addition to the decrease in skull size, the MO-treated zebrafish did not have a swim bladder, had deformed and missing pharyngeal arches, and significantly smaller eyes, without a change in other body dimensions. Interestingly, co-injection with lower doses of human

NUP107 rescues the eye size earlier than while higher doses rescued the size of the brain and the jaw (data not shown). These points further suggest that the expression of NUP107 is dose- dependent in different cell types.

Normally, we would use both a translation blocking and splice blocking MO to confirm our results. Unfortunately, the maternal transcript is long-lived within the embryos and would not be affected with the splice-blocking MO. A recent report confirmed all of our zebrafish results using a nup107 mutant zebrafish. In addition to confirming our MO phenotype, Zheng et al. also showed that NUP107 expression is confined to highly proliferative cells during embryogenesis.

Furthermore, reduction of nup107 resulted in widespread apoptosis within proliferative tissues, in the absence of cell cycle defects.

Many studies have now linked the nuclear pore components to kinetochore and spindle stabilization during mitosis, resulting in the activation of the SAC cell cycle checkpoint. All known

MCPH genes also regulate some aspect of spindle formation during mitosis. These studies suggest that the mechanism of MCPH formation in family MIC-670 could be due to the reduced levels of NUP107 during mitosis.

The in vivo NUP107 knockdown results do not support this conclusion. The zebrafish experiments and cell cycle analysis in primary fibroblasts do not reveal disruptions to the cell cycle.

Unsuccessfully, we tried to generate NUP107 knockout mice, via NUP107 gene trapped cell lines generated by EUCOMM. Further attempts to generate conditional NUP107 mice, with loxP site flanking exons 2 and 3, have yet to yield germ line transmission, although the targeting construct has been successfully generated and electroporated into mESCs.

Overall, the data presented within this chapter suggests that the expression, protein levels, and stability of the nuclear pore are affected by the c.G303A transition within NUP107.

Furthermore, the zebrafish experiments suggest a role of NUP107 in brain size formation.

MATERIALS AND METHODS

Dermal punch biopsies. All biopsies were collected with appropriate informed consent and IRB approval. For individuals U1, U2, A2, and A4, a 4 mm dermal punch biopsy was collected, by a licensed physician, using sterile Sterling Medical Skin Biopsy Kits (Delasco). For individual A1, a fibroblast sample was collected during an elective surgery for clinodactyly. All tissue was collected, placed into human fibroblast media (DMEM containing L-glutamine (Gibco),

20% benchmark FBS (Gemini), and 1X P/S (Gibco)) in 15mL conical vials (BD Biosciences), sealed with parafilm (Parafilm), and shipped to our laboratory.

Primary fibroblast isolation and culture. Primary fibroblast cultures were established from a punch biopsy as described previously23. Cultures were expanded by dissociating confluent samples with 0.25% trypsin-EDTA (Gibco) and subcultivated at a ratio of 1:3. Media was changed every 2-3 days. For cryopreservation, 5x105 fibroblasts were slowly cooled overnight in a solution of 90% human fibroblast media + 10% DMSO (Sigma) in a “Mr. Frosty” container (Sigma) to -

80°C. Within 24 hours, CryoTubes (Nunc) were transferred for long-term storage into liquid nitrogen vapor.

Total RNA extraction. Total RNA from 5x105 fibroblasts was extracted with 1 mL TRIzol

(Invitrogen) and reverse-transcribed with SuperScript III First-Strand Synthesis (Invitrogen) using oligo(dT) primers according to the manufacturer’s directions.

Reverse transcriptase PCR. Semi-quantitative real-time polymerase reactions (RT-

PCR) were performed in biological triplicate using a primer set that amplified a fragment of

NUP107 between exons 3-5 (primer sequences are listed in Appendix III). RT-PCR products were resolved on a 1.5% agarose gel with ethidium bromide, imaged with a ChemiDoc (Bio-Rad) and pixel intensity for each band quantified using ImageJ software (NIH Image). Pixel intensities were normalized to expression levels of GAPDH.

Cloning for identification of mRNA splicing. An RT-PCR reaction was performed with

Taq polymerase to amplify the NUP107 cDNA transcript between exons 3-5. The PCR product was cloned into the pCR2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen) according to manufacturer instructions. Ten individual colonies were cultured overnight in 2ml LB medium containing 50 µg/ml ampicillin. Plasmid DNA was isolated using a Plasmid Mini Kit (Qiagen).

Protein extraction and western blot analysis. Protein was extracted, from exponentially growing fibroblast cells, in RIPA lysis buffer (50mM Tris-HCl, pH7.5; 150 mM NaCl;

1mM Na2EDTA; 1% NP-40; 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) supplemented with protease and phosphatase inhibitor cocktails (Sigma). Protein concentrations were measured at 570 nm using the microtiter plate protocol in the Bio-Rad Protein Assay kit

(Bio-Rad). Samples were boiled with 4X Laemmli sample buffer and 35 µg total proteins resolved on a 4-15% linear gradient polyacrylamide gel (Bio-Rad) and transferred onto PVDF membrane

(Chemicon). In addition to the standard colored recombinant protein standards, the WesternC protein standard (Bio-Rad), contains Strep-tag proteins that can be detected with a StrepTactin-

HRP secondary and visualized using chemiluminescence substrates. Primary antibodies were diluted in 5% non-fat milk and membranes incubated overnight at 4°C. After washing in PBS containing 0.1% Tween 20 (Sigma) (PBS-T), the membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (PIERCE) for 1 hour at room temperature.

After washing with PBS-T, the HRP signals were detected using SuperSignal West Dura chemiluminescent substrate (Thermo) and imaged with a ChemiDoc (Bio-Rad). ImageJ gel quantification was used to compare the intensity of the bands in each lane as normalized to the intensity of the α-tubulin band. Antibodies used are listed in Appendix V.

Immunocytochemistry of primary fibroblasts. Fibroblasts were seeded at a density of

1x105 cells/slide onto 2-well Lab-Tek II Permanox chamber slides (Cole Parmer) coated with poly-L-lysine (Sigma). After 3 days, slides were fixed in 4% paraformaldehyde for 20 min at room temperature and blocked in 2.5% normal donkey serum (Invitrogen) and permeabolized with

0.5% Triton-X-100 (Sigma). Primary antibodies were incubated for at least 1 hour at room temperature or overnight at 4°C. Slides were washed at least 3 times in PBS before secondary antibodies (Molecular Probes) were applied for 1 hour at room temperature. Antibodies used are listed in Appendix V. DNA was stained with Hoescht-33342 (Molecular Probes) for 5 minutes.

Slides were mounted for imaging in Mowoil (Sigma).

Flow cytometry for cell cycle analysis. Exponentially growing fibroblasts were dissociated in Accutase and fixed dropwise in 70% ethanol (-20°C) and stored at -20°C until analysis. FACS analysis was performed at the Flow Cytometry Research Core (Veterans

Medical Research Foundation) using a FACscan flow cytometer (BD Biosciences). Cells were treated with 50 µg/ml propidium iodide (PI), 50 µg/ml RNAse A and 2mM EGTA on ice for 30 minutes. Cell cycle phase was determined with ModFit LT V3.0 software (Verity Software House).

Zebrafish maintenance and breeding. Zebrafish experiments were approved by the

University of California, San Diego IACUC Animal Ethics Committee and maintained as described previously69. Embryos were kept in embryo medium (E3; 5mM NaCl, 0.17 mM CaCl2, 0.33 mM

MgSo4) at 28°C and changed to E3 containing 0.0045% 1-phenyl-2-thiourea (PTU) as described previously70. The wildtype line AB was used for all experiments (www.zfin.org).

Morpholino-mediated knockdown of nup107. A transcription-blocking antisense morpholino (MO) oligonucleotide was designed against the ATG start site of nup107 (GeneTools,

LLC; 5’ TCA ACC AAG TCT GAC TCC ATT CCA T 3’), diluted in sterile distilled H2O to a

38 concentration of 1mM, aliquot, and stored at -20°C. Before use, the MO was denatured for 5 min at 65°C and diluted to 5 ng/nl in a mixture containing 20% (v/v) phenol red (Sigma). Using a

Picospritzer III (Parker), a volume of 2nl was injected into 1- to 2- cells stage zebrafish embryos.

Embryos were maintained in E3 at 28°C.

Cloning and generation of human NUP107 RNA for zebrafish rescue. For rescue experiments, the full-length human NUP107 cDNA was amplified with vectors containing at least

20 base pare overhang complimentary to PCS2+ vector (Forward: 5’-TGC AGG ATC CCA TCG

ATT CGA TGG ACA GGA GTG GCT TTG G-3’; Reverse: 5’- CTA TAG TTC TAG AGG CTC

GAC TAT AAC TGA ATT TCA TAC CCT AAT GGG TCA-3’). The PCS2+ vector was cut with

EcoRI and XhoI and the human NUP107 open reading frame PCR fragment cloned into the space by isothermal assembly78. The PCS2+-hNUP107 vector was linearized with SnaBI and the mRNA transcribed using the T7 mMessage mMachine kit (Ambion) according to manufacturer’s directions. Rescue was performed with co-injection of 10 pg MO and approximately 100 pg of capped human NUP107 mRNA.

Zebrafish protein extraction for western blot. MO knockdown efficiency was assessed

60-hour post fertilization in AB, MO-injected and MO+RNA rescued embryos. Embryos were anesthetized in o.1% tricane (Sigma), dechorionated in an eppendorf tube with 50 µg/ml pronase

(Sigma) and de-yolked by pipetting 3-5 times through a P200 pipet tip. Then, the embryos were washed in PBS, suspended in 2X Laemmli SDS sample buffer (20% glycerol, 4% SDS, 10% β- mercaptoethanol, 0.004% bromophenol blue, 0.125M Tris-HCl (Sigma)) containing phosphatase and protease inhibitors (Sigma) at 2 µl/embryo. The embryos were homogenized by passing through a 25G needle. Resulting homogenates were boiled at 95C for 5 min and centrifuged at

15,000 x g for 5 minutes at 4°C. 10 µL (5 embryos) were loaded into each lane for analysis.

Imaging of quantification of fibroblast nuclear pores. Images of immunofluorescent stained cells were obtained at 100X on the Deltavision Deconvolution Microscope system

(Applied Precision, Inc.) with 5-10 rounds of deconvolution. Images were edited using Adobe

Photoshop CS. For quantification of nuclear pores, circles were manually drawn around pore

39 located within a 2µm x 2µm square box near the center of each nucleus. The total number of pores was counted from at least 30 nuclei from each fibroblast cell line (U1, U2 or A2).

Acridin orange staining. Dechorionated embryos were anesthetized with tricane and incubated in 10 ug/mL acridine orange (Sigma) for 30 minutes at 28°C. After staining, embryos were washed 3X in E3 for 5 minutes each wash. Zebrafish embryos were embedded in 1.5% low melting agarose and overlaid in embryo medium containing tricane.

Morphological analysis of zebrafish. At 5 days post fertilization, zebrafish were anesthetized in o.1% tricane (Sigma) and mount in 3% methylcellulose (Sigma) in the center of a glass depression slide (Fisher). The fish were imaged under a Zeiss compound microscope. For body measurements, lateral images were collected at 80x. For skull and eye quantifications, lateral and dorsal images were collected at 140X. Distance measurements were calculated in

Adobe Illustrator CS5. Rostral/caudal body length was measured from the anterior tip of the snout to the posterior caudal peduncle of the body. Dorsal/ventral (D/V) body width was measured from the genital pore to the dorsal surface. From the D/V image, the interocular distance between the eyes was measured between the most medial pigments. The head width was calculated between the most dorsal points of the lenses. The zebrafish eye is an ellipse, so the eye area was calculated using the height and width of an ellipse: A = Πab; a= ½ width, b= ½ height. The dorsal skull area was measured by tracing the visible boundary of the skull in ImageJ (NIH)l.

Statistical analysis. Mean values and standard deviations were calculated with

Microsoft Excel. Statistical analyses were performed using a 1- or 2-tailed Student’s t-test with equal variance. Data are represented as mean +/- standard error of the mean (SEM). *p<0.05,

**p<0.01, ***p<0.001.

ACKNOWLEDGEMENTS

Chapter 2 contains work submitted for publication by authors Bethany N. Sotak,

Stephanie A. Bielas, Jennifer L. Silhavy, Shu Tu, Ozgur Rosti, Hulya Kayserilli, Amanda D.

Yazguirre, Sofia Infante, Whitney Thuong, Neil Chi, Joseph G. Gleeson. The dissertation author was the primary investigator and author of this material.

CHAPTER 3.

Human MCPH modeling in a dish: Generation and characterization of an induced-

pluripotent stem cell model of MPCH from family MIC-670

ABSTRACT

Our understanding of human autosomal recessive microcephaly is limited by our inability to obtain human neural progenitors from individuals diagnosed with MCPH. Although animal models are useful for studying mechanisms of neurogenesis, the transcription patterns during human fetal development are unique. It may be possible to overcome these limitations by reprogramming human primary fibroblasts from affected MCPH patients into induced-pluripotent stem cells.

Here, we report the generation of patient-specific iPSCs from patient fibroblasts harboring a mutation in NUP107. These MCPH-iPSCs express pluripotent markers, have silencing of the viral transgenes, reactivation of the endogenous pluripotency genes, maintain karyotype and differentiate into all three embryonic germ layers. Additionally, the MCPH-iPSCs retain the nuclear pore phenotypes seen in the original parental fibroblast lines, including altered exon 4 splicing, decreases in NUP107 and NUP133 proteins, and fewer nuclear pores. When directed to differentiate towards the neuronal lineage, MCPH-iPSCs and control iPSCs both express markers on the neuroepithelium within 5 days. But, The formation of organized neuronal rosette structures is drastically delayed. This is the first report of an iPSC model of a neurodevelopmental disease, such as MCPH.

BACKGROUND AND SIGNIFICANCE

One of the major limitations of human neurological research is access to the cell type of interest, most notably the neurons and their progenitors. Theoretically, human pluripotent stem cells can differentiate into all cell types that make-up the embryo. These cells have the ability to

41 42 provide researchers with an unlimited supply of live human cells, such as neurons, that can be manipulated and used to study and test specific scientific questions. Therefore, pluripotent cells provide a powerful tool to identify human disease pathogenesis and for drug discovery and drug toxicity screening246,247.

In 2006, a Japanese group, led by Shinya Yamanaka, identified a set of factors that could reprogram somatic cells into pluripotent-like cells called induced-pluripotent stem cells (iPSCs) 248 that are morphologically and phenotypically similar to embryonic stem cells. In 2007, Yamanaka’s group reprogrammed the first human somatic cells into iPSCs249. Reprogramming requires expression of OCT4 and SOX2, whereas other factors Nanog, KLF4, c-myc and Lin28 have been identified as supporting factors that increase the efficiency of the reprogramming process250-252. In

2012, Yamanaka, along with John Gurdon, was awarded the Nobel prize for Physiology or

Medicine for his technological advancement of somatic cell reprogramming.

Over the last 5 years, the Yamanaka method of retroviral integration into the human genome for reprogramming is no longer necessary. Alternative approaches, which do not add genetic modifications, include non-integrating lentiviral vectors253, protein transduction254, miRNA inhibition255. Reprogramming can also be enhanced with the use of small molecules256-259.

One of the greatest strengths of iPSC technology is that iPSCs are patient-specific, containing the exact genetic make-up of the original somatic cell, making for a powerful application to study genetic diseases. Previously, genetic modifications had to be introduced into hESCs by a process called homologous recombination. But, homologous recombination (HR) is a rare event260 and there have only been a handful of publications reporting successful HR260-271.

Another strength of the iPSC technology is that polygenic diseases, involving multiple known and unknown genetic mutations and modifiers can be studied in addition to recessive monogenic diseases. In order to study a recessive disorder in hESCs, both gene alleles had to be independently targeted in the same hESC, which has only been reported twice262,263. Therefore, the iPSC technology provides an ideal model in order to generate neuronal-specific cell types for the study of MCPH.

Using molecular growth factors and small molecules to stimulate specific signaling cascades, pluripotent cells can be directed to differentiate towards a specific cell type. Dual-

SMAD inhibition, using dorsomorphin, an activin receptor-like kinase (ALK) ALK2/3/6 inhibitor that blocks SMAD 1/5/8 phosphorylation272, and SB431542, an ALK5/4/7 inhibitor the blocks

Lefty/Activin/TGFβ pathways, prevents signaling cascades necessary for differentiation to trophectoderm, me endoderm and ectoderm. Within 7 days, >80% of the cells lose OCT4 expression and become PAX6+273, an early marker of neuroectoderm differentiation274. These early PAX6+ cells express markers of neuroectoderm, such as Nestin and PLZF275, and will

Figure 3.1 Neuronal rosette model of cortical neurogenesis. A) Fluorescent image (left) and cartoon (right) of human neuronal rosette. B) Fluorescent image (left) and cartoon (right) of early mouse embryonic cortex at E15.5. Both of the human neuronal rosettes and mouse embryonic cortex is composed of a similar structure. The apically-located proteins ZO-1 and N- Cadherin represent the ventricular surface. Located at the apical surface are the neuronal progenitors (Pax6, green), with more differentiated cells, such as intermediate progenitors (TBR2, red) and post-mitotic neurons (DCX, blue) moving further away. As in mouse corticogenesis, cells secreting reeling (RELN) are located towards the basal surface. Adapted from Au and Fishell, 20081.

44 eventually self-polarize to form neuronal rosette structures, indicating un-polarized PAX6+ cells as an early neuronal lineage progenitor276.

Numerous studies have demonstrated a specific role of the interactions between the cell and its surrounding microenvironment during embryonic development277-282. Therefore, recapitulation of the developmental niche may be necessary to study molecular interactions within the developing cortex. The formation of the self-polarizing neuronal rosette is not well understood, but has been proposed as a model of early neurulation and corticogenesis283-285. Importantly, signaling pathways important for progenitor fate decisions, such as differentiation self-renewal, are active in the neuronal rosette structure286,287.

Mouse ESC-derived neuronal rosettes follow a pattern of neurogenesis similar to that of the developing embryonic mouse cortex285. Human neuronal rosette formation also follows a similar pattern of apical-basal polarity. The lumen of the neuronal rosette resembles the ventricle with apically-polarized proteins, ZO-1 and N-cadherin, at the apical surface275,285. The neuronal progenitor cells (PAX6+) reside at this apical surface similar to the ventricular zone; the progenitor cells form a pseudostratified neuroepithelium and the nuclei undergo interkinetic nuclear migrations before mitosis occurs at the apical surface288. Basal progenitors (TBR2+) reside toward the periphery of the rosette while immature neurons (TUJ1+, DCX+) migrate away from the apical surface (Figure 3.1) forming neuronal subtypes from each layer of the developing cortex in a fixed temporal order288.

Currently, mouse models may not be adequate for studies of human corticogenesis. In the mouse, cortical neurogenesis lasts around 6 days while human corticogenesis can span over

100289. Anther difference, as discussed in Chapter 2, in that humans generation of neuronal progenitors that reside in the outer subventricular zone (OSVZ)290,291. Like their VZ counterparts,

OSVZ progenitors can self-renew and differentiate to form basal progenitors and neurons. But, the VZ progenitors are polarized in a apical/basal axis and this axis controls the symmetry of the division 39,292. The differences between the OSVZ and VZ progenitors is not well understood, but the addition of the OSVZ progenitors appears to affect the size of the brain and complexity of its

45 folding293. Overall, studies on cortical development in mouse have provided the basic knowledge of structure and function, there is a necessity of a developmental model that can better recapitulate human corticogenesis if we are to better understand human neurodevelopmental diseases.

Currently, iPSC models have been generated for some neurological diseases:

Parkinson’s Disease (PD), Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA),

RETT syndrome, Huntington’s disease (HD), Friedreich ataxia, Familial dysautonomia, schizophrenia, and Alzheimer’s Disease (AD)294-301. In the cases of PD, ALS, HD, no obvious cell defects were identified294-296,302,303 stressing the need for an identifiable cellular defect in cellular models. To date, a neurodevelopmental iPSC model has yet to be reported.

In this chapter, we test the hypothesis that fibroblast, from unaffected and affected individuals in family MIC670 can be reprogrammed into iPSCs with the ability to differentiate into all germ layers. Additionally, we test the hypothesis that MCPH patient-derived iPSCs can make neurons, but inefficiently differentiate along the neuronal lineage using markers of neuroepithelial cells, radial glia, basal progenitors, and immature neurons. This chronological and spatial differentiation along the neuronal lineage can be observed using the self-organizing neuronal rosette as a model of human corticogenesis.

RESULTS

MCPH-iPSCs are pluripotent, have retroviral silencing, and can differentiate into all germ layers

Primary fibroblasts from an unaffected individual (U1) and an affected individual (A2) were reprogrammed into clonal iPSC lines using retroviruses expressing OCT4, SOX2, C-MYC, and KLF4. Both fibroblast lines were transduced at passage 3. Three unique clones (U1.1, U1.2,

U1.3 and A2.1, A2.2, A2.3) were expanded and analyzed for all further experiments.

Colonies were manually selected that had morphological resemblance to human embryonic stem cells (hESCs); good colonies had sharp borders, were densely packed, had a

Figure 3.2 Maintenance of euploidy and expression of pluripotency markers in iPSCs. Euploid karyotypes of iPSC lines (left). Representative brightfield images showing human embryonic stem cell-like morphology including tight borders, high nuclear/cytoplasmic ratio, and a continuous monolayer of cells. Representative immunofluorescence images showing expression of pluripotency marks TRA1-60/OCT4, TRA-1-80/SOX2, and SSEA4/NANOG. Representative images showing alkaline phosphatase expression (right). Three clones each from an unaffected (U1.1, U1.2 and U1.3) and an affected individual (A2.1, A2.2, and A2.3) from family MIC-670 were used for these studies.

47 consistent flat monolayer of cells, and a high nucleus to cytoplasm ratio. From 1 x 105 fibroblasts, approximately 100 colonies were produced and 30 colonies isolated for clonal expansion on

Matrigel. After 5 passages, this was narrowed to 3 different clonal lines per individual for expansion to passage 10.

At passage 10, iPSC colonies were analyzed for stability of the euploid karyotype, hESC- like morphology, expression of pluripotency markers, silencing of the retroviral transgene, activation of endogenous genes necessary for pluripotency, and their ability to differentiate into all three germ layers.

All colonies resembled hESC and had tight borders, a densely packed monolayer, and high nuclear to cytoplasmic ratio (Figure 3.2). Colonies also had little or no spontaneous differentiation. In addition, the iPSC clones were analyzed by immunohistochemistry and express markers of pluripotency: OCT4, SOX2, TRA-1-80, TRA-1-61, SSEA-4, Nanog, and alkaline phosphatase (AP) (Figure 3.X). TRA-1-80, TRA-1-61, SSEA-4, Nanog, and AP are endogenous pluripotency markers and were never introduced into the cells by retroviral expression.

To confirm that the iPSC lines were genetically stable, they were sent for G-band karyotype analysis. All lines maintained euploid karyotypes and no rearrangements or deletions was observed in more than 1/20 cells analyzed (Figure 3.2). We allow the cutoff of 1/20 abnormal cells to account for cytological artifacts.

Although the iPSCs express OCT4 and SOX2 protein, as visualized by immunohistochemistry, the endogenous genes must be reactivated and the retroviral genes silenced. Transgene silencing allows differentiation of iPSCs and also provides evidence the pluripotent network has been re-activated in these cells. Primers were designed to detect RNA transcript from the retroviral transgenes. The forward primer recognized a region common to all transgenes while the reverse primers recognized sequences specific to OCT4, SOX2, C-MYC, or

KLF4. An additional control reaction that did not contain reverse transcriptase (-RT) was included as a negative control and fibroblast 7 days post transfection as a positive control. Primers recognizing the endogenous housekeeping gene, GAPDH, were included for a loading control. All

Figure 3.3 Retroviral transgene silencing and endogenous expression in iPSCs. A) RT- PCR data comparing the transgene-specific expression in iPSC cultures versus fibroblast cells 3 days post transduction (4F). Primers amplified a region between to the transgene 3’UTR and the gene of interest. B) RT-PCR data comparing endogenous expression in iPSC cultures versus transduced fibroblasts. Primers amplified regions specific to the endogenously expressed gene. A null reverse transcriptase (-RT) control was added, number of amplification cycles is listed to the right. iPSC lines expressed reduced of undetectable levels of transgenes relative to the transduced fibroblasts (Figure 3.3A).

Additionally, all IPSC lines must show a re-activation of the endogenous genes required for pluripotency. Primers were designed to specifically recognize the endogenous expression of

OCT4, SOX2, KLF4 and C-MYC using the 3’ UTR that is not expressed in the transgene. All iPSC lines show robust activation of endogenous OCT4 and SOX2, while levels of KLF4 and C-

MYC were expressed at much lower levels, but still above endogenous levels in fibroblasts

(Figure 3.3B). This is consistent with published expression profiles of hESCs and suggests that iPSC lines have re-activated the pluripotency program.

Lastly, a “bone fide” pluripotent stem cell should be able to differentiate into every germ layer. In mice, the gold standard of pluripotency is tetraploid complementation in which mouse

ESCs are injected into a blastocyst and contribute to all tissues of the developing mouse. In the past, the gold standard for pluripotency in human cells has been the teratoma formation assay. In this assay, hESCs or iPSCs are injected into an immuno-compromised mouse where a tumor is allowed to develop spontaneously in vivo. Then, the tumor is removed and histology performed to confirm the presence of all germ layers. Tetraploid complementation in humans is unethical and teratoma formation is becoming obsolete and unnecessary for studying differentiated cell types, such as neuronal progenitors. To determine if the iPSCs are capable of differentiating into mesoderm, endoderm, and ectoderm in vitro, iPSCs were spontaneous differentiated using embryoid body (EB) formation.

For EB formation, colonies from all iPSC lines were cut into pieces and placed in non- adherent conditions. All growth factors were removed and EBs allowed to spontaneously differentiate. After 7 days of non-adherent growth and 7 days on Matrigel-coated slides, slides were analyzed for differentiation in the germ layers. Immunocytochemistry identified markers of endoderm (α-fetoprotein; AFP), mesoderm (smooth muscle actin; SMA) and ectoderm (β-III tubulin; TUJ1) within each slide (Figure 3.4)

These data suggest that all of the iPSC lines used for this study expressed markers of pluripotency, closely resembled hESC morphology, downregulated the retroviral transgenes, and could differentiate 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 and nuclear pore density

Figure 3.4 Differentiation of iPSCs into all germ layers. Representative images of iPSC lines differentiated into endoderm (AFP, alpha fetoprotein), mesoderm (SMA, smooth muscle actin), and ectoderm (TUJ1, beta-III tubulin).

In chapter 2, we showed that the c.G303A transition in fibroblasts resulted in the reduction of the splice donor affinity of exon 4. To confirm that our iPSCs still maintain that phenotype, we performed quantitative RT-PCR using primers that amplify along the NUP107 transcript, using GAPDH as a control. Primers amplifying transcript between exons 2-3, 3-4, 4-5, and 27-28 show that normal 3à4à5 splicing occurs in the affected individual iPSCs (A2.1, A2.2,

A2.3) at ~17% of the levels of the unaffected iPSCs (U1.1, U1.2, U1.3) (Figure 3.6A). Low levels of amplification between exons 2-3 and 27-28 further suggest nonsense mediated decay of the

3à5 transcript.

Previous studies from Beohmer et al. suggest that depletions of NUP107 effects the protein levels in all components of the NUP107-160 complex, but not the levels of the transcripts.

Indeed, RT-PCR comparing iPSCs generated from affected or unaffected individuals verify the reduction in NUP107 transcript in without a change in NUP133, NUP358, NUP214 or P62

(NUP62) transcription (Figure 3.6B).

To verify the predicted reduction in NUP107 and other components of the NUP107-160 complex, we used specific antibodies against NUP107 and NUP133, and the MAB414 antibody recognizing P62, NUP153, NUP214, and NUP358. α-tubulin was used as a loading control.

We see a reduction in all NUP107-160 complex members without any changed to P62

Figure 3.5 NUP107 protein expression and quantification in iPSCs. A) Western blot showing decreased expression of NUP107 and NUP133 (arrows) in iPSCs generated from affected individuals A1, A2 and A4 compared to the unaffected individual, U1. Additionally, using MAB414, an antibody that recognizes NUP358, NUP214, and P62 (NUP62), we see a decrease in NUP358 and NUP214 (arrows), but not in P62. B) Quantification of bands by pixel intensity, compared to GAPDH. Data represented as mean +/- SEM.

52 expression (Figure 3.5A). The intensity of each band on western blot was calculated using densitometry software (ImageJ) and normalized to α-tubulin intensity (Figure 3.5B). This is consistent with previous data.

As with the fibroblast data, MAB414 immunocytochemistry was used to visualize nuclear pores. Fluorescent nuclei were imaged using a 100X objective; Z-stack images were obtained and projected using a maximum intensity projection. For nuclear pore quantification, a 2µm x 2µm square images were selected from an internal region of each nucleus, representing the number of nuclear pores present on the top and the bottom of the nucleus through that area. Relative

*** *** *** ***

Figure 3.6 Decreases NUP107, but not other NUP107-160 components, is regulated by transcription. A) qRT-PCR using primers that amplify NUP107 between exons 2-3, 3-4, 4-5, and 27-28. Like in fibroblasts, the splice affinity of exon 4 is decreased and inclusion of exon 4 occurs ~16%. Data represented by mean +/- SEM. ***p<0.001 B) RT-PCR suggests that NU107 (arrow), but not other components of the NUP107-160 complex, is regulated by transcription. Data are represented as mean +/- SEM. *** P<0.001 fluorescent intensity was calculated with ImageJ (Figure 3.7B). In addition, a mean intensity filter of 1.2 pixels was applied to the images to necessitate manual nuclear pore counting (Figure

2 3.7C). By both methods, there was a 30% decrease in the number of pores per 4 µm area in iPSCs generated from affected vs. control individuals. Representative images are displayed as a

Z-plane cross section, maximum intensity projection, a zoomed image of the region used for counting, and the zoomed image after the maximum filter was applied (Figure 3.7A) This data suggests that a decreased NUP107 protein expression leads to reduction of number of nuclear pores.

***

Figure 3.7 Nuclear pore quantification in MCPH-iPSCs. A) Representative fluorescent images of 100X nuclei. Nuclear pores are visualized with the MAB414 antibody, which recognizes a subset of nuclear pore components. Images are displayed top to bottom as a single Z-stack (top), maximum intensity projection, a zoom of a selection from the middle of a nucleus, and with a maximum intensity filter (bottom). Visually, nuclei from affected individuals have fewer nuclear pores. B) Quantification of nuclear pores by relative fluorescence and C) manual count of pores show a significant 30% decrease in nuclear pore number in iPSCs generated from affected individuals. Data are represented as mean +/- SEM. *** p>0.001

MCPH-iPSCs can differentiate along the neuronal lineage but have delayed organization of neuronal rosettes

As pluripotent cells differentiate, the cells become progressively more restricted in differentiation potential and will change expression patterns. Pluripotent (OCT4+) cells can differentiate into all three major germ layers: mesoderm, endoderm, ectoderm. Cell destined for neurogenesis first become neuroectoderm (NE; OCT4-, PAX6+). Radial glial (RG; PAX6+,

BLBP+) cells, a more restricted NE subtype will give rise to the cell of the cortex. During corticogenesis, the RG cells can self-renew and divide symmetrically to generate expand the RG population, or divide asymmetrically forming a RG and a neuron (TUJ1+, DCX+) or a basal progenitor cell (BP; TBR2+, PAX6-). BP cells divide symmetrically to generate two neurons. RG cells that lose their apical attachment, but not their differentiative capacity, are called outer RG cells (ORG). (Figure 3.8A)

In order to study human cortical development, we used dual-SMAD inhibition to drive neuroepithelial differentiation. By qRT-PCR, iPSCs consistently lost pluripotency and began to express markers of neuroepithelial lineage around 5 days post differentiation (dpd). At 14 dpd, the NE/(O)RG cells started to express markers of BP and immature neurons. When compared to pluripotent iPSCs form the unaffected individual, MCPH-iPSCs express more PAX6 (8, 14 and 17 dpd) and less BLBP (14 and 17 dpd) (Figure 3.8B). When the change in expression is analyzed by cell genotype over the differentiation time, the BLBP expression is not significantly different after 5 dpd in MCPH-iPSCs but significantly increases over time in iPSCs from the unaffected individual (Figure 3.8C). This suggests that the RG population is reduced during the differentiation of iPSCs from affected MCPH individuals.

Interestingly, within 7 days, ZO-1+ neuronal rosettes were observed in iPSCs from the unaffected individual. This was true for all three clones analyzed. In contrast, ZO-1+ rosette structures were not visible until ~day 30 in iPSCs from the affected individual (Figure 3.9A). ZO-

1+ puncta is an average of three biological replicates from each of three clones (Figure 3.9B).

Figure 3.8 Directed differentiation of iPSCs into neurons A) Schematic time course of directed differentiation of iPSCs into neurons. IPSC express pluripotency marker OCT4. Within 5 days post differentiation (dpd), cells lose OCT4 expression and express PAX6 as neuroepithelial (NE) progenitors. NE progenitors expressing BLBP are radial glial (RG) cells, which can self-renew and expand, or can self-renew and differentiate into TBR2+ basal progenitors (BP) and TUJ1+ neurons. Basal progenitors will divide to yield two neurons. B) Quantitative RT-PCR comparing expression between iPSCs from unaffected and affected individuals at 0, 5, 8, 14, and 17 dpd. All data is an average of three biological replicates from each of three clones per individual. iPSCs from affected individuals have increase PAX6 expression at 8, 14 and 17 dpd and decreased BLBP expression at 14 and 17 dpd. C) Quantitative RT-PCR comparing the changes in expression within each genotype during differentiation. Cells from U1 are on the left, A2 on the right. OCT4 expression decreases within 5 dpd, PAX6 begins a reciprocal increase, as well as BLBP. Although, BLBP expression does not increase after 8 dpd in affected-individual iPSCs. All data are represented as mean +/- SEM. *p<0.05. Figure continued on next page.

Figure 3.8 Directed differentiation of iPSCs into neurons, Continued. TBR2 and DCX expression begins around 14 dpd. All data are represented as mean +/- SEM. *p<0.05

The directed differentiation experiments suggest that the RG population plays a role in the development of neuronal rosette structures in affected MCPH-iPSCs.

Decreases in NUP107 do not affect the cell cycle length or Aurora B localization during mitosis

*** *** *** **

Figure 3.9 Directed differentiation of control and MCPH-iPSCs into neuronal rosettes. A) Representative images of neuronal rosette formation. Neuroepithelial cells express PAX6 (green), but rosettes have a highly polarized apical domain expressing ZO-1 (red). In control cultures (U1), rosettes begin to form around day 7. In affected patient cells, rosettes rarely form before day 30. B) Quantification of ZO-1+ lumen per 10X optical field. Data are represented by the mean +/- SEM. ** p<0.01, ***p<0.001

Based on literature suggesting a role for both MCPH genes and NUP107 in cell cycle control, we performed flow cytometry in iPSCs to analyze DNA content (Figure 3.10A). Single cell iPSC cultures were stained with propidium iodide (PI), which binds to DNA by intercalation with no preference for DNA sequence. Therefore, one dye binds per 4.5 base pairs; based on fluorescent intensity of the PI, DNA content in a cell can be measured. Cells in G0/G1 are diploid and have 2N DNA. Cells in S phase will begin to duplicate their DNA, binding more PI. Cell in

G2/M should have twice the PI fluorescent intensity (4N). The fractional DNA content due to DNA degradation can identify Apoptotic cells by the apoptosis-associated endonuclease304,305. iPSCs from unaffected (U1) or MCPH-affected (A1 and A2) individuals had similar amounts of cells in

Figure 3.10 Cell cycle analysis in MCPH and control-derived iPSCs. A) Representative cell-cycle distribution and ModFit plots of propidium iodide (PI) concentration for one unaffected (U1, left), and two affected patients (A1, A2). Cell cycle phases are calculated by the concentration of PI. B) Quantification of the percent of cells in each phase of the cell cycle (G1, G2/M or S). MCPH-iPSCs have more cells in S phase C) Quantification of the percent of cell population undergoing programmed cell death (apoptosis). *p<0.05

G1/G0 and G2 phases of the cell cycle (Figure 3.10B). But, MCPH-iPSCs had more cells within S phase and also a higher population of apoptotic cells (Figure 3.10C)

With reports of Aurora B Kinase mislocalization during mitosis in MCPH patients33 and a known role of NUP107 in recruitment of the chromosomal passenger complex to the centromere197, we investigated the anaphase localization of Aurora B in dividing iPSCs. The CPC proteins play a role in the correct alignment of chromosomes and show dynamic localization during mitosis 283,306,307. First, the CPC localizes to all chromatin and becomes more localized to the centromere at prometaphase. At anaphase, the CPC is transferred to the midzone of the central spindle and then at the midbody during cytokinesis308. In our MCPH iPSCs, we could not detect any mislocalization of Aurora B during anaphase or telophase (Figure 3.11). These results suggest that the contribution of NUP107 to MCPH may not be related to cell cycle kinetics.

Lentiviral expression of NUP107 is silenced in MCPH-iPSCs

In order to rescue the delayed formation of neuronal rosettes, we generated a lentivirus containing a NUP107-ORF-IRES2-EGFP to over-express the 3à4à5 spliced NUP107 transcript

Figure 3.11 Anaphase distriubtion of Aurora B expression in iPSCs. Representative immunofluorescent images of anaphase Aurora B distribution in iPSCs from affected and unaffected individuals. At anaphase, Aurora B is transferred to the midzone of the central spindle and then to the midbody during cytokinesis. There are no visual localization differences between iPSCs.

59 in MCPH-iPSCs. Within 24 hours, we saw robust GFP expression within the iPSC cultures. But, after two passages for expansion, only 6.1% of the cells were EGFP+ based on flow cytometry.

After 5 passages, this was further reduced to 0.3% of the cells indicating that transduced cells may be under some kind of growth disadvantage.

A pure population of transduced EGFP+ cells (A2+Lenti) was generated by FACS; after one passage (passage 23; P23), colonies were consistently EGFP+ (Figure 3.12A) But, at passage 28, large portions of the colonies had lost EGFP expression (Figure 3.12B). This closely

Figure 3.12 Lentiviral-mediated expression in affected MCPH-iPSCs. A-B) Representative fluorescent images of induced pluripotent stem cell colonies (iPSCs) colonies expressing NUP107-IRES-EGFP2 at passage 23 and 28. Cell nuclei are stained with Hoechst 33343 (left) and express EGFP (right). Note that all cells express EGFP at passage 23, but have mosaic expression by passage 28. C-D) QRT-PCR of iPSCs derived from one unaffected (U1), and two affected patients (A1, A2). The NUP107 expressing lentivirus was used to rescue patient line A2 (A2+Lenti). Using primers that amplify a product between exons 3-4 and 3-5, the lentivirus restore NUP107 expression levels to 1.5X that of control at passage 23. By passage 28, the lentiviral expression has been reduced. E-F) Representative flow cytometry results showing that 6.1% of the cell population was GFP-positive and passage 25, but the same population at passage 28 only contained 0.3% of cells.

61 corresponded to loss of NUP107 expression. Using primers amplifying a product between exons

3-4 and 4-5, we determined that the A2+lenti cells express NUP107 at 1.7 fold that of U1, iPSCs from the unaffected individual, at passage 23 (Figure 3.12C). By passage 28, this expression was reduced to 60% of the control cell line indicating silencing of the viral transgene (Figure 3.12D).

Additionally, as analyzed by flow cytometry, 6.1% of the cells were GFP+ after initial transformation at passage 25 (Figure 3.12E) while 0.3% of the same, unsorted cells, were positive five passages later at passage 28 (Figure 3.12F).

CONCLUSION AND DISCUSSION

In this chapter, our data suggest that we have successfully generated human iPSCs from

MCPH patient fibroblasts. We have generated three clones each from one affected and one unaffected individual for our studies. Our iPSC lines can be expanded indefinitely, express critical pluripotency markers, silence the retroviral transgenes, maintain euploid karyotypes, and have the potential to differentiate in vitro into mesoderm, endoderm and ectoderm. This suggests that these MCPH-iPSCs can be directed into the neuronal lineage for generation and study of the properties of neuronal progenitors.

MCPH patient-iPSCs can be differentiated synchronously into neuroepithelial cells and self-organizing neuronal rosettes. Using dual-SMAD inhibition, OCT4, a marker of pluripotency, has been downregulated and an early marker of neuroepithelial cells, PAX6, is expressed consistently within 5 dpd in all iPSC lines. Neuronal rosettes, which resemble early cortical neurogenesis in vitro, consistently appear within 7 days post differentiation in control iPSCs, but not until 30 dpd in iPSCs from an affected individual. Further studies are required to find the mechanistic cause of this phenomenon. Our data suggests that the RG cell expansion may be one possible delay in the polarization of the lumen. Alternatively, polarization of the lumen may control the expansion of the RG population. Little is understood about the generation of the polarized rosette structure. Another possibility could be a delay in the differentiation and generation of radial glial-like cells from the early neuroepithelial precursors. This could either

62 indicate a change in gene regulation or a slowing of the cell cycle, which could decrease the amount of time the RG cells expand before differentiation, or increase premature differentiation of

RG cell into post-mitotic cells. Our RT-PCR experiments suggest a smaller RG population without premature expression of post-mitotic markers.

To rescue the rosette generation phenotype, we used a lentivirus expressing a NUP107-

ORF-IRES2-EGFP. Unfortunately, after initial transformation, 6.1% of the cells were GFP+ after initial transformation while 0.3% of the cells were positive five passages later. This indicates that cells expressing the NUP107 lentiviral transgene may be under some kind of growth disadvantage. GFP+ cells were collected by FACS and NUP107 expression measured to have increased 1.5-2 fold over control unaffected iPSCs, as seen by qRT-PCR, suggesting that cells incorporating multiple lentiviral insertions may be selected against or undergo apoptosis.

Additionally, GFP+ cells isolated by FACS silenced the viral transgene over time. When considered along with the zebrafish data, where phenotypic rescues were dose-dependent in the eye and head, our data suggest that NUP107 expression is tightly regulated within cell types. Too much or too little expression can cause developmental changes within the tissue. For a successful rescue experiment, lentiviral overexpression experiments may not be adequate.

Instead, genetic rescue using zinc finger of TALEN-mediated gene repair would be ideal.

Here, we have demonstrated a model in which iPSC technology can be used to study human neurodevelopmental diseases, not just neurodegenerative disease. For MCPH, most likely is a disease affecting the generation and maintenance of the neuronal progenitor population, which can be modeled using human neuronal rosettes. Because iPSC lines are patient-specific, these cells harbor the genetic makeup, including the disease-causing mutation and any genetic modifiers. Therefore can use these cells to investigate the mechanisms of disease. To our knowledge, this is the first iPSC model of MCPH.

MATERIALS AND METHODS

Retroviral production and iPSC generation. Retroviral pMXs vectors expressing the

Yaminaka factors (OCT4, SOX2, C-MYC, and KLF4) were separately transfected into 293T cells, along with the VSV-G pseudotype and pCMV-Gag-Pol (Addgene) packaging vectors using polyethylenimine (25kD linear, Polysciences, Inc.). Transfected cells were grown and 10% CO2,

37°C in fibroblast media. Viral supernatants were collected at 48 and 72 hours after infection,

5 combined, filtered and concentrated (19500 RPM, 2hr, 4°C). Fibroblasts were seeded at 8x10 cells/6 well plate and allowed to attach overnight. Three wells from each line were transduced with equal volumes of the four viral supernatants. After an overnight incubation with virus, the human fibroblast media was replaced. Two days after transduction, the fibroblasts were re-plated on a 10cm dish containing MEFs. The hESC media was also supplemented with 1mM valproic acid (Sigma) between days 2-8. Individual colonies were manually isolated onto Matrigel-coated

96-well plates in MEF-CM ~21 days after transduction. Three iPSC clones were selected each from one unaffected (A2; MIC-670-II-VI-1) and one affected (U1; MIC-670-I-VI-3) patient for further analysis.

IPSC culture. IPSCs were maintained on Matrigel-coated plates (BD Biosciences) in

MEF-CM supplemented with 20 ng/ml basic FGF (bFGF, Invitrogen). MEF-CM was produced from conditioning hESC media (containing 20% knockout serum replacement (KSR), 1% non- essential amino acids (NEAA) and 0.2% b-mercaptoethanol (BME)) in DMEM/F12 media (Gibco)) with mitomycin-C treated mouse embryonic fibroblasts (MEF, Millipore) for 24 hours. IPSCs were passaged manually after 1 hour pre-treatment with 10 mM Y-27632 ROCK inhibitor (Abcam

Biochemical).

Immunocytochemistry and imaging. IPSC colonies were plated on 2-well LabTek II

Permanox chamber slides (Cole Parmer) coated with Matrigel (BD Biosciences). After 5 days, slides were fixed in 4% paraformaldehyde for 20 min at room temperature and blocked/permeabolized in 2.5% normal donkey serum (Invitrogen) with 0.5% Triton-X-100

(Sigma). Primary antibodies were incubated for at least 1 hour at room temperature or overnight

64 at 4°C. Slides were washed at least 3 times in PBS before secondary antibodies (Molecular

Probes) were applied for 1 hour at room temperature. Antibodies used are listed in Appendix V.

DNA was stained with Hoescht-33342 (Molecular Probes) for 5 minutes. Alkaline phosphatase

(AP) was visualized with the Alkaline Phosphatase Detection Kit (Millipore) according to manufacturer’s directions. All slides were mounted for imaging in Mowoil (Sigma). Images for nuclear pore quantification were obtained on the Deltavision OMX super-resolution microscope

(Applied Precision). Images for all other experiments were obtained on either the FV-1000

Confocal or the ix51 inverted microscopes (Olympus).

Karyotype analysis of iPSCs. G-band karyotyping was performed by Cell Line Genetics

(Madison, WI) on live, undifferentiated iPSCs. 20 cells were analyzed per cell line. Karyotype was considered normal if at least 19/20 cells had no chromosomal aberrations.

Reverse transcriptase PCR. RT-PCR was performed with standard methods described in Chapter 1. RT-PCR products were resolved on a 1.5% agarose gel with ethidium bromide, imaged with a ChemiDoc (Bio-Rad) and pixel intensity for each band quantified using ImageJ software (NIH Image). Pixel intensities were normalized to expression levels of GAPDH. Primers for retroviral expression are listed in Appendix IV, endogenous gene expression in Appendix III.

Directed differentiation via dual-SMAD inhibition. IPSCs were rinsed with PBS and dissociated to a single cell suspension and suspended in MEF-CM containing 20 ng/mL bFGF

2 and 10 µM Y-27632. IPSCs were seeded at 18,000 cells/cm on Matrigel and allowed to grow to confluence with daily media changes. Once confluent, cells were fed with hESC medium. Each successive media change contained a higher percentage of N2 media (0%, 25%, 50%, 75%,

100%) until cells were cultured exclusively in N2. Media was changed daily and supplemented with 2.5 µM dorsomorphin and 10 µM SB431542 for 5 days started with the initial change to hESC medium.

Western immunoblotting. Proteins from non-confluent iPSC cultures were collected in

RIPA buffer and immunoblot using standard methods. Antibodies are details in Appendix V.

Germ layer differentiation through embryoid body formation. IPSC cultures were enzymatically passaged using 1 mg/ml Collagenase IV (Invitrogen) and embryoid bodies generated by plating cultures into 6 well plates and shaking at 95 RPM overnight. Starting on day

2, media was changed to DMEM containing 15% fetal bovine serum (FBS) with media changes every other day. After 7 days, EBs were plated onto Matrigel (BD bioscience)-coated plates or 2- well Permanox slides (Cole Parmer) and cultured for an additional 7 days before analysis.

Nuclear pore quantification in iPSCs. Images of immunofluorescently stained cells were obtained at 100X with an Applied Precision DeltaVision OMX super-resolution microscope

(Applied Precision). Using SoftWoRx software (Applied Precision), Z-stack images were aligned and deconvolved. Images were edited using Adobe Photoshop CS. Quantification of nuclear pores was calculated in a 2 µm (100 pixel) x 2µm square box near the center of each nucleus by two approaches: manually, or by relative fluorescence. For manual counting of pores, a mean intensity filter of 1.2 pixels was applied to the image. For total fluorescence, images were processed in a batch using ImageJ (NIH). First, a binary threshold was applied to the images in order to reflect the presence of fluorescent per pixel. Then, the percent of positive cells was determined. At least 30 x 160 nm2 images were used for quantification.

Quantitative RT-PCR. RNA was purified and reverse transcribed as described above.

Quantitative RT-PCR (qtr.-PCR) was performed on using 2X SYBR Green Master Mix (Roche) and primer pairs for genes listed in Appendix III. 10 µl Reactions were performed in 96 well plates

(Roche) on the LightCycler480 II Real Time PCR system using the ΔΔCt method. All expression levels were normalized to the mean levels of GAPDH.

Lentiviral generation and infection of iPSCs. A 3rd generation HIV-based lentiviral vector containing a NUP107ORF-IRES-EGFP2 insert driven by a CMV promoter (Ex-W1361-

Lv114, Genecopoeia) was used for lentiviral rescue of the IPSCs. The lentiviral construct was transfected, using polyethylenimine (25kD linear, Polysciences, Inc.), into HEK293T cells along with the 3rd generation packaging vectors pMDLg/pRRE and pRSV-Rev and the envelope pseudotyping plasmid pCMV-VSVG. Transfected cells were grown in 10% CO2, 37°C in

66 fibroblast media. Viral supernatants were collected at 48 and 72 hours after infection, combined, filtered and concentrated (19500 RPM, 2hr, 4°C). For lentiviral infection, iPSC colonies were dissociated in 1 mg/ml Dispase (Invitrogen), triturated to produce small colonies of 10-100 cells.

The cells were incubated at 37C for 1 hour while suspended in high-titer lentiviral-containing medium supplemented with 10 µM Y-27632 and 4 µg/ml polybrene (Sigma). The cell suspension, containing the lentivirus, was diluted 10-fold in MEF-CM and cells distributed onto Matrigel- covered plates. The MEF-CM was replaced daily and cells passaged as normal. To produce pure a pure GFP+ cell population, cultures were dissociated using Accutase (Invitrogen) and sorted in media containing 2mM EGTA on a FACSAria II cytometer (BD biosciences. Sorting was done under strict conditions to collect cells with high GFP expression.

Neuronal progenitor cell generation. For NP generation, IPSC cultures were enzymatically passaged using 1 mg/ml Collagenase IV (Invitrogen) and embryoid bodies generated by plating cultures into 6 well plates and shaking at 95 RPM overnight. Starting on day

2, media was changed to hESC medium. Each successive media change contained a higher percentage of N2 media (0%, 25%, 50%, 75%, 100%) until cells were cultured exclusively in N2.

Media was changed daily and supplemented with 2.5 µM dorsomorphin and 10 µM SB431542 for

5 days started with the initial change to hESC medium. After 7 days, EBs were plated in 6 well dishes coated in O/N successively in 20 µg/ml poly-l-ornithine (POL) and 5 µg/ml laminin

(POL/laminin). EBs were allowed to spread and grow for 4 days before neuronal rosettes were manually collected with a needle. The neuronal rosettes were dissociated by incubation in

Accutase and seeded onto POL/laminin-coated dishes at 500,000 cells/well in N2 medium supplemented with 20 µg/mL bFGF.

Statistical analysis. Mean values and standard deviations were calculated with

Microsoft Excel. Statistical analyses were performed using a 1- or 2-tailed Student’s t-test with equal variance. Data are represented as mean +/- standard error of the mean (SEM). *p<0.05,

**p<0.01, ***p<0.001.

ACKNOWLEDGEMENTS

Chapter 3 contains work submitted, in part, for publication by authors Bethany N. Sotak,

Stephanie A. Bielas, Jennifer L. Silhavy, Shu Tu, Ozgur Rosti, Hulya Kayserilli, Amanda D.

Yazguirre, Sofia Infante, Whitney Thuong, Neil Chi, Joseph G. Gleeson. The dissertation author was the primary investigator and author of this material.

APPENDIX I. RECIPES FOR CELL CULTURE MEDIA

Media Name Plate Base Serum Supplements Growth factors coating media

MEF Gelatin DMEM 10% FBS L-glutamine None

HEK293T None DMEM 10% FBS L-glutamine None

P/S*, L- Human None DMEM 15% FBS glutamine, None fibroblasts NEAA L-glutamine, Matrigel DMEM/ 10% KO serum iPSC NEAA, - 20 ng/mL bFGF or MEFs F12 replacement β mercaptoetha nol 600 nM 1X N2 dorsomorphin Differentiation None DMEM supplement, 1X NEAA (Sigma), 10 M B27 µ SB431542 (Tocris) 1X N2 NPCs Matrigel DMEM supplement, 1X NEAA 20 ng/mL bFGF B27 supplement MEF, mouse embryonic fibroblasts; FBS, fetal bovine serum; P/S, Penicillin/Streptomycin; NEAA, non-essential amino acids; MEF, mouse embryonic fibroblasts; iPSC, induced-pluripotent stem cells; KO, knockout; NPCs, neuronal progenitor cells *P/S was only used during biopsy shipping and fibroblast derivation

APPENDIX II. PRIMER SEQUENCES FOR GENOTYPING

Gene Application Orientation Sequence (5’-)

Genomic PCR F GGC GAA TGG GTG AGT AAC ACG CCR for mycoplasma R CGG ATA ACG CTT GCG ACC TAT G Genomic PCR F GGC CAA GGT CAT CCA TGA GADPH for mycoplasma R TCA GTG TAG CCC AGG NUP107 F AAA CGC GGT AGC TAA ACT GC Genomic PCR exon 1 R TTC ACC TAT TTC GTT TCG CC

NUP107 F AGG CTT TGA AGA ATA AGC AGG Genomic PCR exon 2 R ACC AAA TGA CAG CTC TGT GG

NUP107 F GGTAGCCCTAAAATACTTGTTCTTG Genomic PCR exon 3 R TGCTTATTGATATCGTAACCAAATAC

NUP107 F TTG ATA ATG AAT GCT CAC Genomic PCR exon 4 R TAA GCC ACC ACT CCC AGC

NUP107 F CCACTCAGTGACAATAACTCCATC Genomic PCR exon 5 R GAAATGGTTTCCTTCATCTTGC

NUP107 F CAGTGAATTTATAAAAGACTTGTAGCC Genomic PCR exon 6 R ACGCAGGGATGCTCTAGTTG

NUP107 F CACCTTGTTCCAAATTAGATCC Genomic PCR exon 7 R ATTTCCAAGCACGACCTACC

NUP107 F TCTCTCAGATGCTCAGTGGC Genomic PCR exon 8 R GAAACTCAAGATGGAAGGCTG

NUP107 F TTGGAATTTACACAATAGATACCTTTC Genomic PCR exon 9 R TGCATGTCATTAACCTTTTCTCC

NUP107 F TGAAACTGTTTGAATTAAGGGTAGTAG Genomic PCR exon 10 R TGCTCACACACCAAACAAAG

NUP107 F TCATCTGAGCTAAAATGTACTTCTG Genomic PCR exon 11 R CCATCAGATTCCAATGCAAC

NUP107 F CAATTTATTTCAGCCAGGCG Genomic PCR exon 12 R GTGCCTTATCACCGGAAATG

NUP107 F TGGGATGGATGGAACATTTG Genomic PCR exon 13-14 R TTTCACAGCCCCTGAATAGG

69 70

NUP107 F TTCTTCCTTGTTATTACCCCAC Genomic PCR exon 15 R TCAAATTAAGCAGAACAAGTCACTATC

NUP107 F TCATTGGATGTAATTGCTTTTCC Genomic PCR exon 16-17 R TTTTCCTTTCAGCTACAGTGC

NUP107 F CTAAGGCCCTTCATTTCCAG Genomic PCR exon 18 R CTGAGACTGAGCCACTGCAC

NUP107 F CAGTGACTTGAAAGCACTATAGGAG Genomic PCR exon 19 R ATGGAAGACTTTTGATTCCTTATC

NUP107 F TCAGGCAGTAGATGAACTTTGG Genomic PCR exon 20 R AATCCCTGCATTATTCAAAGG

NUP107 F CAAATCCACATCTGTATGAACCTC Genomic PCR exon 21 R ATTCCTCTTTTCCCCTCAAC

NUP107 F TTCAGGGAATTTCTGTTTTAGGAC Genomic PCR exon 22 R AAAGGTCTGGGCTGACAAAG

NUP107 F TGATGTTTACTGGAAGTACAGGTG Genomic PCR exon 23 R AGTGGCTGGTTTTCTCCTCC

NUP107 F TTTAACAGAGTGCCTTGGGC Genomic PCR exon 24 R TCCCCTTGTGGATGAGTCAG

NUP107 F ACAGTTTTATTTCTTGATGCTGC Genomic PCR exon 25 R AGGACATTAACAGGGAGATTACC

NUP107 F TTGGCAGACTGAAACATCAAC Genomic PCR exon 26 R AGATGCTCCCATTTAGAGACAG

NUP107 F TGGGTTTGATTCAGTACCTGG Genomic PCR exon 27 R GCTTACAAATATACACACATTTTGGG

NUP107 F TGGCTACATAAATCTTAAAGAGGC Genomic PCR exon 28 R CAAATGTCTAAATTTTACATGGTAATG

D16S3137, Genomic PCR F GCAAAGAATAATTGCACATATACG chr 16 for genotyping R AGTACAAAGAGATCCCCTGTAAC

GATA151F03, Genomic PCR F CTGGACAGTCAGTTGGAGCT chr 15 for genotyping R AAGGACTTACATGCACACTGC

D13S1851, Genomic PCR F ACATAGCTAGTCTGCATCTCA chr 13 for genotyping R GCAAGCAGCCGGATTCCATT

D6S292, Genomic PCR F AATTCACAAGACACAATCTCAG chr 6 for genotyping R AGAACTAAAGTTGCCTGTTCNTGTA

D3S2462, Genomic PCR F TTAATCTGCCAACTTGTCTGG chr 3 for genotyping R TTTTCACCTGTGCTGTTGCT

APPENDIX III. PRIMER SEQUENCES FOR RT-PCR AND/OR QRT-PCR

Gene Application Orientation Sequence (5’-)

NUP107 F CGG GAG GCA GAG GTG ACA q/RTPCR exon 2-3 R AAA AGA GTT TTA CTT CAG GCA TCT CA

NUP107 F TAT CCT CTG AGC GCC ACA AA q/RTPCR exon 3-4 R AAG GAA GTT GCT GCA GAA GCT C

NUP107 F TCC CTC GAA CTC CTA GCT CAT T q/RTPCR exon 4-5 R CAA CAA GCC GAA GCT TAC TAA GG

NUP107 F CTT CAG GGT TCT TTG GAA ATC TCT RT-PCR exon 3-5 R GTA ACT GGG CAG CTG CAT TTT

NUP107 F CCC TCG AAC TCC TAG CTC ATT TC RT-PCR exon 27-28 R GAT GAC AGT AAC TGG GCA GCT G F AAG GTG AAG GTC GGA GTC AA GAPDH q/RTPCR R AAT GAA GGG GTC ATT GAT GG F TCC AAA TCA GAG TGA GAG GAA AA NUP133 q/RTPCR R GAA AGC TGC CTT TCT GCA AT F GTA GCC ACC CTG GCA CAA TA NUP153 q/RTPCR R GGG CCA ATT AAG CCT TAC CA F CGC TAC CAC TGG AAG AGG AT NUP214 q/RTPCR R CAA GCT TCA GGT TTT GGG TC

NUP358 F ACA GCT TTG CAA AAT AGA ATC C q/RTPCR (RANBP2) R CTG ACG TGG AGC GGT ACA T

P62 F CTC CCA AAG CAA ATC CGT C q/RTPCR (NUP62) R CGG TTA CTC ACT CCA TGG CT F AAG GTG AAG GTC GGA GTC AA GAPDH q/RTPCR R AAT GAA GGG GTC ATT GAT GG

F TGG GCT CGA GAA GGA TGT G OCT4 q/RTPCR R GCA TAG TCG CTG CTT GAT CG

F GTT GGT ATC CGG GGA CTT C PAX6 q/RTPCR R TCC GTT GGA ACT GAT GGA GT

F CGC CAC CAA ACT GAG ATG AT

TBR2 q/RTPCR R CAG CAC CAC CTC TAC GAA CA

72 73

F AGA CCG GGG TTG TCA AAA A DCX q/RTPCR R TCA GGA CCA CAG GCA ATA AA

F ACA GAA ATG GGA TGG CAA AG BLBP q/RTPCR R AAC AGC AAC CAC ATC ACC AA

F CTA AAG ATG CAG GTT GTG CG ASCL1 q/RTPCR R GGA GCT TCT CGA CTT CAC CA

F CCC TCC AGC AGC TCA AGT TA ASCL2 q/RTPCR R GGC ACC AAC ACT TGG AGA TT

F GCA CTT GAG GAA GTG GCT CT BRN1 q/RTPCR R CAC AAG CAT CGA CAA GAT CG

F TC ATT TCC CCC AAT GAA TGT BRN2 q/RTPCR R AAA GGC CAG TTC CCA TAC CT

F GAC CGG TGC AAT CTT CAA A CDH1 q/RTPCR R TTG ACG CCG AGA GCT ACA C

F CCA CCT TAA AAT CTG CAG GC CDH2 q/RTPCR R GTG CAT GAA GGA CAG CCT CT

F TTC ACA TTG CAC AAG GCA CT CEBPA q/RTPCR R GAG GGA CCG GAG TTA TGA CA

F CAA GAG GGA GAA GAC CAC GA CRB3 q/RTPCR R GCA CTG TTT TGC CTT CAT CC

F CAA ATG GAC TTG TGT TCC CA CRBBP q/RTPCR R TGA GAC CCT AAC GCA GGT TT

F GCT CAG AAG CGA CGG AAT TA CUX2 q/RTPCR R GTC TCG ATA GCC CCC AAG AT

F CCA GGG TTG CAC ACT TTC TC DLL1 q/RTPCR R CTA CTA CGG AGA GGG CTG CT

F TCC GAG TCT GCC TCG AGT T DLX4 q/RTPCR R GAC TCC TAC CTG TCC TGC CA

F TTT GCC ATT CAC CAT TCT CA DLX5 q/RTPCR R CGC TAG CTC CTA CCA CCA GT

F AAA CAG ATT TTC CCC CAA CC DLX6 q/RTPCR R AAT GCA GGA GTC CAA AAT GC

F CGC CTT CGA GAA GAA CCA C EMX1 q/RTPCR R GGT TCT GGA ACC ACA CCT TC

F AGC CAG GTA ACG GTT AGC AC FGF2 q/RTPCR R GGA GAA GAG CGA CCC TCA C

F TTA CTA TCT AGC GCC GCC AT GLAST q/RTPCR R TTC CTG GGG AAC TTC TGA TG

F TCA GCA CTT AAA AGA TTC CGT G ID2 q/RTPCR R GAC AGC AAA GCA CTG TGT GG

F CTT CCG GCA GGA GAG GTT ID3 q/RTPCR R AAA GGA GCT TTT GCC ACT GA

F ACT GTC AGG TTG AAC GGT GTC JAG1 q/RTPCR R ATC GTG CTG CCT TTC AGT TT

F TGC TTG CTT TCC CAA AAT TC LIX1 q/RTPCR R TCT GAG ACA CAT CAT TGC CC

F GTT GGG GTC CTG GCA TC NOTCH1 q/RTPCR R GGT GAG ACC TGC CTG AAT G

F CTT CAG GGT CTG ATG CGT CT OTX1 q/RTPCR R GAC AAG GTT GGC TGC CC

F TTG GAG GTT CCA GTG CTT TC RELN q/RTPCR R TCA CGT GAG AGG CTA CCA CA

F GGG TCT GAG TCC CCA GTT G SPANXA1 q/RTPCR R AAT GGA CAA ACA ATC CAG TGC

F CGG GTA AAA GCA AGA GCA GA STMN2 q/RTPCR R TCT GCA CAT CCC TAC AAT GG

F GGA GGA GGC TAC GTT CAC AA WNT1 q/RTPCR R TTT CTG CTA CGC TGC TGC T

F AGT TGC TTG GGG ACC AGG WNT3 q/RTPCR R CTC GCT GGC TAC CCA ATT T

F ATG AGC GTG TCA CTG CAA AG WNT3A q/RTPCR R GTG GAA CTG CAC CAC CGT

F CAG TTC ATG TTC TCC TCC AG WNT7A q/RTPCR R AAG GTC TTT GTG GAT GCC C

F ACT CGT TGA TGC CCA TCT G WNT7B q/RTPCR R GCA ACA AGA TTC CTG GCC TA

F TCG AAG TCA CCC ATG CTA CA WNT8A q/RTPCR R ACC CAC AAC AGG CTG AGA AG

F CAT CAC AAT GAT CAC AGC CA ZNF572 q/RTPCR R AGA GGG AGA GGG GGT ACC TT

F GAG AGC CTC CGT CAT CTG G ZNF750 q/RTPCR R GTA CTG CTT CCT GAG CAC CG

F GTC CAA TGG CCT GGT GC SIX3 q/RTPCR R CAC TCC CAC ACA AGT AGG CA

APPENDIX IV. PRIMERS FOR RETROVIRAL EXPRESSION

Gene Application Orientation Sequence (5’-)

Tg-pMXs RT-PCR F TTA TCG TCG ACC ACT GTG CTG Tg-C-MYC RT-PCR R AGA GTC TGG ATC ACC TTC TGC TG Tg-KLF4 RT-PCR R TCT CTT CGT GCA CCC ACT TG Tg-OCT4 RT-PCR R GAG AAC CGA GTG AGA GGC AAC Tg-SOX2 RT-PCR R CTT GGC TCC ATG GGT TCG F TGA TTG TAG TGC TTT CTG GCT GGG CTC C hKLF4 RT-PCR R ACG ATC GTG GCC CCG GAA AAG GAC C F TTG AGG GGC ATC GTC GCG GGA GGC TG hMYC RT-PCR R GCG TCC TGG GAA GGG AGA TCC GGA GC F CTT CCC TCC AAC CAG TTG CCC CAA AC hOCT4 RT-PCR R GAC AGG GGG AGG GGA GGA GCT AGG F TTG CGT GAG TGT GGA TGG GAT TGG TG hSOX2 RT-PCR R GGG AAA TGG GAG GGG TGC AAA AGA GG

APPENDIX V. PRIMARY AND SECONDARY ANTIBODIES

Antibody Conjugation Species Vendor Dilution Application -fetoprotein Immunocytochemi α Unconjugated Rabbit DAKO 1:400 (AFP) stry (ICC)

Aurora B Unconjugated Rabbit Chemicon 1:1000 ICC

1:1000 Western Blot (WB)

β-Catenin Unconjugated Mouse BD Bioscience 1:1000 ICC

brain lipid binding Unconjugated Rabbit Millipore 1:100 ICC (BLBP)

BrdU Unconjugated Rat Abcam 1:100 ICC

cyclin D1 Unconjugated Rabbit Santa Cruz 1:500 ICC

cyclin D3 Unconjugated Mouse Cell Signaling 1:200 ICC

DCX Unconjugated Goat Santa Cruz 1:500 ICC

ERK (total) Unconjugated Rabbit Cell Signaling 1:1000 WB

p-ERK (p44/p42 Unconjugated Mouse Cell Signaling 1:1000 WB MAPPK)

GFAP Unconjugated Rabbit Dako 1:1000 ICC

Molecular GFP Unconjugated Mouse 1:250 ICC Probes

PH3 Unconjugated Rabbit Upstate 1:500 ICC

Ki67 Unconjugated Rabbit Novocastro 1:500 ICC

Lamin A Unconjugated Mouse 1:1000 WB

77 78

MAB414 Unconjugated Mouse Covance 1:500 ICC

MAB414 Unconjugated Mouse Covance 1:2500 WB

MAP2a/b Unconjugated Mouse Millipore 1:1000 ICC

Nanog Unconjugated Rabbit Santa Cruz 1:200 ICC

Nestin Unconjugated Mouse Chemicon 1:2000 ICC

NUP107 Unconjugated Rabbit Bethyl Labs 1:2500 WB

NUP133 Unconjugated Rabbit Bethyl Labs 1:2500 WB

OCT4 Unconjugated Rabbit Santa Cruz 1:1000 ICC

Developmental PAX6 Unconjugated Mouse Studies 1:75 ICC Hybridoma Bank

PAX6 Unconjugated Rabbit Covance 1:500 ICC

smooth muscle actin Unconjugated Goat Sigma 1:400 ICC (SMA)

SOX2 Unconjugated Rabbit Chemicon 1:1000 ICC

SSEA-3 Unconjugated Rat Chemicon 1:200 ICC

SSEA-4 Unconjugated Mouse Chemicon 1:200 ICC

tubulin, α Unconjugated Mouse Sigma 1:5000 ICC

tubulin, III β Unconjugated Mouse Covance 1:500 ICC (TUJ1)

tubulin, III Alexa Fluor β Mouse Covance 1:500 ICC (TUJ1) 488

TRA1-60 Unconjugated Mouse Chemicon 1:200 ICC

TRA1-81 Unconjugated Mouse Chemicon 1:200 ICC

ZO-1 Unconjugated Mouse BD Bioscience 1:100 ICC

anti-mouse Alexa Fluor secondary Donkey Invitrogen 1:500 ICC 488 antibody anti-mouse Alexa Fluor secondary Donkey Invitrogen 1:500 ICC 555 antibody anti-rabbit Alexa Fluor secondary Donkey Invitrogen 1:500 ICC 488 antibody anti-rabbit Alexa Fluor secondary Donkey Invitrogen 1:500 ICC 555 antibody anti-mouse secondary HRP Thermo 1:1000 WB antibody anti-rabbit secondary HRP Thermo 1:1000 WB antibody Alexa Fluor Phalloidin Invitrogen ICC 488

StrepTactin HRP Bio-Rad 1:10,000 WB

Hoescht 1:10,000 ICC 33342

APPENDIX VI. QUANTITATIVE RT-PCR FOR DIRECTED NEURONAL DIFFERENTIATION

80 81

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