sign in sign up
<<
Home , APBB3, ASCC3L1, ASCL1, ATF6, BICD2, CBY1

EPIGENETIC REGULATION AND FACTOR

PROGRAMMING ENHANCES NEUROGENESIS IN NEURAL STEM CELLS

by

CHRISTOPHER L. RICUPERO

A Dissertation submitted to the

Graduate School-New Brunswick

Rutgers, The State University of New Jersey

and

The Graduate School of Biomedical Sciences

University of Medicine and Dentistry of New Jersey

In partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

Graduate Program in Neuroscience

Written under the direction of

Dr. Ronald P. Hart

And approved by

______

______

______

______

New Brunswick, New Jersey, OCTOBER, 2011

ABSTRACT OF THE DISSERTATION

EPIGENETIC REGULATION AND

PROGRAMMING ENHANCES NEUROGENESIS IN NEURAL STEM CELLS

By

CHRISTOPHER L. RICUPERO

Dissertation Director:

Ronald P. Hart

In this thesis, we questioned how neuronal and glial phenotypes become specialized.

Epigenetic chromatin modifiers and transcription factors were investigated for their roles in programming and maintaining neural lineage restriction. A relatively homogeneous population of cells was generated by deriving immortalizing neural clones from embryonic rat forebrains. Three phenotypes; neuronal, glial and multipotential (GE6,

GE2, CTX8), provided contrasting lineages to probe the factors responsible for shaping cell fate. One particular clone, GE6, differentiated into a functional inhibitory like interneuron. expression analysis showed several such as Ascl1, Dlx1, Dlx5, may be responsible for the interneuronal specificity. Epigenetic regulation through histone modifications is believed to be an essential component within the developing , ultimately affecting cell fate. Testing chromatin signatures on specific neural genes with permissive and repressive histone “marks” shows that chromatin state in undifferentiated precursors correlates with current and predicts downstream . These results suggest that cell fate may already be predetermined.

Furthermore, ChIP sequencing reveals global differences between the representative clones. Extrinsic growth factors, such as BMP2 promotes the neuronal and glial

ii

phenotypes in the multipotential cell CTX8. BMP2 asserts its phenotypic response in part by regulating global acetylation enrichment in specific neural gene networks, providing a mechanism to promote and maintain cell fate. Directly altering chromatin marks using a histone deacetylase inhibitor, valproic acid (VPA), globally acetylates the chromatin of CTX8 cells and enhances neurogenesis. VPA treatment was also confirmed to maintain and increase acetylation in specific neuronal genes, such as

Ascl1. In addition, several thought to play a role in neurogenesis were epigenetically regulated after VPA treatment. Finally, through the combination of gene expression and epigenetic analyses, direct programming through exogenous expression of Ascl1, Dlx1 and Dlx5 enhanced neurogenesis in CTX8. Gene expression and

epigenetic signature mapping provides us with a deeper understanding of how lineage

restriction occurs. Learning the programming rules will assist in directing homogeneous

populations of neuronal cells to further probe the mechanisms of neurodegenerative

diseases.

iii

PREFACE

Shortly after entering graduate school, the goal seemed simple enough; dive in, study development, and then then fix it when things go awry. For example, the order of operations is to generate a hypothesis, design and then test the predictions.

Revise and repeat until proven or disproven. A fairly straightforward path to the scientific method we are all taught. However, one of the earliest lessons learned during my graduate experience has been that this path is usually never straightforward. In addition, one of the more surprising things I discovered throughout my training was the many shades of grey that I would encounter. These shades of grey can lead to new and exciting areas to pursue but at the same time can cause distractions and loss of focus. I would like to thank my mentor, Dr. Hart for providing the environment to gain experience and pursue new ideas. He has always been supportive of new projects, while at the same time requiring me to know “what is the biological question I am trying to answer?”

With the risk of sounding cliché, I have learned that becoming an expert in one’s field is not just about the answers, but also being aware and responsive to the newer questions that arise.

The work presented within this thesis describes the independently designed experiments exploring my ideas on lineage restriction programming during neural development. Overall, I have had the support of many people over the years, and they will be described in detail in the next section. However, I can state that throughout my graduate career at Rutgers, I have been very independent in learning and developing the techniques necessary to answer the biological questions at hand. I have also had the

iv

privilege to collaborate and contribute to multiple scientific endeavors leading to several co-authorship publications. Some of these will be described below.

Before the development of the newly reprogrammed neural clones described in detail throughout this thesis, I worked on multiple studies using the previously published neural precursor clones, L2.2 and L2.3. Collaborating with Dr. Hedong Li from Dr.

Grumet’s lab, we investigated the role of activated Notch1 and how it maintains the phenotype of radial glial cells, inhibits their differentiation, and promotes their adhesion and migration. Specifically, I compared the levels of cell adhesion on Notch expressing

NL2.3 cells compared to controls using cell culture and immunocytochemistry (Li et al.,

2008A).

In the L2.2 study, where it was confirmed to represent a precursor capable of generating GABAergic like interneurons, my contribution was the separation of radial glia from primary embryonic ventral forebrains by magnetic bead sorting for a neuronal restricted marker PSA-NCAM. This was useful to confirm that within the dissociated ventral regions, 5A5+/A2B5− cells are more likely to be enriched compared to BLBP+ radial glial cells (Li et al., 2008B). This magnetic bead sorting method was not used in the lab previously. I independently refined the technique using consecutive sorting runs using cell surface markers.

More recently, I collaborated with the chemical engineering department, applying another radial glial precursor cell clone, RG3.6, to an engineered nanofibrillar substrate

(Delgado-Rivera et al., 2011). Using a novel micro-patterning technique (µPIP), the goal

v

of this project was to access the adherence and alignment of neural precursor cells on an electrospun polymer scaffold, patterned with laminin-1. The work demonstrated that these textured surfaces could be micro-patterned to provide external chemical cues for cellular organization, and could be potentially applied towards areas such as spinal cord injury.

The Hart lab has identified a neurogenic set of microRNAs that enhance neurogenesis in a multipotential neural clone, L2.3 (Dr. Loyal Goff, Dr. Jonathan Davila).

My contribution was to perform most of the cell culture in these studies, where I over expressed and knocked down a set of these microRNAs to confirm their modulating roles during neurodevelopment (manuscript in preparation).

In staying with the microRNA theme in the Hart lab, I had the opportunity to collaborate then fellow graduate student Dr. Loyal Goff (Rutgers) and Dr. Uma

Lakshmipathy from Life Technologies. This study highlighted how specific microRNAs are regulated in multipotent mesenchymal stromal cells (MSC) during differentiation (Goff et al., 2008). Specifically, the PDGF pathway was examined and found to be regulated during osteogenesis by microarray. A set of microRNAs were bioinformatically predicted to respond to PDGF signaling. My contribution experimentally confirmed the microRNA regulation by preparing the human MSCs for differentiation followed by qPCR.

Lastly, the most recent ongoing collaboration has been was with Dr. Jiali Li, from the Herrup lab at Rutgers, investigating the role of histone deacetylase 4 (HDAC4) in

vi

ataxia-telangiectasia (A-T) (revised manuscript in prep). In addition to the existing knowledge that ATM, the gene mutated in A-T and is catalytically activated in response to DNA damage, the Herrup group has discovered that Atm deficiency also causes nuclear accumulation of histone deacetylase 4 (HDAC4) in and promotes neurodegeneration. My contribution was to analyze and compare HDAC4 chromatin immunoprecipitation sequencing (ChIP-seq) data from Atm mutants and wild type samples. We conclude from out part of this this study that there is widespread binding of

HDAC4 across the Atm mutant genome that differed substantially from the wild type.

This suggested a negative regulation of multiple genes because of the nuclear accumulation of HDAC4. I also assisted in the ChIP-qPCR validation of the ChIP-seq results by designing primers with Dr. Li within the identified HDAC4 peaks, as well as in two sites flanking each peak.

During some of my graduate years, I was an NSF funded IGERT fellow for both the Biointerfaces and the Integrated Science and Engineering of Stem Cells themes.

This has been a privilege and has taught me to describe my research in a tight package understood by a diverse audience. The IGERT program has also opened many avenues to collaboration, some of which have resulted in the publications described above. These collaborations, both formal and informal, were integral to my education and made the doctoral process much more exciting and satisfying. I plan to learn from these experiences and hope to collaborate as much as possible in my future research.

vii

ACKNOWLEDGEMENTS AND DEDICATION

This thesis represents the hard work and sacrifice of many people, not just me during my graduate career at Rutgers University. Having the full support of colleagues, friends and family enabled me to push on during the most trying times of my research.

First, I would like to thank my thesis advisor and mentor, Dr. Ronald P. Hart. Providing a stimulating scientific environment along with comfortable working conditions is not always easy to find, and Dr. Hart provided both. Throughout my years in the lab, Dr. Hart was always verbally and financially supportive of my ideas and provided the structure necessary to pursue them. He has been one of the most hands on advisors I have had the privilege to work with and learn from. I am grateful to have been part of the Hart lab over the past six years.

I would like to thank Dr. Martin Grumet for being a co-advisor throughout my thesis research. This project would not have been possible if it not were for Dr. Grumet's cell clone system. He has always made time to discuss and give me advice throughout my time at The Keck Center. I would also like to thank Dr. Hedong Li from the Grumet groups as he was the first to train me on cell culture and provided all of the cell clones described in this thesis.

I would like to thank the additional members of my committee, Dr. Bonnie

Firestein and Dr. Li Cai. Both of them have been extremely supportive during my initial years of pre-proposal up to the final few months of this project. Their constructive feedback has been and will be useful for the future publications stemming from this work.

viii

Mavis Swerdel has been such an integral part of my graduate experience, it is tough to list all of the ways she has assisted me throughout the years. In addition to making the lab run as smoothly as possible, Mavis played a large role in many of the gene expression assays described in this thesis, along with preparing all of the ChIP-seq libraries for sequencing. Mavis is really the cornerstone of the lab, and I will always appreciate the interest in both my professional and personal life that she has shown me.

She is a good friend and I value her advice.

Petronio Zalamea was a great asset to have in the lab as he assisted me in all of the ChIP-seq experiments. His speed and attention to detail greatly improved our results and I am greateful for his help.

Alana Toro-Ramos assisted in some of the western blot assays along with the cloning of multiple expression constructs. I want to thank her for this assistance along with being such a good friend over the years. Sitting next to her in the lab has made my experience much more interesting.

I would like to thank Chendong Zhang for providing his bioinformatic and programming skills to assist in the early ChIP-seq data sets. His patience and knowledge was an asset when we needed to anlalyze the immense amount of data from the sequencing experiments.

It has been such a pleasure working with Joanne Babiarz from the Grumet lab over the years. I would like to thank Joanne for provided me with very good advice and for her assistance in many of the cell culture and immunostaining assays described in this project.

I would like to thank Dr. Loyal Goff for my first exposure to the world of microRNAs and neural stem cells. From these early discussions between Loyal and

ix

Jonathan was where I first started to organize my thoughts on how epigenetics may play a role in neural development.

I would like to also thank my good friend Dr. Jonathan Davila for being essentially my partner in crime for the bulk of my stay at the Hart Lab. Jonathan and I have had so many fruitful and also useless conversations about science both in and out of the lab over the years. He has been a good friend and colleague and the lab is not the same without him.

Dr. Jennifer Moore has been a great asset to the Hart Lab since she joined a few years ago. Jen has always provided me with constructive feedback and has been extremely supportive of my research. I would like to thank her for all of her advice, especially on my presentation preparations.

I would like to thank Dr. Maria Barrero from the Center of Regenerative Medicine in Barcelona for graciously hosting me in her lab as part of the NSF IGERT internship in the summer of 2008. It was here that she mentored me in the field of epigenetics which allowed me to pursue the core of my thesis research.

Dr. Mark Plummer’s lab group consisting of Anna Hadar, Eli Nazar and Bavahuk

Ghupta performed all the electrophysiology assays and were extremely helpful in collaborating on this project and I am grateful for the advice and assistance.

Thank you to Dr. Cindy Camarillo for being a good a friend in the lab and for training me to properly perform Western Blots. In addition, my newer colleagues in the

Hart lab, Eileen Oni and Mike D’Ecclessis, assisted in the recent VPA treatment assays and will be continuing to collaborate in future experiments.

x

I would like to thank Dr. Jian Chen for being such a good friend over the years.

Although we did not collaborate on any projects together, his friendship was important in providing balance to my research studies.

I would like to also thank Yuwen Chang for being one of my first friends when I joined the Keck Center. Her years of experience and advice were priceless for my navigation as a new graduate student in the Department of Neuroscience.

Finally, I would like to thank my parents in the constant support throughout this process. My mother and father have been the most positive people I know during my graduate years. They have shown such patience and I forever grateful to have them in my life. Furthermore, I would like to thank my extended family in their constant interest and support over the years. My goddaughter, cousins, aunts and uncles were always in my court and have shown me the true meaning of family.

Last but not least, I would like to thank Lara for standing by me all of these years.

By sharing in this sacrifice with me over the years, I dedicate this to you because a piece of you is also in this thesis. You have provided me with advice and support that has given me balance in life. I love you every day for this and much more. I am truly lucky and grateful to have you by my side.

xi

TABLE OF CONTENTS

ABSTRACT OF THE DISSERTATION ...... II

PREFACE ...... IV

ACKNOWLEDGEMENTS AND DEDICATION ...... VIII

TABLE OF CONTENTS ...... XII

LIST OF FIGURES ...... XVI

LIST OF TABLES ...... XIX

ABBREVIATIONS USED ...... XX

I. INTRODUCTION ...... 1

THE PROMISE OF STEM CELLS: CAN IT BE KEPT? ...... 2

NEURAL DEVELOPMENT – NEURONS FOLLOWED BY GLIA ...... 5

NEUROGENESIS ...... 6

GLIOGENESIS ...... 7

THE REGULATION OF NEURAL PATHWAYS BY EXTRINSIC AND INTRINSIC SIGNALING ...... 8

CROSS‐TALK BETWEEN NEURONAL AND GLIAL TRANSCRIPTIONAL NETWORKS ...... 9

WHAT IS EPIGENETICS? ...... 10

DNA METHYLATION ...... 11

MICRORNA ‐ A SMALL NON‐CODING RNA ...... 12

HISTONE MODIFICATIONS ...... 14

FUNCTION OF HISTONE MODIFICATIONS? NOT JUST FOR ANNOTATION ...... 15

HOW ARE HISTONE MODIFICATIONS DEPOSITED? ...... 16

INVESTIGATING THE EPIGENOME BY CHROMATIN IMMUNOPRECIPITATION ...... 17

EPIGENETICS IN EMBRYONIC STEM CELLS ...... 18

xii

EPIGENETIC MECHANISMS REGULATE NEURAL DEVELOPMENT ...... 19

SECTION A...... 22

REPROGRAMMED PRECURSORS REPRESENT SPATIAL AND TEMPORAL STAGES OF DEVELOPMENT ...... 22

IN VITRO MODELS FOR DISTINCT NEURAL STEM CELL PHENOTYPES...... 22

LONG TERM DIFFERENTIATION IDENTIFIES DIFFERENT PHENOTYPE CAPACITIES BETWEEN THE REPRESENTATIVE PRECURSORS..26

GENE EXPRESSION PROFILES IDENTIFY UNIQUE REGULATORY PATHWAYS ...... 28

QPCR VALIDATION OF MICROARRAYS ...... 31

MYC IS DOWN REGULATED, BUT NOT SILENCED, DURING INDUCED DIFFERENTIATION ...... 32

GE6 LONG TERM DIFFERENTIATION IDENTIFIES SYNAPTIC COMPONENTS SUGGESTING FUNCTIONAL ACTIVITY...... 33

SUMMARY OF SECTION A: REPROGRAMMED PRECURSORS AS AN IN VITRO MODEL FOR NEURAL STEM CELL ...... 34

SECTION B...... 36

EPIGENETIC REGULATION OF NEURAL PRECURSORS ...... 36

NEURAL CLONES DISPLAY UNIQUE HISTONE SIGNATURES ...... 37

GROWTH FACTORS RAPIDLY ALTER NEURAL PHENOTYPE ...... 46

EPIGENETIC MODIFIERS ALTER CHROMATIN STRUCTURE LEADING TO ENHANCED NEUROGENESIS ...... 49

CHIP SEQUENCING IDENTIFIES NEURONAL REGULATORY REGIONS ...... 50

NEUROGENIC TRANSCRIPTION FACTOR ASCL1 IS EPIGENETICALLY REGULATED BY VALPROIC ACID...... 52

MICRORNAS ARE EPIGENETICALLY REGULATED BY VALPROIC ACID ...... 53

EXOGENOUSLY EXPRESSED MICRORNAS INCREASE NEUROGENESIS ...... 55

SUMMARY OF SECTION B: EPIGENETIC REGULATION OF NEURAL PRECURSORS ...... 57

SECTION C...... 59

ENHANCED REPROGRAMMING OF NEURAL PRECURSOR CELLS TOWARDS INTERNEURONAL SUBTYPES 59

ASCL1 EXPRESSION IS INCREASED IN NEURONAL AND MULTIPOTENTIAL CLONES ...... 61

REPROGRAMMING THROUGH TRANSIENT TRANSFECTION IS RAPID AND EFFICIENT ...... 62

xiii

EXOGENOUS EXPRESSION OF ASCL1 INCREASES NEURONAL PROGENY AND ENHANCES MORPHOLOGY ...... 63

DLX FAMILY MEMBERS ACTIVATE INTERNEURONAL NETWORKS ...... 64

INTERNEURONAL SUBTYPES BETWEEN NEURONAL RESTRICTED CLONES L2.2 AND GE6 DISPLAYS DIFFERENCES IN

MATURATION AND FUNCTION ...... 65

CO‐CULTURES PROVIDE ADD TREATED PRECURSORS WITH AN ENVIRONMENT FOR NEURONAL MATURATION...... 67

ADD TREATED CO‐CULTURES ENHANCE NEURONAL FUNCTION AND INCREASE SYNAPTIC ACTIVITY ...... 69

ADD TRANSCRIPTION FACTORS BIND TO NEURAL COMPONENT REGULATORY REGIONS ...... 70

III. DISCUSSION ...... 72

HOMOGENEOUS MODELS OF NEURAL FOREBRAIN DEVELOPMENT USING CELL CLONES ...... 75

THE BENEFITS OF IMMORTALIZATION ...... 77

NEURAL CLONES HAVE UNIQUE IDENTITIES ...... 79

DO EPIGENETIC MARKS DISTINGUISH CORTICAL NEURAL PRECURSORS WITH DISTINCT FATES? ...... 83

GROWTH FACTORS AFFECTING CELL FATE REGULATE EPIGENETIC MARKS ...... 89

CHANGING EPIGENETIC MARKS ALTERS CELL FATE ...... 90

TRANSCRIPTION FACTORS REGULATED DURING GABAERGIC DIFFERENTIATION CAN EXOGENOUSLY ALTER CELL FATE BY

REGULATING NEUROGENIC GENES ...... 97

IV. FIGURES ...... 106

V. TABLES ...... 144

...... 144

...... 145

VI. METHODS ...... 155

RAT CELL CULTURE AND DIFFERENTIATION ...... 155

WESTERN BLOT ...... 155

GENE EXPRESSION PROFILING ...... 155

CHROMATIN IMMUNOPRECIPITATION ...... 156

xiv

CHIP ENRICHMENT AND ANALYSIS ...... 158

CHIP SEQUENCING AND ANALYSIS ...... 158

ADD TRANSFECTIONS ...... 160

ELECTROPHYSIOLOGY ...... 161

VII. REFERENCES ...... 162

xv

LIST OF FIGURES

Figure 1. L2.2 and L2.3 NSC clones - An early in vitro model for neural development.

Figure 2. Isolation and immortalization of GFP positive neural precursor clones from the dorsal and ventral regions of the forebrain.

Figure 3. Differentiated Fischer clones display distinct markers of neuronal and/or glial cell lineage.

Figure 4. GE2 represents a glial restricted precursor.

Figure 5. CTX8 is multipotential, but produces primarily non-neuronal cells.

Figure 6. GE6 represents a neuronal restricted precursor.

Figure 7. Microarray gene expression profiles display clear differences between Fischer clones.

Figure 8. Heat map of expression profiles identifies a cascade of regulated transcription factors during critical stages of cell fate maturation.

Figure 9. qPCR of additional Dlx family members, GAD-2 and v- mRNAs.

Figure 10. Differentiated GE6 cells generate primarily GABAergic phenotypes.

Figure 11. Gene expression profiles display lineage differences between Fischer neural

clones.

Figure 12. Chromatin immunoprecipitations of distinct neural precursor clones identify

fate-specific marks.

xvi

Figure 13. ChIP-Seq generates widespread genomic coverage across all .

Figure 14. ChIP sequencing correlates with previous ChIP-qPCR results, highlighting permissive and repressive histone enrichment..

Figure 15. ChIP sequencing of epigenetic acetyl marks highlights differences between clones in their undifferentiated states.

Figure 16. CTX8 fate can be affected by factors present as gradients in developing cortex.

Figure 17. BMP2 treatment promotes the progression of both astrocytes and neurons.

Figure 18. Valproic acid alters global chromatin and enhances neurogenesis.

Figure 19. Valproic acid increases neurogenesis and neurite process length.

Figure 20. Valproic acid alters the chromatin in a network of nervous system and specific genes.

Figure 21. Valproic acid induces epigenetic acetylation markings and gene expression in neuronal transcription factor Ascl1 (Mash1).

Figure 22. Select microRNAs are epigenetically regulated by valproic acid.

Figure 23. Exogenous expression of a select group of microRNAs increases the

neurogenic capacity of a multipotential NSC clone L2.3.

Figure 24. Verifying co-transfection of plasmids using CAG-GFP and CAG-DsRed.

Figure 25. Transfection of Ascl1 enhances neurite length and branching.

Figure 26. Transfection of Ascl1 alone enhances neurogenesis.

xvii

Figure 27. Combination transfection of ADD transcription factors enhances neurogenesis.

Figure 28. Co-Culture of ADD transfected CTX8 and Hippocampal Neurons enhances neuronal morphology.

Figure 29. Co-Culture of ADD transfected CTX8 and Hippocampal Neurons enhances total neurite length and branching.

Figure 30. Transfection of Ascl1, Dlx1, and Dlx5 enhances neurogenesis and synaptic activity.

Figure 31. Transfection of Ascl1, Dlx1, and Dlx5 enhances neurogenesis by generating

VGAT positive progeny.

Figure 32. Transfection of Ascl1, Dlx1, and Dlx5 enhances neurogenesis by generating

Vglut-1 positive progeny.

xviii

LIST OF TABLES

Table 1. Epigenetically regulated genes after VPA treamtment

Table 2. Epigenetically regulated microRNAs after VPA treamtment

Table 3. GO analysis of epigenetically regulated categories in response to ADD over

expression

xix

ABBREVIATIONS USED

bHLH Basic Helix-Loop-Helix

Bp cDNA Complementary DNA

ChIP Chromatin Immunoprecipitation

CNS Central nervous system

Dnmts DNA methylation and methyltransferases

FACS Fluorescence activated cell sorting

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GFAP Glial fibrillary acidic

GFP Green Fluorescent Protein

GO

GRP Glial restricted precursor

H3K4me3 Histone Lysine #4 tri-methyl

H3K9/14me3 Histone Lysine #9/14 acetyl

H3K27me3 Histone Lysine #27 tri-methyl

HDAC Histone Deacetylase hESC Human embryonic stem cells

xx

IP Immunoprecipitation miRNA MicroRNA mRNA Messenger RNA

NRP Neuronal restricted precursor

NSC Neural stem cell

OPC Oligodendrocyte precursor cells

RISC RNA-induced silencing complex

RFP Red Fluorescent Protein

RRQ Relative relative quantity

TSS Transcription start site

VPA Valproic acid

xxi

1

I. INTRODUCTION

The loss of neural cells from spinal cord injury or brain trauma can be devastating for many patients and most often results in chronic loss of function.

Similarly, neurodegenerative diseases may progress through different mechanistic routes but still end in debilitating loss of function due to the loss of healthy neurons or glia. Physical therapy and therapeutic intervention using pharmacological drugs have made great strides in easing pain, reducing disease symptoms and have even extended the lives of some terminally ill patients. However, in many cases these approaches are prolonging the inevitable as the injury or disease will progressively deteriorate resulting in incapacitation or death. The suffering endured by the patients and their families is immense and they are in desperate need for cures, not temporary solutions that eventually lead back to prior conditions. Stem cell transplantation is a potential paradigm shift in that it may offer bona fide cures for many neural injuries or neurodegenerative diseases. It has already achieved early successes in the field of hematopoiesis. Bone marrow transplants have saved thousands of lives since its first implementation in 1957

(Thomas et al., 1957). However, just as there are many types of , there are also multiple strategies for stem cell transplantation. In addition, each approach has its own set of complexities such as proliferation, migration, integration and ultimately functionality within its host environment. The combined execution of each one of these components is critical for a successful transplantation “cure.”

Our objective is to learn the programming rules a neural cell undergoes during its development into a functionally mature neuron or glial cell. By understanding the effectors of cell fate, we can then engineer a cell that contains and expresses the required components for proper function. Obtaining specific cell subtypes is critical for

2

transplantation because incorrect cell types can frequently do more harm when implanted into a patient. Tumors, widespread pain or exacerbation of symptoms can arise if improper cell phenotypes are introduced into the injury site. In collaboration with Dr. Martin Grumet’s lab at the Keck Center for Collaborative

Neuroscience, we are developing new strategies to both isolate and program neuronal subtypes for neurotrauma applications such as spinal cord injury.

The promise of stem cells: can it be kept?

Recently, the stem cell field has made strides in two broad categories: clinical application of human embryonic stem cells (hESCs), and the development of induced pluripotent stem cells (iPSCs). For example, Geron has initiated the first human embryonic stem cell clinical trial in the United States

(http://clinicaltrials.gov/ct2/show/NCT01217008, 2010). It is a safety trial with the ultimate goal of remyelinating injured axons in acute spinal cord injury patients. Originally, this was based on Dr. Hans Kierstead’s studies in the rat spinal cord (Keirstead et al., 2005).

His team carefully developed a differentiation protocol to generate specific oligodendrocyte precursors derived from human ES cells. When transplanted into injured spinal cords, they matured into proper oligodendrocytes where they myelinated injured axons and dramatically improved motor function. To apply this research in human patients, Geron has been working for many years on protocols to specifically generate human oligodendrocyte precursors (OPCs). It is critical to only generate OPCs and no other cell type because of the inherent risks mentioned above. These studies illustrate the need for learning cell fate programming of specific neural lineages.

3

The second category that has revolutionized the stem cell field in recent years is induced pluripotent stem cells (iPSCs). Originally reported by Dr. Shinya Yamanaka back in 2006, his group discovered that mouse fibroblasts could be successfully reprogrammed into an embryonic-like pluripotent state with the addition of just four transcription factors (Takahashi and Yamanaka, 2006). His group, along with Dr. James

Thompson’s group, shortly thereafter applied the method to reprogram human fibroblasts into a pluripotent state (Nakagawa et al., 2007; Yu et al., 2007). A cell is considered pluripotent when it has the potential to differentiate into any of the three germ layers; endoderm, mesoderm, or ectoderm. Essentially, a stem cell with this capability can differentiate into just about any cell type within the proper context.

Human embryonic stems cells are considered pluripotent and have been the gold standard in stem cell research since they were isolated from a human blastocyst and derived into the first human embryonic stem cell line (Thomson et al., 1998). Although human embryonic stem cells remain the primary models in pluripotency research, there are some serious drawbacks. The first being a lack of inventory, as these cells can only be extracted from the embryonic blastocyst stage. Therefore, in vitro fertilization clinics have been a major contributing source in the creation of these stem cell lines. Second, any successful transplantation from the existing human stem cell lines would require immunosuppressive drugs to avoid immune rejection. Lastly, since the embryos are destroyed for proper isolation, there is an existing ethical dilemma that has permeated the political landscape of the past 12 years.

Therefore, the creation of pluripotent like stem cells from adult fibroblasts has

been revolutionary within the stem cell field. This discovery is intriguing for many

reasons; first, it opens the door for autologous patient specific stem cell transplantation

without immune rejection, and second, it has started a new era of iPS disease modeling

4

using patient specific iPS cells. This has provided researchers and clinicians access to patient cells that have never been previously available. Therefore, this paradigm shift that has opened many avenues into obtaining previously unavailable cell types, but is now faced with its next challenge: how to generate the proper cell type? This question has always been a concern, but is now even more apparent due to the accessibility of patient derived stem cells.

In this thesis we set out to identify critical roles for some key regulators during the path towards neuronal maturation. There are several hundred broadly defined cells in the human body, and hundreds of subtypes within the nervous system (Alberts et al.,

2002). A fundamental aspect of neural development is the sequential generation of neurons followed by glia. This temporal sequence requires crosstalk between extrinsic and intrinsic regulatory pathways to properly coordinate precise and rapid cell fate decisions. Extrinsic signals such as growth factors and cell to cell contact induce a variety of cascades. Meanwhile, intrinsic transcription factors and epigenetic modifiers regulate the competency of the cell, controlling its reaction to these extrinsic signals. Learning the mechanisms that regulate lineage restriction is essential for the goal of engineering neural subtypes for disease modeling or cell transplantation.

To begin the search for neural regulatory mechanisms, we took a three tiered approach that first isolates contrasting neural phenotypes with varying levels of lineage restriction.

Using these as models of neural development, we then investigate both the epigenetic and gene expression profiles throughout their differentiation life cycle. Finally, we apply reprogramming methods to alter the epigenetic state and drive the neural cells towards a desired neuronal phenotype.

5

Neural Development – Neurons followed by glia

The mammalian subventricular zone is where neural stem cells and lineage restricted precursors were first discovered (Davis and Temple, 1994; Doetsch et al.,

1999; Guillemot, 2007; Kriegstein and Noctor, 2004; Morshead et al., 1994; Noctor et al.,

2004). A fundamental aspect of neural development is the sequential generation of neurons followed by glia (Guillemot, 2007; Temple, 2001). The total number of cell divisions during these periods is tightly regulated to ensure proper ratios and size of the central nervous system (Nowakowski and Hayes, 1999). Crosstalk between extrinsic

(extracellular factors) and intrinsic (epigenetic modifiers, transcription factors) mechanisms coordinate these precise and rapid cell fate decisions. In addition, it has been shown that the same extrinsic signal can produce different results depending on the temporal and anatomical context of the organism (Takizawa et al., 2001). The developing forebrain is a strong model system for studying cell fate because it is amenable to both in vitro progenitor cultures and in vivo fate tracking studies (Guillemot,

2007).

However, there are drawbacks to studying neural precursors directly isolated from the telencephalon. First, freshly isolated precursors can rapidly differentiate towards their phenotypic fate during primary cell culture. This can be tempered slightly by supplementing the cultures with mitogenic factors, however the consistent lineage progression can severely limit consistency between sequential passages. Second, there is a scarcity of unique markers to properly distinguish neural stem cells from slightly more restricted precursors. During the stages of development, the telencephalon contains a variety of precursors containing distinct levels of phenotypic potential. The lack of unique markers makes it is difficult to properly isolate and segregate these precursors for analysis. Therefore, most protocols that dissociate certain regions of the

6

forebrain result in the culturing of a heterogeneous population of cells (Miller and

Gauthier, 2007). This complicates future studies and may conceal some underlying mechanisms responsible for neuronal and glial differentiation. To improve upon these limitations, stably transformed multipotential NSCs have been previously generated and used as models of neural development (Ryder eta al., 1990, Synder et al., 1992). These clones remain more stable than primary cells and can potentially represent the stages of neuronal or glial development. We employed this method for our own research into neural lineage commitment. By analyzing a less heterogeneous set of restricted precursors, we could potentially discover unique transcriptional networks that were masked within a mixed population.

Neurogenesis

During neurogenesis, the basic Helix Loop Helix (bHLH) family of transcription factors plays a major role and is known to be conserved (Bertrand et al., 2002;

Guillemot, 2007; Ross et al., 2003). Transcription factors in the bHLH family include

Ascl1 (also known as Mash1), Neurogenin 1/2, NeuroD1 and others. These are spatially

and temporally regulated throughout development and may have dual roles depending

on when and where they are expressed. Certain transcription factors have been shown

to promote subclasses of neurons. For example, Ascl1 has been shown to be involved in

the specification of interneuronal subtypes (Jakovcevski et al., 2011), whereas other

transcription factors such as Neuogenenin2 and NeuroD1 inhibit that process by

targeting Ascl1 (Roybon et al., 2011). Also, the Dlx family is known to be expressed in

interneurons, and one of the functions of these transcription factors is to promote the

tangential migration from ventral to dorsal regions of the telencephalon (Anderson et al.,

1999; Eisenstat et al., 1999; Marin and Rubenstein, 2003). By understanding the

7

transcriptional networks that are active in specifying neuronal subtypes, we predict that we will be able to alter or engineer the desired cell fate.

Gliogenesis

The transcriptional network of glial cells is as complex as in neuronal cells.

Subsets of the Sox family have been implicated to have important roles in gliogenesis.

Specifically, the Sox E subset (Sox8, Sox9, Sox10) has similar expression patterns during the generation of oligodendrocytes and Sox8 and Sox10 continue to maintain

expression in mature oligodendrocytes (Guillemot, 2007). Sox9 has a direct role in the

commitment of both oligodendrocytes and astrocytes and has also been shown to inhibit

neuronal fate (Stolt et al., 2003). Notch expression and downstream activity of Hes1 and

Hes5 have also been shown to play large roles in the promotion of glial fate (Gaiano and

Fishell, 2002). The parental lineage of oligodendrocytes has been controversial and two

current hypotheses prevail (O2A and GRP), one suggesting that oligodendrocytes and

motor neurons share a common ancestor and the other stating glial restriction is

separate from any neuronal restrictive lineage (Lee et al., 2000; Liu and Rao, 2004;

Noble et al., 2004; Richardson et al., 2000). In both models, the bHLH Olig genes play a

significant role in the development of oligodendroglial fate. Furthermore, Olig2 has been

implicated in the specification of both oligodendrocytes and a variety of neuronal

subtypes including motor neurons and interneurons (Guillemot, 2007) and has also been

shown to inhibit astrogliogenesis (Gabay et al., 2003; Zhou et al., 2000).

Recently a transcriptome database for astrocytes, neurons and oligodendrocytes

was established (Cahoy et al., 2008). Results suggest that the oligodendrocyte

transcriptional profile is just as different from astrocytes as they are neurons.

8

Surprisingly, the authors go on to suggest that oligodendrocytes should be classified into their own distinct category of neural cells. Therefore, understanding the intrinsic similarities and differences of glial cells is necessary to determine the levels of lineage restriction. For example, it may be critical that a particular glial restricted precursor cell differentiate solely into oligodendrocytes when transplanted into an injured spinal cord. If the cell is bi-potential, it could also differentiate into astrocytes and could be detrimental or even fatal to the patient. Previous studies have shown that unwanted astrocytes can have a detrimental effect by initiating inflammatory responses (Popovich et al., 1998).

The regulation of neural pathways by extrinsic and intrinsic signaling

Specification of neuronal and glial phenotypes requires the cell to react appropriately to spatial and temporal gradients of extrinsic growth factors. This relies in part by the current intrinsic state of the cell that reacts by sequentially expressing the network of transcription factors needed for rapid and robust gene expression in order to develop the cell’s identity. How these transcription factors and other intrinsic factors such as enhancers are regulated is of great interest.

Much progress has been made uncovering the various molecular pathways driving cell fate restriction and new transcriptional networks are frequently being discovered and refined. Some of these include: Wnt signaling for promoting neuronal fate, the Notch pathway for radial glial and astroglial fate, and many others (Jak-Stat,

BMP, SHH, PDGF) (Guillemot, 2007). Both the expression and cellular response to these factors are integral to the correct patterning of the telencephalon. However, depending on when they are expressed may produce very different results. For example,

9

a neural precursor’s response to leukemia inhibitory factor (LIF) treatment at embryonic day 11 and 16 are quite different with respect to GFAP expression (Song and Ghosh,

2004). Knowledge of intrinsic state and how the cell may respond to various growth factors is critical for future cellular engineering and regenerative medicine therapies.

Cross-talk between neuronal and glial transcriptional networks

Until recently, attempts to elucidate regulators of neuronal and glial pathways were usually completed separately. However, these pathways cross-talk regularly and their interactions with each other are now starting to be revealed. A recent study illustrated dynamic feedback interactions between committed neuronal precursors and precursor cells that will acquire the potential to become astrocytes, through the activation of the (Chenn, 2009; Namihira et al., 2009). Briefly, the neuronal precursors that expressed Neurogenin1 also expressed notch ligands DLL1 and Jagged (Tokunaga et al., 2004; Xue et al., 1999). This triggered a signaling cascade of the Notch pathway that subsequently induced binding of the STAT3 binding site on both the GFAP and S100B promoters, thus promoting astrocytic differentiation. The up regulation of Notch also induced expression of nuclear factor 1A (NFIA) transcription factor (Deneen et al., 2006; Gronostajski, 2000). This transcription factor binds to the

GFAP and protects it from further DNA methylation leading to constitutive expression and differentiation of astrocytes (Chenn, 2009; Namihira et al., 2009). This cross-talk and regulation of diverse pathways is one example of how the field is uncovering the mechanisms behind cell fate transitioning from one progenitor cell to the next (Guillemot, 2005). However, less is known about the mechanisms that regulate the intrinsic players such as transcription factors and enhancers. Epigenetic modifiers such

10

as DNA methylation, histone modification and non-coding are vital to the rapid and precise response of these intrinsic effectors.

What is epigenetics?

Every cell in an organism generally contains the exact sequence and amount of

DNA, excluding any somatic or mosaicism. One important factor that distinguishes the identity of each cell is its pattern of gene expression. The expression pattern must be inherited through each cell division to ensure proper maintenance of identity. Epigenetics is the study of this non DNA sequence-related heredity (Feinberg,

2008). The epigenetic landscape was originally described by Conrad Waddington

(Waddington, 1953; Goldberg et al., 2007). He described the interaction between what we now know as genes and the environment, and the different pathways the cell can take towards differentiation. He visualized the differentiation process and described it as a canalization where the cell encounters hills and valleys, representing a sort of “energy hill”, as its phenotype becomes more specialized. Currently, the strictest definition labels epigenetics as any meiotically or mitotically heritable change in gene function that is not explained or results from changes in the underlying primary DNA sequence (Bird, 2007;

Hamby et al., 2008; Wu and Sun, 2006). Recently, this definition has been expanded to include epigenetic modifications that may not be only be heritable, but still result in gene expression changes (Hamby et al., 2008). These modifications are dynamic and have been shown to regulate gene expression both before and after cell division takes place.

To fully appreciate the mechanisms behind epigenetic modifications, it is important to understand the substrates that these chemical moieties act upon. The human nucleus contains over two meters of DNA, and it is tightly wrapped around

11

nuclear histones in approximately 200 base pair increments. Chromatin consists of units of DNA with histones and non-histone that promote the proper packaging of

DNA, thus regulating gene expression (Kouzarides, 2007; Mehler, 2008). The smallest fragment of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around the octomer histone core. The nucleosomes are connected together by variable lengths of DNA plus linker H1 and other variant histones. The higher order architecture of the nucleosomes provides the proper compaction or unfolding of the chromatin fiber. These dynamic structural changes, termed chromatin remodeling, lead to variable levels of DNA accessibility for transcription factors and other co-factors promoting gene expression.

The underlying machinery of epigenetic modifications is of great interest because of the wide ranging clinical implications. Mis-regulation of gene expression due to epigenetic abnormalities has been linked to complex genetic disorders, psychiatric illness and cancer (Feinberg, 2008; Mehler, 2008). However, a benefit of these defects is that it may be clinically possible to reverse epigenetic mis-regulation since there are no mutations within the underlying DNA. Epigenetic modifications can be broadly classified into DNA methylation, pre/post transcriptional modifications of non-coding

RNAs, and covalent modifications of histone tails. All of these can be used to describe the current chromatin state that in turn correlates with gene expression.

DNA Methylation

A well-known epigenetic mechanism with a wide range of effectiveness is DNA methylation. DNA methylation occurs by the addition of a methyl group to the cytosine in a CpG dinucleotide. The reaction is catalyzed by DNA methyltransferases and results in

12

repression of both local and genome wide transcription. DNA methylation occurs in different regions of the genome (intergenic, intronic, promoter) and its functions range from genome integrity, parental imprinting and preserving cell identity throughout cell division (Schones and Zhao, 2008). De novo methylation of promoters and transcription factor binding sites can significantly impact binding and repress gene expression (Tate and Bird, 1993). In addition, atypical DNA methylation patterns of both hyper and hypomethylation states have been a hallmark of cancer and other diseases (Feinberg,

2008). DNA methylation has been one of the primary epigenetic mechanisms investigated throughout neural development. One example is the temporal expression of

GFAP (astrocyte marker) in neural precursors. Here, DNA methylation is a crucial component of altering the competency of the cell to react to the same extrinsic factors during different temporal stages in development (Takizawa et al., 2001). Understanding the mechanisms and repercussions of DNA methylation will continue to elucidate the regulatory pathways of neural development and will also lead us to potential reprogramming strategies for engineering desired neural subtypes.

microRNA - A small non-coding RNA

microRNAs are single-stranded RNA species of ~21 nucleotides that derive from a ~70-100 nucleotide precursor and are found in a wide variety of organisms, from plants to insects to (Ambros, 2001; Bartel, 2004). The primary transcripts of microRNA

(pri-microRNA) are processed by the Microprocessor (Drosha-DGCR8) complex resulting in a stem-loop precursor microRNA (pre-microRNA) (Han et al., 2004; Zeng et al., 2005). In animals, the pre-microRNAs are exported from the nucleus by Exportin-5 and cleaved by , resulting in the mature double stranded microRNA (Bartel, 2004;

Zeng and Cullen, 2004). One of the strands is finally then loaded into the RNA-induced

13

silencing complex (RISC) where it targets the sequence of 3’ of mRNAs. The predominant form of microRNA regulation in animals is mediated by imperfect pairing to a 3’ UTR element in an mRNA target (Bartel, 2004). Once bound, the microRNA either degrades the mRNA or suppresses the translation (Carrington and

Ambros, 2003; Dykxhoorn et al., 2003; Pickford and Cogoni, 2003; Vasudevan et al.,

2007). This action results in one of several identified mechanisms resulting in attenuation of protein production (Bartel, 2004; Liu et al., 2004; Meister et al., 2004;

Yekta et al., 2004), rapid mRNA de-adenylation (Giraldez et al., 2006; Wu et al., 2006), or mRNA sequestration to P-bodies (Behm-Ansmant et al., 2006). Estimates suggest there are ~400 microRNA genes in each invertebrate species, and ~1000-1500 genes in mammals (Lewis et al., 2005.

Cell lineage restriction has been found to correlate with regulated changes in microRNA expression patterns, presumably to promote differentiation or to assist in maintaining the cell's identity. MicroRNAs have been found to be essential during neural development. Conditional Dicer knockouts in neural progenitor cells show that neural progenitors undergo cell death and abnormal differentiation in the cortex and striatum

(Kawase-Koga et al., 2009). Previous work identifying microRNAs and their expression profiles has established a distinct subset of microRNAs with enriched or specific expression in neural tissues. Several embryonic stem cell specific microRNAs are down- regulated during RA-induced differentiation (Song and Tuan, 2006) of neuronal precursor cells. Concurrently, brain-enriched microRNAs such as mir-9, mir-124, mir-

125, and numerous others, are induced in developing neural tissues and predicted to target hundreds of mRNAs, thereby modulating cell fate (Krichevsky et al., 2003; Miska et al., 2004). It is of interest to investigate how this subcategory of epigenetics coordinates with DNA and histone modifications to affect cell lineage restriction.

14

Histone modifications

The amino terminal tails of histones are subjected to a wide array of post- translational covalent modifications known as ‘marks.’ In the 1960s and 1970s many histone modification discoveries were made such as acetylation, methylation, phosphorylation, ubiquitination and ribosylation (Mehler, 2008). The histone marks are orchestrated by chromatin modifying that catalyze the addition or removal of certain chemical moieties, such as acetylation or methylation. However, it is now widely accepted that histone modifications are not just annotation marks that reflect mRNA expression profiles. Correlating histone modification state to gene transcription and also elucidating their biological roles remained elusive for many decades (Marmorstein and

Trivel, 2009). It is now known that they actively participate in the regulation of gene transcription by altering chromatin structure. Moreover, histone modifications are dynamic during cellular restriction or differentiation to stably program different gene networks of activation or repression/silencing. These induced networks give rise to various cell lineages that now have an epigenetic memory for retaining cellular identity.

It is now widely accepted that the wide variety of histone modifications can be used to describe the current chromatin state of the cell and is termed the “histone code”

(Jenuwein and Allis, 2001). This chromatin state correlates to local, current or potential gene expression. The combination of these marks has led to a unique chromatin signature that is read by chromatin modifying enzymes and components of the transcriptional machinery. This histone code is then properly interpreted and leads to robust and proper gene regulation.

15

Function of histone modifications? Not just for annotation

In recent years, it has become clear that histones do far more than just acting as a scaffold for chromatin assembly. They actively participate in the regulation of gene transcription by directly altering chromatin structure (Gan et al., 2007). Histone acetylation and methylation are the two most well-known histone modifications (Allen,

2008). The addition of an acetyl group to the N-terminal tails of histones, by histone acetyl transferases (HATS), modifies the chromatin structure and results in a neutralization of positively charged histone and negatively charged DNA. This electrostatic event results in the loosening of the underlying chromatin. Ultimately, the relaxed chromatin can lead to an increase in transcription factor binding and eventually increases in gene expression (Schones and Zhao, 2008). The reverse effect is possible via histone deacetylases (HDACS) which remove the acetyl group from the histones resulting in chromatin compaction and eventually transcriptional repression or silencing

(Hamby et al., 2008).

The covalent addition of a methyl group to specific residues residing on the histone tails is another well characterized epigenetic modification. Methyl groups added to lysine or arginine residues are used as binding sites for non-DNA binding chromatin proteins. These ‘molecular beacons’ attract domain specific chromatin modifying factors to directly adjust chromatin structure. These changes will then modulate gene expression and various cellular processes (Mehler, 2008). For these additions the relative abundance of methylation does not predict transcriptionally active or repressed regions as it does with acetylation. Instead, the particular residues that are covalently modified are correlated with permissive or repressive gene expression. For example, a tri-methyl group on histone #3 (H3) at lysine residue #4 (H3K4me3) generally correlates with transcriptionally competent regions in the promoters of genes. In

16

contrast, tri-methyl group on histone #3 (H3) at lysine residue #27 (H3K27me3) and tri- methyl group on histone #3 (H3) at lysine residue #9 (H3K9me3) generally signify repressed gene activity (Hamby et al., 2008; Wu and Sun, 2006). Each residue can be mono-, di- or tri-methylated and may signify a different result either alone or in combination with other histone marks.

How are histone modifications deposited?

The large varieties of marks are orchestrated by a wide array of chromatin modifier enzymes that catalyze the addition or removal of certain covalent histone modifications. Many of these modifications stem from the polycomb complex or trithorax group of proteins, well known for their antagonistic binding patterns correlating with gene activation and repression, respectively (Lim et al., 2009; Schuettengruber et al., 2007).

In some cases, the histone modifications and the chromatin patterns they create can also propagate and spread over kilobase lengths of genomic DNA. These patterns can then be passed on and inherited from parent to daughter cell.

These findings (Cavalli and Paro, 1999; Gan et al., 2007; Hall et al., 2002) led to the idea that histone modifications may be used during cellular differentiation to stably program different gene networks of activation or repression/silencing. These networks eventually give rise to various cell lineages that have an epigenetic memory for retaining cellular identity. We embrace this idea by proposing that neural precursors contain various levels of restriction, even before differentiation is induced. We hypothesize that epigenetic mechanisms, specifically histone modifications, play a direct role in altering the chromatin structure in the neural clones, which in turn affects downstream transcriptional networks responsible for specific cell lineages.

17

Investigating the epigenome by chromatin immunoprecipitation

The epigenome is defined as the combination of all epigenetic modifications in any cell type. In contrast to the genome, there are multiple dynamic epigenomes showing extensive differences per tissue type and developmental stage (Schones and

Zhao, 2008). The field is still in its infancy in elucidating how epigenetic modifications coordinate on a global scale to properly regulate downstream gene expression. These modifications can be broadly classified into DNA methylation, covalent modifications of histone tails and pre/post transcriptional modifications of non-coding RNAs (Bird, 2002;

Goldberg et al., 2007; Hamby et al., 2008; Wu and Sun, 2006). The underlying mechanisms of each modification class are very different and past research has discovered many pathways converging to regulate gene expression. Only recently have new studies revealed that these different epigenetic mechanisms cross-talk to achieve both stable and dynamic gene expression patterns (Cedar and Bergman, 2009).

Tracking DNA methylation, histone modifications and non-coding RNA expression have drastically improved from focusing on a single genomic region to global views of epigenetic state (Barski et al., 2007). There are many worthy techniques to investigate the epigenetic state of cells. However, most are beyond the scope of this thesis. Instead we will focus on the tracking of histone modifications in our neural development models. Researchers first interrogated local gene structures for various histone modifications using chromatin immunoprecipitation (ChIP) (Solomon et al., 1988;

Hebbes et al., 1988). The enrichment level of multiple marks was then used to describe and correlate the activity of the interrogated gene(s).

ChIP is a technique used to isolate and enrich chromatin fragments using antibodies to specific features of the chromatin. These elements can be DNA binding proteins such as transcription factors, or a particular covalent histone modification such

18

as H3K4me3. The chromatin is sheared into small fragments (200-500 bp) to allow for proper resolution of location on the genome. Initially, the analysis of an enriched region was confirmed using PCR or quantitative PCR (qPCR) where specific primers were designed for each genomic region. Although this method is precise and consistent, it is limiting because one needs to know where and what gene(s) they are probing. To overcome some of these limitations, this technique has been scaled to genome wide maps of chromatin state. Combining ChIP with microarrays (ChIP-Chip) or next generation deep sequencing methods (ChIP-Seq) has allowed for to the global viewing of chromatin state for the entire genome (Ren et al., 2000; Lieb et al., 2001; Iyer et al,

2001; Johnson et al., 2007; Robertson et al., 2007; Barski et al., 2007). There are many advantages to these ‘chromatin state maps’, some of which include providing a snapshot of certain activation or repression marks genome wide. These methods can display unique patterns of histone modifications that were previously unknown and lead to novel transcriptional networks. Future application of these chromatin maps will look to compare normal versus diseased tissue, and temporal assays to study lineage differentiation. These comparisons may illuminate new chromatin patterns, leading to future strategies for altering histone marks, and eventually modifying downstream gene expression.

Epigenetics in embryonic stem cells

Observing the chromatin in embryonic stem cells has been beneficial in recent years because it provides an un-paralleled snapshot of chromatin before most lineage restriction pathways are induced. In this state, most pluripotency genes are active and the lineage restriction genes are either still repressed or primed to be rapidly activated

(‘bivalent’). The ‘bivalent state’ is described as the enrichment in both histone

19

modifications H3K4me3 and H3K27me3 simultaneously (Bernstein et al., 2006). The presumption of this double activation and repression signal is that the gene is considered to be in a poised state. Following an induction signal for lineage differentiation, one of these ‘bivalent marks’, will become resolved resulting in either an active or repressed signal. Although ‘bivalency’ observations have been made primarily in embryonic stem cell models, there have been recent reports in other stem cell models, notably in the hematopoetic and nervous systems (Cui et al., 2009; Mohn et al., 2008).

We predict that both neural stem cells and restricted precursors will contain varying levels of histone modifications including ‘bivalency.’ Tracking these marks and their resolution over time in both neuronal and glial transcriptional networks may assist in predicting the future phenotypes of undifferentiated neural precursors.

ES cells have been used as a benchmark to compare chromatin state. With the recent revolution of iPS technology where somatic cells are reprogrammed to an ES cell like state, strict epigenetic analysis must be employed to ensure proper reprogramming.

This state may be very similar to ES cells in morphology, gene expression, germ layer potential and teratoma formation. However, there have been reports of partial reprogramming where the epigenetic state of both DNA methylation and/or histone modifications were quite different than ES cells. Improper reprogramming can have devastating effects from tumor formation to improper tissue differentiation (Stadtfeld et al., 2008; Takahashi and Yamanaka, 2006; Yamanaka, 2009). Appropriate epigenetic diagnostics can highlight inconsistencies or improper reprogramming that may go unnoticed with previous morphology and gene expression assays.

Epigenetic mechanisms regulate neural development

Not all neural precursor cells have the capacity to generate both neurons and glia during all developmental time periods. There are intrinsic differences that restrict

20

phenotype potential during development and some are more malleable than others

(Miller and Gauthier, 2007). Epigenetic mechanisms are responsible for many aspects of neural cell fate and by observing the current epigenetic state, one may predict its phenotypic potential. Recently, DNA methylation is one epigenetic mechanism that has been investigated throughout neural development. As mentioned previously, one example is the temporal expression of GFAP (astrocyte marker) in neural precursors.

When rodent precursors are exposed to Leukemia Inhibitory Factor (LIF) at embryonic day 14 (E14), the JAK-STAT pathway is activated. This leads to STAT3 binding to its response element in the GFAP promoter followed by the up regulation of GFAP (Song and Ghosh, 2004). However, at E11 when neural precursors are subjected to the same extracellular environment above, LIF treatment does not up regulate GFAP. The reason for this lack of gene expression lies in the methylation of DNA within the STAT3 binding site. Once methylated, STAT3 cannot bind to the GFAP promoter regardless of extrinsic

factors such as LIF. Although DNA methylation studies have been the featured

epigenetic mechanism to describe neural lineage restriction, there are many other

possibilities on how the underlying chromatin regulates downstream gene expression.

By uncovering when and where the epigenetic modifications occur and then tracking

their consequences, we will extend our existing knowledge of neural development and

begin to engineer specific neural subtypes for future regenerative therapies.

Neural development is a useful model to examine epigenetic mechanisms because

lineage restriction begins early and proceeds through birth. The objective is to discover

unique patterns of histone modifications at key regulatory genes responsible for cell fate

decisions. This will lead to specific chromatin modifiers or lineage genes to target with

the goal of engineering cell fate. To study these epigenetic mechanisms, our colleagues

have developed multiple immortalized precursor clones from embryonic rat forebrains to

21

serve as in vitro models of cell fate determination (Li et al., 2004; Li et al., 2008; Li et al.,

2011). These clones possess unique phenotypic properties and are advantageous due to their homogeneity, compared to primary cultures, and relative stabilities in culture. Our hypothesis states that epigenetic factors, specifically histone modifications, are dynamic regulators of chromatin structure leading to progressive neural lineage restriction. They are also reliable markers of current chromatin state and can be used to predict gene expression.

22

II. RESULTS

Section A.

Reprogrammed precursors represent spatial and temporal stages of development

To uncover novel regulatory pathways responsible for programming neural

phenotypes of the forebrain, we developed in vitro models representing several stages

of neural development (Li et al, 2011). A set of three immortalized clones cover three

general categories of forebrain development: one generating primarily neurons, another

producing only glial cells, and finally one with a more mixed potential identity.

Furthermore, multiple characterization and functional assays utilizing gene expression

profiles, immunocytochemistry and electrophysiology, highlight specific transcription

factors and neuronal components likely responsible for the maturation of neuronal and

glial progeny. The primary benefit of the immortalized clones compared to primary cell

isolation is that the starting population and resulting progeny contain less heterogeneity,

unmasking key regulatory pathways responsible for lineage restriction. We have

interrogated these neural clones to identify genetic and epigenetic factors responsible for

driving neuronal or glial networks. Ultimately, the results presented herein are a stepping

stone for the creation of directed cellular subtypes.

In vitro models for distinct neural stem cell phenotypes.

Our plan was to investigate the regulatory pathways of neural stem cells during

their progression towards more mature phenotypes. Previously, clones of embryonic

neural precursor cells were created by expressing an immortalization gene, one very

23

similar to the pluripotent Yamanaka factors myc (v-myc) (Li et al., 2004; Li et al., 2008).

Transduced neural stem cell clones were derived from embryonic day 14.5 (E14.5) dissociated cortex of Sprague Dawley rats. These clones were characterized and differentiated into distinct neural phenotypes. One clone, L2.2, is neuronally restricted and exhibits GABAergic properties (Li et al., 2008). In contrast, L2.3 maintains a more mixed phenotype that is multipotential and has the ability to develop into glutamatergic neurons, astrocytes and oligodendrocytes (Figure 1) (Li et al., 2004; Goff et al., 2007).

L2.2 was one of the first reported stable clones with the ability to differentiate with interneuronal properties. The creation of this L2.2 clone was a stepping stone towards the isolation and stabilization of neural precursor cells that still maintain the ability to divide and will differentiate into a more homogenous neuronal population compared to primary cultures. However, they were unable to form functional synapses on their own.

Since interneurons are largely known to originate within the ventral forebrain before migrating into the cortex, the Grumet lab predicted that isolating both dorsal and ventral regions of the forebrain before immortalization would yield precursors with varying restrictive capabilities. Specifically, a neural precursor cell with a ventral origin would have a reasonable chance to generate an interneuronal phenotype. Therefore, a new set of immortalized clones were produced with the goal of isolating precursors and assaying for neuronal capability (Li et al., 2011). By obtaining stable cell populations of functional precursors, they could then be applied towards modeling different stages of neural development..

Using a GFP positive Fischer rat (Figure 2A) (Marano et al., 2008), 17 additional clones from either the cortex or ganglionic eminences were selected and reprogrammed with the intent of obtaining spatially distinct precursors (Li et al., 2011). Benefits of using the inbred GFP Fisher rats include transplantation into non GFP Fisher rats should not

24

require immunosuppressive drug treatments. Furthermore, the transplanted cells can be easily tracked using epi-fluorescence microscopy. Briefly, Dr. Hedong Li created the clones by dissection and dissociation of rat embryonic forebrains at E14.5 into single cells. The Fischer rat forebrains were further subdivided into dorsal (cortex) and ventral regions (ganglionic eminences) (Figure 2B). Cells were chosen based on neurosphere growth and were again dissociated and cultured for two days. They were then reprogrammed using the v-myc by infection with PK-VM-2 retrovirus (Villa et al., 2000). Cells were infected twice and selected by resistance to G418.

From these clones, we obtained varying degrees of cell fate potential, including both neuronal and glial restricted cells along with additional multipotential precursors that were similar to the L2.3 clones (Figures 2 and 3). There are many benefits of using these clones as an in vitro model of neural development. Since they are clonally derived, they are less heterogeneous than isolated primary cells, remain “undifferentiated” and mitotically active in the presence of basic fibroblast growth factor (bFGF) and can be rapidly expanded without extensive differentiation (Li et al., 2004). Therefore, clones cultured with bFGF are said to be in an undifferentiated state since they maintain expression of the neural stem cell marker and do not yet display any neuronal or glial differentiation markers.

The 17 Fisher clones were initially screened in their undifferentiated state using a handpicked set of genes that were previously expressed in the original neuronally restricted clone L2.2. This initial screening highlighted relative differences between clones and shows that one ventrally derived clone, GE6, is the most similar to L2.2

(Figure 2C). Two additional clones, GE2 and CTX8, were chosen based on the previous screening and western blot results (Figure 3). Their phenotypes had contrasting

25

properties as GE2 differentiated into primarily glial cells and CTX8 produced a more mixed phenotype generating both neurons and glial cells.

These clones do show some similarities in their undifferentiated state (+bFGF).

For example, as shown by immunostaining, all are positive for Nestin, a well-known marker for neural precursors (Wiese et al., 2004) (Figure 4, Figure 5, Figure 6). In addition, the young neuronal marker TuJ1 and astrocytic marker GFAP were both negative in the presence of bFGF. However, when differentiated by bFGF withdrawal,

L2.2 and one of the Fischer clones (GE6) display primarily neuronal phenotypes, as assayed by western blot (Figure 3). In contrast, L2.3 and CTX8 have the potential to generate neuronal and glial markers (Li et al., 2004, Li et al., 2011). We have also isolated a glial restricted clone (GE2) that develops solely into astrocytes but is incapable of generating neurons under standard differentiation conditions. This is of great interest because it provides us with a contrasting phenotype in which we can investigate the underlying regulatory pathways involved in defining each of the clone’s cell fate.

We chose to focus on three representative clones from both the dorsal and ventral forebrain: GE6 as neuronal restricted, GE2 as glial restricted, and CTX8 as multipotential. The advantage of choosing three contrasting phenotypes is that it allows us to methodically address the genetic and epigenetic differences between clones while maintaining a relatively stable cellular model system. We hypothesized that these differences would lead to underlying regulatory pathways that have been previously difficult to uncover using heterogeneous primary cultures.

26

Long term differentiation identifies different phenotype capacities between the representative precursors

To learn more about the phenotypic potential of these reprogrammed clones, we designed long term differentiation studies where multiple time points were used to assess the current cellular fate of each neural clone. We predicted that the more restricted precursors would maintain or enhance their phenotype but would not switch cell fates as has been described during neural development (Temple, 2001). Cells were propagated in suspension as neurospheres for two to three days. Neurospheres were dissociated into single cells and then plated onto laminin coated glass coverslips.

Differentiation was induced by bFGF withdrawal and cells were cross-linked with 4% paraformaldehyde at each of the four pre-determined times ranging from undifferentiated

(Day 0) to two weeks (Day 14). Fixed cells were then immunostained with antibodies against well-known markers to neural stem cells (Nestin), early neurons (TuJ1) and astrocytes (GFAP). Cultures were also imaged under DIC optics to assess morphology and GFP fluorescence to observe the maintenance of signal.

While in the presence of bFGF, clones were mitotically active and maintained their neural stem/precursor state as verified by morphology and the maintained expression of Nestin. When initially plated, the glial only clone (GE2) displayed a flat morphology with short process lengths compared to the other two precursors. Upon differentiation, these cells became progressively flatter and exhibited a cobblestone like morphology (Figure 4). Prior to differentiation, the GE2 cells were all Nestin positive, a neural precursor stem marker that was reduced once the cells began differentiating

(Figure 4). One observation was that GE2 cells hd reduced GFP fluorescence intensity upon differentiation. This may be a concern for future transplantation or co-culture experiments where it is necessary to track cellular integration and migration. The

27

astrocytic filamentous protein marker GFAP was expressed after bFGF withdrawal within three days and became more intensely discernable throughout the time course study.

This observation indicates an astrocytic phenotype progressive throughout GE2 differentiation. As predicted, the neuronal marker TuJ1 remained absent during all temporal periods confirming that GE2 is a precursor restricted towards the glial family of cells (Figure 4).

Next, CTX8’s phenotypic capacity was analyzed through a similar time-course protocol (Figure 5). In contrast to GE2, CTX8 generally maintained a bi-polar and a phase bright appearance that strikingly resembled radial glial cells (Pollard and Conti,

2007). Similar to the other clones, CTX8 also began as Nestin positive, which was down regulated post bFGF withdrawal. Opposing the glial clone’s loss of fluorescence, GFP increased upon differentiation of CTX8. The extended time course study confirmed the gene expression and western blot observations presented earlier. Specifically, immunostained CTX8 cells were positive for both the glial marker GFAP and neuronal marker TuJ1 (Figure 5), justifying the multi-potential categorization.

However, we observed a surprising pattern throughout the time course. First,

GFAP expression was present by day three and became progressively more intense throughout the entire differentiation series. Also, early on by day three, many TuJ1 positive neuronal cells were present. However, they became progressively outnumbered by GFAP positive cells throughout the rest of the differentiation time course. In addition,

their morphology displayed short neurite processes that did not seem to increase in

length, even up to two weeks in culture. This may indicate a young neuronal cell that is

refractory to neuronal maturation. On the other hand, the short processes may represent

a definitive neuronal subtype since Nestin was rapidly downgraded, a sign of

progression out of the neural stem cell state towards a more differentiated phenotype

28

(Hockfield and McKay,1985, Dahlstrand et al., 1995, Ma et al., 2005). To investigate whether the TuJ1 positive cells were either immature or a specific neuronal subtype displaying short processes, we stained for MAP2, a well-known marker that defines more mature neurons. We also stained for synaptophysin, a protein known to play a role in synapse formation in maturing neurons. Although we did confirm MAP2 positive cells in culture, all time points were negative for synaptophysin (data not shown) suggesting that CTX8 cells do develop into neuronal cells, however they do not progress into the next stage of maturity, at least within their current differentiation environment.

Lastly, in the primarily neuronal clone category, GE6 cells were cultured in a similar temporal series and their progression was observed (Figure 6). Unlike the GE2 and CTX8 clones, GFAP expression was minimal in most of the cells, and in all time points up to 14 days. There was a large increase in GFAP at day 14 and these appeared to co-localize with Tuj1 positive cells. This result was surprising and may have resulted in a de-differentiation of the clones. Another possibility is that the neuronal cells did not survive well in the serum free long term culture conditions. Their appearance was not healthy, as there was substantial cell death and short process lengths when observed with the DIC optics. Tuj1 staining was strong after induced differentiation at days three and six. Unique to this clone, the processes appeared highly branched and long compared to the CTX8 positive neurons. Also surprising was that Nestin expression did not appear to diminish throughout the differentiation time series.

Gene expression profiles identify unique regulatory pathways

To extend the initial characterization experiments, we asked how the mRNA expression profiles of the clones differ and if their gene expression patterns correlate

29

with our initial classifications from the immunocytochemistry and western blot results.

We hypothesized that the expression profiles would further extend our knowledge of overall similarities and differences between clones. Furthermore, we predicted they would reveal interesting networks of genes that contribute to or regulate the neuronal or glial phenotypes. To test this, we chose the three Fischer contrasting clones representing a neuronal restrictive (GE6), glial restrictive (GE2) and a multipotential phenotype (CTX8), in addition to the previously characterized clones L2.2 and L2.3 (Li et al., 2008). All five clones were subjected to a three point temporal differentiation series where total RNA was extracted at each time point for mRNA expression profiling by microarray.

Clones were thawed and grown as previously described in the presence of bFGF. All five clones were plated with biological replicates (n=3) and time points were chosen to represent undifferentiated (Day 0) and differentiated phenotypes (Days 3, 7).

Differentiation was induced by bFGF withdrawal with the addition of 0.5% Fetal Bovine

Serum (FBS). At each temporal period, cells were harvested and total RNA was extracted, quantified and analyzed for quality. Approximately 11 μg from each sample was supplied to our collaborators, Myles Fennel and John Corradi (Bristol-Myers Squibb,

Wallingford, CT) for Affymetrix Rat Focus microarray analysis.

Since the microarrays generated thousands of variables per sample, in this case mRNA expression levels, it is difficult to visualize the differences between cell clones.

Principle component analysis (PCA) was run to reduce the dimensionality of the data into a few components, representing the maximum variation in the data set. This was then plotted to visually analyze the similarities and differences between clones (Figure

7A) (Ringer 2008). PCA indicated similar patterns for the two multipotential clones L2.3 and CTX8, along with clone L22, at three days and seven days respectively.

30

Interestingly, the GE6 clone segregated from these clusters indicating that although both

GE6 and L22 are primarily neuronally restricted, there are several differences between the two in their gene expression profiles. Strikingly, the GE2 clone was very different than the rest and suggests that the resulting phenotype will be quite different as well. To visualize the data set an alternate way, hierarchical clustering was performed using correlation as the metric, drawn as a dendrogram displaying the relationships between all samples (Figure 7B). Results demonstrate tight replicate distribution with the neuronal and glial phenotypes having the least similarity.

Turning our attention towards the expression of individual genes, a select set of transcription factors, synaptic proteins and was chosen to learn about the short and long term fate characteristics of each Fischer clone. A heat map consisting of cell clones and their respective temporal periods was constructed from the microarray data (Figure 8). Strikingly, we observed cell type specific transcript levels throughout the entire differentiation series. For example, Ascl1, also known as Mash1, was expressed

in both the multipotential (CTX8) and neuronal (GE6) clones. However, its expression

was only maintained in CTX8. This continued expression of Ascl1 but lack of any

recognizable neuronal subtype or synaptic gene expression during later stages suggests

that this particular multipotential clone may be locked in an immature neuronal state.

From the primarily neuronal clone GE6, we observed robust changes at mid to later

stage differentiation. More specifically, during days 3 and 7, the neuronal clone GE6

displayed an increase in expression of well-known transcription factors, (Dlx1 and Dlx5)

linked for the regulation of interneurons (Panganiban and Rubenstein, 2002). Also,

during the later stages of differentiation, multiple transcripts involved in

neurotransmission (Npy, Sst) and presynaptic proteins (Snap25, Stxbp1 and Syt1, Syt4,

Syt5 , Syt6) were expressed. The up-regulation of these later stage genes indicates the

31

neuronal maturation of this clone and sets it apart from CtX8, the multipotential clone that does not express any of these mature genes during the later stages of differentiation. In contrast, the glial clone GE2 did express Pax6 but showed no expression or up regulation of Ascl1 nor any of the synaptic component genes observed in GE6. These results agree with the previous immunostaining and protein analysis concluding that GE2’s cellular fate is non-neuronal under current conditions.

qPCR validation of microarrays

In order to investigate further, the expression profiles of the three Fischer clones, qPCR was performed on additional genes whose probes were not available on the

Affymetrix Rat Focus microarrays. These included additional members of the Dlx family,

Dlx2 and Dlx6 and also v-myc. In addition, it also provided the opportunity to biochemically validate the existing microarray expression profiles. Using the same biological replicate RNA sample for each time point (n=3) that were previously used in the microarray studies, total RNA was reverse transcribed into cDNA and used for qPCR. Results confirm the immunostaining and western blots previously described

(Figure 9). As previously mentioned, the microarray data suggested to classify GE6 as a maturing neuron that expresses select interneuronal genes. We confirmed the increases in these genes with Dlx1 and Dlx5 in GE6 and only slightly in CTX8. In addition, we were also curious if other members of the Dlx family were induced upon GE6 differentiation as these probes were not included in the custom microarrays. qPCR results conclude that

Dlx2 and Dlx6 were also preferentially expressed in the neuronal clone. Next, we looked at a gene responsible for the early development of interneurons, Gad2 (Kunkel et al.,

1986). Similar to the Dlx family transcriptional profiles, Gad2 was also increased upon induced differentiation in the neuronal restricted precursor GE6. Together, these

32

temporal expression patterns point towards a network of regulatory factors driving the neural precursor cell towards its pre-programmed interneuronal phenotype.

Myc is down regulated, but not silenced, during induced differentiation

Myc is a family of that has been implicated in the formation of tumors

(Meyer and Penn, 2008). c-Myc is one of four genes required for reprogramming fibroblasts into induced pluripotent stem cells (Takahashi and Yamanaka, 2006). The benefit of transducing clones with v-myc is the ability to keep them mitotically active while maintaining a relatively stable phenotype throughout multiple cell passages

(Synder et al., 1992). Each clone will continue to divide and maintain its neural stem cell

“undifferentiated” state when cultured in the presence of bFGF. This allows us to rapidly amplify clones for in vitro cell fate assays or transplantation studies. We have previously observed some clones producing tumors post transplantation, while others do not (non- published, The Grumet lab). Therefore, it is a valid concern to choose a neural precursor where v-myc expression is reduced upon bFGF withdrawal.

There have been reports that v-myc is either down regulated or silenced once

reprogrammed clones are differentiated (Synder et al., 1992; De Filippis et al., 2007).

Therefore, we asked if v-myc expression levels diminish during differentiation in our model system. Since there were no probes specific for v-myc on the microarrays, we

generated primer pairs corresponding to the protein coding sequence (CDS) region of

myc and analyzed this by qPCR (Figure 9). Results show a significant down regulation

following differentiation in both GE2 and GE6 clones. We conclude that although v-myc reduced expression during differentiation, it is not completely silenced. Any future transplantation experiments utilizing these clones will need to be aware of the potential

33

activity of this oncogene. These results also make a case for an inducible silencing method such as a lox/cre recombinase or a tetracyline system that can excise v-myc from transduced cells. These proposed methods will ensure silencing and may reduce potential tumor formations post transplantation.

GE6 long term differentiation identifies synaptic components suggesting functional activity

Consistent with a more mature neuronal identity, and unlike L2.2 cultures, differentiated GE6 cultures were intensely positive for TuJ1 and synaptophysin within ten days (Figure 10). These positive immunostaining results indicate that there are synaptic components present and the cell may be capable of synaptic activity. Since the gene expression studies discussed earlier displayed strong interneuronal subtype characteristics, we predicted that GE6 would display the proper protein markers associated with interneuronal signaling pathways. Therefore, differentiated cultures were stained for interneuronal specific markers and VGAT (Craig et al., 1996). Both showed strong expression after 10 days of differentiating, building a case for a functionally mature interneuron. As a control, GE6 cells were also stained for the glutamatergic marker Vglut-1, a vesicular glutamate transporter that mediates glutamate uptake into synaptic vesicles of excitatory neurons (Nunzi et al., 2003; Nakamura et al.,

2005). Vglut-1 staining was negative for all cells concluding that our reprogrammed clone GE6, isolated from the ganglionic eminence, is capable of generating cells resembling interneurons.

In collaboration with Dr. Mark Plummer’s lab, we further investigated the GE6 clones and asked if they were capable of producing electrophysiologically functional

34

interneurons (Li et al., 2011). Cells were differentiated by bFGF withdrawal and tested for electrical activity. To determine if GE6 clones would integrate with other mature neurons, they were co-cultured with hippocampal neurons prepared from E18 rat embryos. GE6 neurons could be identified by their GFP florescence and displayed depolarization-induced action potential firing when recorded in current-clamp mode. In voltage clamp mode after 12-15 days in culture, ongoing synaptic activity in GE6 cells were recorded that included both excitatory post synaptic currents (EPSCs) and inhibitory post synaptic currents (IPSCs). The co-cultures show that GE6 neurons can produce action potentials and also receive both excitatory and inhibitory synapses.

Because the expression profiles and immunostaining results suggested that neurotransmitters and synaptic proteins were being robustly expressed, GE6 clones were tested for synaptic activity when cultured alone. After 14 days of differentiation, spontaneous postsynaptic currents were recorded. All events that were recorded suggested that all currents were IPSCs. These results demonstrate that the GE6 clone is capable of producing functional neurons in isolation and that the resulting progeny are functional inhibitory neurons.

Summary of Section A: Reprogrammed precursors as an in vitro model for neural stem cell

In this section we described the isolation and reprogramming of neural precursors from different spatial regions of the rat embryonic forebrain. From this we have identified three clones representing the diversity of neural precursors in the developing embryonic forebrain. These include; a glial restricted precursor, a primarily neuronal clone with a preference for generating interneurons, and finally a multipotential precursor that is inherently more flexible and maintains the capability for both neuronal

35

and glial phenotypes. Some of the benefits of using these as models of neural development are that they are relatively stable over time by maintaining Nestin expression and in the presence of bFGF and are less heterogeneous than primary cultures by generating consistent neuronal or glial progeny. The gene and protein expression data support the hypothesis that contrasting phenotypes can lead to the identification of downstream regulatory pathways responsible for cellular fate. In the next section, we continue our investigation into the inherent genetic and epigenetic factors responsible for driving neuronal or glial networks.

36

Section B.

Epigenetic regulation of neural precursors

Our hypothesis states that certain chromatin marks contribute to an epigenetic signature representing the state of the cell, and these signatures can correlate with current and predict future gene expression. The goal is to discover novel regulatory pathways responsible for programming the neural phenotypes of the forebrain. The in vitro models we obtained represent various stages of neural development and are predicted to be useful for identifying factors responsible for driving cell fate. One area having significant influence on phenotype but has historically lacked in attention until recently is the field of epigenetics. The highly specific chemical modifications to both

DNA and amino acid residues of histone tails directly alter chromatin architecture. These changes can lead to widespread regulation of transcription and ultimately to the restriction or reprogramming of cell lineages. Only recently have the tools to robustly investigate the epigenetic state become available (Buck and Lieb, 2004; Ren et al.,

2000; Barski et al., 2007).

We investigated the chromatin state from our three reprogrammed cell clones in both their undifferentiated and differentiated states. We hypothesized that certain histone modifications would be enriched near lineage-specific genes and would correlate with current expression and also predict future mRNA expression. Furthermore, we predicted that chromatin markings were dynamic and would be altered by certain growth factors

known to be expressed throughout neural development and by chromatin modifiers such

as histone deacetylases (HDACs). This in turn would activate or repress certain lineage

genes, ultimately directing the neural precursor towards a particular fate. To test these

predictions and to uncover epigenetically regulated genes, select clones were treated

37

separately with BMP2 and certain HDACs to induce chromatin changes. Taking a genome wide approach, enriched chromatin marks were sequenced and aligned to the rat genome for analysis. Results presented herein first describe chromatin signatures that existed before differentiation, suggesting histone markings that may already limit future phenotypes. In addition, we present chromatin signatures that are dynamically altered after differentiation, suggesting levels of flexibility that can be modified with extrinsic factors and chromatin modifiers. Each of these findings provides us with a clearer understanding of how lineage restriction occurs through epigenetic modifications, and highlights specific genes that are regulated during neural development.

Neural clones display unique histone signatures

Whether an undifferentiated clone will ultimately become neuronal or glial cannot always be confirmed by morphology, immunocytochemistry or western blot. Depending on the assay used, some of the gene or protein expression differences that distinguish the phenotypes only present themselves after induced differentiation. For example, all three clones were positive for the neural stem cell marker Nestin, but negative for the well-known glial and neuronal markers. Only after differentiation was induced followed by a certain period of time did they generate the classic phenotypic markers of differentiated neural cells. In addition, only certain gene transcripts are expressed in an undifferentiated state, potentially masking cell fate potential. It is true that morphology may yield clues in some cases, such as the differences in neurite length or phase brightness. However, this method is not always reliable and should not be the only method to choose neural clones when a particular subtype is needed.

38

There are multiple epigenetic modifiers affecting gene expression and some contributing to structural changes in chromatin. Some of these include DNA methylation, histone modification and microRNAs. We first focused on histone modification marks because recent studies have shown they are both integral to embryonic cell fate restriction and appear to be dynamic throughout development (Meshorer and Misteli,

2006; Hong et al., 2011). We wanted to apply this knowledge towards neural development and first hypothesized that undifferentiated neural restricted precursors already possess chromatin that is primed for downstream gene expression. We predicted that some histone marks have already been deposited or removed before any induced differentiation, leading to a cascade of chromatin architecture alterations in key lineage genes. The changes coordinate the compacting or loosening of the chromatin by adjusting nucleosome spacing. This will allow for the proper transcription factors or co- factors to bind to the desired underlying DNA sequence initiating gene transcription.

Because of the strong correlation between histone modifications and gene transcription

(Bernstein et al., 2006; Pan et al., 2007), many of the well-known histone modifications have been previously shown to cluster near transcription start sites (TSS) of many genes.

Two specific histone signatures were chosen in an attempt to categorize the neuronal, glial and multipotential clones in their undifferentiated state. For example, we predicted that the neuronal clone GE6 would contain additional histone enrichment in levels of known permissive mark histone H3K4me3 while simultaneously displaying low levels of repressive mark H3K27me3 within specific neuronal pathway genes. In contrast, we predicted that the glial clone GE2 should contain an opposing profile displaying low levels of H3K4me3 and increased levels in H3K27me3 leading to a repressed chromatin state for certain neuronal genes. It has been previously shown that

39

“proper” chromatin state is necessary for the cell to react accordingly when presented with various maintenance or differentiation environments (Feinberg, 2008; Hamby et al.,

2008; Mehler, 2008). Our intent was to first probe for enrichment near the promoter regions of both well-known neuronal and glial genes and then uncover novel key regulators of cell fate using a genome-wide approach.

To test these predictions, we cultured all three neural clones (GE2, CTX8 and

GE6), in their undifferentiated state and performed ChIP with two histone marks correlating with gene activation (H3K4me3) or repression (H3K27me3). Histone marks were chosen based on previously described studies and antibody availability (Bernstein et al., 2006; Barski et al., 2007). We prepared neurospheres from each cell clone by culturing them in suspension in the presence of bFGF for three days. The neurospheres were then dissociated and distributed into aliquots. Each aliquot was cross-linked with fresh 1% formaldehyde and stored at -80° C until the sonication and ChIP experiments were performed.

Levels of enrichment were analyzed using qPCR with primers that were designed within 250 baser pairs (bp) of specific neuronal or glial promoter transcription start sites.

Primer sequences to a few well-known transcription factor genes known for directing neuronal and glial development such as Ascl1 (Mash1) and Olig2 were selected based

on results from the mRNA microarray study discussed earlier (Figure 11). Both of these

transcription factors displayed clear gene expression differences between clones in their

undifferentiated state and thus were initially chosen to compare by ChIP enrichment. In

addition, three other genes were chosen also based on their mRNA expression profiles

throughout the differentiation time course. All three genes had relatively the same

expression in their respective undifferentiated state. However, when differentiation was

induced, Erbb4 and Vgat were preferentially expressed in only the neuronal and

40

multipotential clones. Interestingly, Cbs expression increased in only the glial and multipotential clones. Therefore, by choosing contrasting gene expression profiles between clones, we could test our hypothesis that histone enrichment correlates with current and future gene expression.

We predicted that the neuronal clone GE6 would contain a chromatin signature more conducive to the activation of key genes involved in neurogenesis and the glial clone GE2 would display an opposing chromatin state, showing a more repressed signature with regards to the same neuronal genes. Results confirm some of these predictions (Figure 12). For example in GE6, promoter sequences upstream of the neuron-specific transcription factor Ascl1 (Mash1) and the tyrosine- Erbb4, a Neuregulin receptor, displayed enriched levels for a marker associated with gene expression (H3K4me3) and lower levels for gene repression (H3K27me3). In contrast, GE2 chromatin showed an inverse relationship with increased levels of

H3K27me3 and low H3K4me3. The confirmation that the chromatin states of Ascl1 correlate well with the undifferentiated gene expression was not surprising. As previously shown, the mRNA expression in their undifferentiated states also showed clear differences between clones (Figure 11). What is interesting is that the chromatin states and gene expression for Erbb4 do not correlate in their undifferentiated state. All three clones have a very similar level of mRNA expression before differentiation is induced while their chromatin states favor active expression only in the neuronal and multipotential clones GE6 and CTX8. However, the mRNA expression levels for Erbb4 post differentiation only show increases in GE6 and CTX8 after three and seven days of differentiation. As predicted, it appears the chromatin architecture surrounding the transcription start site of Erbb4 was primed for activation in these two clones (Figure

12B). This suggests that cell fate for some neuronal pathways is already predetermined

41

in its chromatin state and a step or two ahead of traditional gene expression studies by altering the architecture for downstream gene expression.

Next, we mined the gene expression profiles of genes that were preferentially induced after differentiation in the glial restricted clone GE2. We were again particularly interested in expression profiles that were similar in all three clones in their undifferentiated state, but were differentially expressed after bFGF withdrawal.

Cystathionine Synthase (Cbs) was one of the genes to fit these criteria as its expression was similar in the undifferentiated states of all clones. It increased in GE2 and CTX8 when differentiation was induced (Figure 11). Based on these mRNA expression values, we predicted that there were chromatin differences in the regulatory regions of this gene.

Similar to the neuronal genes, we performed ChIP on the permissive and repressive histone modifications and analyzed the promoter regions of Cbs in the undifferentiated state. As predicted, we observed clear differences in the repressive histone marker

H3K27me3 (Figure 12). Neuronal clone GE6 had significantly more enrichment in this repressive modification compared to GE2 with a p-value <0.05 using Student’s t-test. It was interesting that we observed opposite enrichment patterns between restricted clones in both neuronal and glial regulatory regions. This finding demonstrates that epigenetic regulation is not limited to the lineage restriction of a particular neuronal or glial phenotype.

These observations lead credence that chromatin structure is playing a role in current or future gene expression and that some of these epigenetic signatures can be used to predict neural lineages. Finally, we chose to investigate epigenetic enrichment in a neuronal gene well known for its involvement in interneuronal cell fate, Dlx5. As presented earlier, Dlx5 mRNA expression was preferentially increased in GE6 cells and slightly in CTX8. We asked if the regulatory region upstream of Dlx5 was also

42

preferentially enriched in either the permissive or repressive histone modifications. A primer set was chosen upstream of the Dlx5 transcription start site (TSS) for qPCR quantitation of enrichment. We did not observe any significant differences for either histone mark or for any of the cell clones (data not shown). Dlx5 may not be regulated epigenetically or specifically by the two chosen histone marks. It also possible that the specific upstream regions chosen for qPCR are not enriched with these histone signatures. The locations near the transcription start site with the best histone enrichment may vary from each gene.

In summary, our initial focus was on well-known histone modifications previously linked to gene activation or repression. The comparison of all three Fischer clones in their undifferentiated states showed some clear epigenetic differences at key transcription factors and other regulators that were active and distinct in the microarray expression profiles. These genes are believed to be involved in neural fate decisions.

Conclusions from these enrichment patterns suggest that the neuronal clone GE6 is either already active or is poised to initiate gene transcription for a host of known neurogenic genes. Contrasting these results in GE2, the same neuronal gene regulatory regions are more enriched in repressive marks H3K27me3 suggesting that this cell clone is refractory and does not favor the transcription of these neurogenic set of genes.

Therefore, combinations of epigenetic modifications can provide interesting snapshots of chromatin state preceding cell differentiation. Cataloging these chromatin marks may be a convenient method for predicting future gene expression. However, tracking the histone differences is not just an annotation exercise. Previous studies have shown that these modifications directly alter the chromatin architecture by recruitment of additional chromatin modifying enzymes (Jenuwein and Allis, 2001; Narlikar et al., 2002;

Kouzarides, 2007). In turn, these alterations lead to gene repression via chromatin

43

compaction or gene activation by the loosening the chromatin and widening the space of nucleosomes for transcription factor binding.

ChIP Sequencing of Fischer clones

Targeted ChIP qPCR at specific regulatory regions was a first step in elucidating histone signatures that may be predictive of downstream gene expression patterns.

However, this approach is limiting because it only targets specific regions of the gene of interest. Furthermore, although both of these particular histone modifications have been documented to be deposited near transcription start sites, the ChIP qPCR method is at best an estimated guess for discovering enrichment patterns. To gain an overall perspective of where histone modifications are enriched and to potentially discover epigenetically regulated genes, a genome wide chromatin map is necessary. We pursued this with the ChIP sequencing (ChIP-seq), of all three Fisher clones in their undifferentiated states. Our prediction was that the epigenetic signatures would be distinct during this temporal period and would highlight additional epigenetically regulated neuronal or glial networks. Widespread coverage of sequence tags was achieved throughout the entire genome as Figure 13 represents the aligned sequence reads across all chromosomes of each Fischer clone in its undifferentiated state.

Enriched fragments were sequenced and aligned to the rat genome. A genomic coverage map of the undifferentiated Fischer clones displays the H3K9/14ac peak densities that were scaled to the total number of peaks per Mb per sample. Genomic coverage, the level of was as follows: GE6-Mean coverage: 1.1, Coverage range: 1 -

49. GE2-Mean coverage: 1.1,Coverage range : 1 - 38. CTX8-Mean coverage: 1.4,

Coverage range: 1 - 58. Results show that the entire sequencing reaction covered the

44

genome at least by 1.1X rate on average. This consistent coverage results showed no or DNA strand biases and was used as a quality control giving confidence that we achieved genome wide coverage.

For the ChIP sequencing datasets, we chose to use the acetyl markings H3K9/14 ac instead of H3K4me3 as in the original ChIP qPCR (Figure 12B). This was due to reports that although the H34me3 histone mark is considered permissive, it can also be co-localized with repressive mark H3K27me3. Regulatory regions containing the combination of these two modifications are said to be in bivalent state, where the expression of the gene may not yet be active (Bernstein et al., 2006). While the topic of bivalency is fascinating as we predict it also plays a role in neural cell fate, we preferred that the first ChIP-seq experiment used more contrasting histone modifications.

Therefore, the acetyl histone modification H3K9/14ac was used as the permissive epigenetic representative. In addition, we reasoned that if the regulatory regions were truly enriched in permissive marks, there was a good chance of observing both

H3K4me3 and H3K9/14 ac near the TSS of the gene of interest. The first samples analyzed were the original genes that displayed such contrast between neuronal and glial clones. We chose to view the CTX8 regulatory regions surrounding the transcription start sites of Erbb4, Olig2, and Slc32a1 (VGAT) during their undifferentiated states (Day

0) (Figure 14). This was another quality control check to verify that the permissive and repressive enrichment correlated with the original ChIP qPCR experiments. Confirming the previous data (Figure 12), we observed permissive enrichment near the TSS in both

Erbb4 and Olig2, while the VGAT regulatory region was enriched only in the repressive histone modification H3K27me3. Although the ChIP-seq permissive and repressive modifications did correlate with the ChIP-qPCR results, the enrichment patterns did not

45

strictly overlap. This highlights the limitations of ChIP-qPCR, whereas ChIP-seq provides a more robust overview of chromatin state throughout the genome.

Next, to get an overview of the networks that were enriched in either the permissive H3K9ac or the repressive H3K27me3 modifications, we performed a gene ontology (GO) analysis of all sequenced peaks for each sample. Using the

ChIPpeakAnno algorithm (Zhu et al., 2010), all peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a Benjamini & Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset. We compared the highest ranked GO terms and then grouped them into broader categories to highlight the most enriched categories per sample. Figure 15 displays the groupings from the top 25 GO terms for each sample. There were differences between clones in multiple categories such as General Differentiation and

Neuron Specific. These results support the hypothesis that the Fischer clones in the undifferentiated states have chromatin state differences that may affect downstream gene expression. One interesting example when analyzing the GO categories was that the CTX8 sample was relatively more enriched in General and Neural Development and less in the more restricted specific Neuronal category. This correlates with what previous characterization studies have shown that CTX8 is a more multipotential cell, not yet fully committed. In contrast, the glial clone GE2 clones were also enriched in the Neuronal category, similar to GE6. This was surprising as we had predicted that GE2 cells might be more enriched in glial like categories. Next, we predicted that differentiation may also induce histone changes over time to modulate cell fate. To further investigate this and discussed in detail in the next section, we induced differentiation in the multipotential clone CTX8 with treatments altering its phenotype and predicted that epigenetic changes would be identified.

46

Growth factors rapidly alter neural phenotype

How exogenous factors influence cell fate through epigenetic mechanisms was one of the next questions we addressed. It is well established that different areas of the developing forebrain produce growth factor gradients that have profound effects on neural development, cellular migration and lineage restriction (Temple, 2001; Briscoe et al., 2001; Panchinson and McKay, 2002; Li and Grumet, 2007). Networks of genes can be rapidly induced or dampened by the introduction of some of these factors. We were interested to observe how the chromatin state responded, so we predicted that epigenetic marks would be deposited or removed near lineage genes after growth factor treatment. Therefore, we next focused our attention on the plasticity of the multipotential clone, CTX8 and how exogenous factors influence cell fate. The gene expression and immunostaining data show that that CTX8 clone can generate both neuronal and glial phenotypes without any exogenous factors. Therefore, we chose this clone as the most malleable and predicted that it would be responsive to multiple of endogenous factors that are expressed throughout neural development.

Three growth factors (CNTF, BMP2, SHH) normally secreted in the medial, dorsal, ventral areas of the forebrain during embryonic development (Li and Grumet,

2007) were introduced to CTX8 and cells were differentiated by bFGF withdrawal for three days. Plated cells were cross-linked and immunostained with antibodies against neuronal and glial markers TuJ1 and GFAP. Increases in both neuronal and glial markers were observed between controls and the multiple growth factor treatments

(Figure 16). CNTF and LIF, normally produced from the choroid plexus (Gregg and

Weiss, 2005) and shown to stimulate the JAK/STAT pathway (Segal and Greenberg,

1996; Stahl and Yancopoulos, 1994) substantially up regulated GFAP expression while

TuJ1 expression appeared diminished. In contrast, the ventrally produced SHH, known

47

to promote Olig2 positive oligodendrocytes and motor neurons (Gabay et al., 2003; Lu et al., 2002; Molne et al., 2000), had the opposite effect of CNTF treatment and appeared to increase TuJ1 positive cells and decreased GFAP expression. Dorsally expressed

BMP2 increased both TuJ1 and GFAP expression after three days of differentiation versus the control samples. In addition, the morphology of the differentiated neuronal cells appeared more mature with increased fluorescence intensities and highly branched processes. This study confirms that CTX8 cells can respond to morphogenic factors normally expressed as gradients in developing forebrain and that phenotypic plasticity is easily and rapidly obtained. It has been suggested that lineage restriction resulting from growth factors BMP2 and CNTF, occurs in part by the regulation of transcription factors, which in turn activate neural restricted markers (NRPs) and glial restricted markers

(GRPs) such as PSA-NCAM and A2B5 (Li and Grumet, 2007).

Next, we tested our hypothesis that morphogenic factors promote neuronal and glial development in part through epigenetic mechanisms. A time course consisting of control and BMP2 treated CTX8 cells were differentiated, immunostained, and harvested for ChIP-sequencing analysis. Figure 17A shows the progression of neuronal marker

TuJ1 and astrocytic marker GFAP at one, three and seven days of differentiation for both the control and BMP2 treated samples. In the controls, we observed both TuJ1 and

GFAP positive cells throughout the cultures by day three. At day seven, however, while

GFAP staining was prevalent, TuJ1 positive cells were diminished compared to day three and were clearly the minority phenotype within the culture. This trend of diminishing TuJ1 positive neurons agrees with the previous long term differentiation study shown earlier (Figure 5). The BMP2 treated cells also showed widespread TuJ1 and GFAP positive cells at day three. In addition, the morphology of TuJ1 positive cells appeared more mature by displaying a more branched structure with longer process

48

lengths. In contrast the control samples, at day seven of differentiation, the levels of

TuJ1 positive cells were maintained and appeared as healthy neuronal cultures, suggesting that the BMP treatment was maintaining and promoting both the neuronal and glial phenotypes throughout the extended differentiation.

ChIP-sequencing was performed with antibodies against the two histone modifications H3K9/14ac an H3K27me3. To get a global snapshot of the functional pathways that may be involved after BMP2 treatment we performed a GO analysis of all sequenced acetylated peaks for each sample during all time points. Similar to the approach taken with the three Fischer clones, using the ChIPpeakAnno algorithm (Zhu et al., 2010), all peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a Benjamini & Hochberg

(BH) adjusted p-value based on the number of enriched counts within the dataset. We compared the highest ranked GO terms and then grouped them into broader categories to highlight the most enriched categories per sample and temporal period. Figure 17B displays the groupings from the top 25 GO terms for each sample. An interesting result was the maintenance of percentages of genes epigenetically marked within both neural development and neuronal gene ontology groups in the BMP treated samples during three and seven days of differentiation (Figure 17C). While the control group had decreases in both of these categories by day seven, the BMP2 treated samples maintained their enrichment in histone acetylation within these categories. These trends mimic the immunostaining results (Figure 17A) and suggest that BMP2 treatment promotes the acetylation of neural and neuronal development genes, leading to downstream gene activation. Discovering that morphogenic factors such as BMP2 have epigenetic effects can be useful for discovering regulated genes and new functional pathways responsible for the generation of neurons or astrocytes.

49

Epigenetic modifiers alter chromatin structure leading to enhanced neurogenesis

In addition to the epigenetic markings present before neural cells develop into their mature phenotypes, we hypothesized that additional histone modifications are deposited or removed from specific neural genes throughout differentiation, molding ongoing lineage restriction. Moreover, we predicted that altering chromatin structure would lead to downstream gene expression changes altering the phenotypic outcome.

We decided to test our predictions using ChIP with the CTX8 clone. CTX8 was chosen due to its inherent multipotential capabilities. In addition, slight alterations in the generation of neurons or astrocytes would be relatively easy to quantitate compared to the more restricted clones.

Histone deacetylase inhibitors have previously shown to alter chromatin structure by interfering with endogenous histone deacetylases, resulting in a net increase of acetylated histones. This in turn, has a direct effect and loosens the underlying DNA making it more viable for transcription. Two well-known HDAC inhibitors Trychostatin A

(TSA) and Valproic Acid (VPA) were chosen as chromatin modifiers and were predicted to alter chromatin structure and influence cell fate. VPA was not as toxic as TSA during initial differentiation assays, although moderate cell death was observed compared to controls (not shown). We tested whether VPA was altering chromatin and performed a western blot on differentiated progeny using the antibody against H3K9/14ac that recognizes acetylated histones on lysine residues 9 and 14. The levels of total histone

H3 were also analyzed and used as a negative control. We did not expect the addition of

VPA to adjust the total amount of histone H3. As predicted, we observed a global increase in acetylation in the VPA treated samples after 3 days of differentiation (Figure

18A). Both short and longer term differentiation studies were completed with the addition of VPA. Cultures were cross-linked and immunostained for neuronal markers TuJ1 and

50

MAP2 along with the glial marker GFAP (Figure 18B). VPA treated cells increased the number of TuJ1 and MAP2 positive cells at 3 and 14 days of induced differentiation. We further quantified both the morphological changes and the increased numbers of TuJ1 positive neurons from VPA treatment. Figure 19A shows the increased percentage of

TuJ1 positive neurons in VPA treated cells compared to controls after 7 days of differentiation and quantified by flow cytometry. Due to the low numbers of differentiated cells, three biological replicates were combined before flow cytometry analysis. VPA treatment also led to increased neurite process length (18%), and total cable length

(20%) which is defined as the combined process lengths per cell (Figure 19B). Fom these experiments, I conclude that the addition of valproic acid alters chromatin resulting in global increases in acetylated histones and also generates enhanced neurogenesis as well as process length. The next goal was to locate specific areas of the genome where the acetylation increases were occurring and test the hypothesis that these changes are near regulatory regions responsible for modulating neuronal cell fate.

ChIP sequencing identifies neuronal regulatory regions

To take an unbiased approach and discover specific acetylation enrichment throughout the entire genome following valproic acid treatment, we employed a global methodology and applied chromatin immunoprecipitation sequencing (ChIP-Seq).

Similar to the ChIP sequencing experiments described earlier all enriched chromatin fragments were sequenced and aligned to the rat genome. To discover novel enrichment patterns and test our predictions that histone modifications are dynamic throughout neural development and are altered by the HDAC inhibitor VPA, we sampled three time points at days 1, 3 and 7. As shown earlier CTX8 treated cells both increased in global acetylation and increased numbers of neuronal progeny. In this context, we believe that

51

this chromatin modifier alters specific regulatory regions in certain genes responsible for neuronal cell lineage restriction and maturation. We predicted that the addition of VPA would both promote and maintain acetylated histones of these genes and promote their gene expression. Using ChIP-sequencing we were now able to test this hypothesis. To get a sense of the functional pathways that may be active following VPA treatment we initiated a GO analysis as described earlier. Briefly, all acetylated peaks were annotated to their nearest gene and ranked by functional category, using the Benjamini &

Hochberg (BH) adjusted p-value that was based on the number of enriched counts within the dataset. Interestingly, within the highest ranked 25 GO terms, many included neural and neuronal functions in both the control and VPA treated samples. Some of these included: formation, neural tube development, neurite outgrowth and morphogenesis. This confirms that the acetylated epigenetic signature of CTX8 cells during this time point identifies with neural development. During the differentiated time course, we observed the maintenance in numbers of both nervous system related

(Figure 20A) and neuron specific gene (Figure 20B) categories in the VPA treated samples. This is contrary to the progressive loss of acetylated neural and neuronal genes by day 7 within the control group. These epigenetic enrichment patterns correlate with the immunostaining results that show a progressive loss in the percentage of TuJ1 positive neuronal cells in controls compared to VPA treated cells. These results suggest that VPA treatment promotes or maintains the acetylation of multiple neural and neuronal development genes, leading to downstream gene activation towards the neuronal phenotype. The next step was to further investigate specific genes that were epigenetically regulated by the addition of valproic acid.

To locate the specifically altered chromatin that may be playing important roles in neuronal development, we decided first filter out all permissive and repressive histone

52

modifications that were not within 3 kilobases (kb) of each gene’s transcription start site.

The 3 kb filter was arbitrarily chosen based on previous histone modification signatures that are usually enriched near promoter regions close to the TSS of each gene (Berstein et al., 2006; Kouzarides, 2007). Although we cannot rule out the importance of these epigenetic marks that remain a significant distance away, such as enhancer regions, we decided to focus on modifications near the TSS. Once the peak lists were filtered, we compared both treatments and temporal periods. Table 1 includes a gene list of all VPA acetylated genes after 3 days of differentiation. Although H3K27me3 may represent genes that are silenced post differentiation and may contain important pathways involved in the inhibition of non-neural or neural stem cell phenotypes, we chose to focus on the enriched acetylation genes. We hypothesized that VPA inhibits endogenous histone deacetylases resulting in acetylation increases near the regulatory or coding regions of neuronal transcription factors and microRNAs. These changes in acetylation alter chromatin structure by loosening the surrounding DNA, making it more amenable for transcription. Therefore, some of these epigenetically regulated genes may be responsible for the increased percentage of neuronal cells that are observed after VPA treatment.

Neurogenic transcription factor Ascl1 is epigenetically regulated by valproic acid

Reviewing the list of genes associated with upregulated acetylated histones from

VPA treated cells at 3 days post differentiation (Table 1), Ascl1 was one transcription factor that was highly enriched in H3K9/14 ac marks. For each ChIP-Seq sample, aligned peaks were converted into tracks that could be easily visualized using a genome browser. For example, Ascl1’s permissive enrichment (H3K9/14Ac) spanned

approximately 2 kb through almost the entire coding region (Figure 21A). In addition,

53

there was also a loss of the repressive mark H3K27me3 in the VPA treatment. This suggested that Ascl1’s gene expression is epigenetically regulated after VPA treatment.

To test this, total RNA was harvested after 3 days of treatment and assayed by qPCR with primers targeting the coding region of Ascl1. Results confirm a significant increase

(p value <0.05 using Student’s t-test) in VPA treated samples compared to controls

(Figure 21B). Although this is one the more well-known neuronal transcriptional regulators, it peaked our interest as a VPA regulated gene. In addition to the existing knowledge that Ascl1 increases neurogenesis, it has also been shown to preferentially regulate the development of interneurons (Poitras et al., 2007; Battiste et al., 2007;

Zhang et al., 2010). Since we observed contrasting expression levels in our three

Fischer clones, we predicted that the exogenous addition of Ascl1 into the multipotential line, CTX8, would lead to enhanced neurogenesis and may also guide them towards an interneuronal fate. This hypothesis was tested and will be discussed in the next section.

microRNAs are epigenetically regulated by valproic acid

We hypothesized that similar to transcription factors and other neural related genes, microRNA regulatory regions would also be affected by VPA treatment, specifically by enhanced acetylated histone modifications. In turn, the resulting relaxed chromatin architecture would then correlate with future transcriptional expression. There are benefits to discovering microRNAs that are epigenetically regulated by chromatin modifiers such as valproic acid. If their expression patterns correlate to the observed histone enrichment, they can be further investigated by testing their abilities to enhance neurogenesis. Novel microRNAs discovered in through increased acetylation enrichment may be useful for future therapeutic strategies where specific neural phenotypes are required. As predicted, enriched acetylated peaks were found near many microRNA

54

genes. Using the ChIP-sequencing dataset, we generated a list of all microRNAs enriched in acetyl marks within 3kb from each TSS after three days of differentiation

(Table 2). Included in the list are some well-known microRNAs (mir-9 and mir-30b) that have been previously identified with brain development and specifically neurogenesis

(Krichevsky et al., 2003; Gao, 2010; Song et al., 2011).

For example, these findings suggest that the chromatin surrounding mir-9 is specifically regulated by addition of an HDAC inhibitor. Previously, this microRNA has also been observed to be induced during neuronal differentiation in other reprogrammed cell clones, L2.2 and L2.3 (Hart Lab - unpublished data). Therefore, to observe that it may also be epigenetically regulated is interesting because it may be useful for future

HDAC inhibitor or other applications where targets of mir-9 need to be dampened down or silenced. Another regulatory region enriched post VPA treatment was mir-125b. Not as well-known as mir-9 and mir-124, this microRNA has been recently getting some attention for its role in neuronal development (Minh et al., 2010). Therefore, it was of interest to observe the epigenetic enrichment of this gene though our peak filtering methods and speculate its involvement within neuronal differentiation.

Since our hypothesis asserts that chromatin state either represents or precedes downstream gene expression, we chose to biochemically validate multiple microRNA genes that were enriched in acetyl markings using qPCR. All cultures were set up similarly to the ChIP-seq experiments. Total RNA for each condition (n=3) was extracted and reverse transcribed into cDNA for qPCR. Using Student’s t-test, we confirmed significant differences (p-value <0.05) between the control and VPA treated samples

(Figure 22). It was also interesting that the star sequences of mir-9, and mir-125b also increased post differentiation with the addition of valproic acid. The microRNA star strand, also known as the passenger strand is the stand not selected for entry into the

55

silencing complex. It is usually known to be degraded, however recent studies have implicated a potential function as a guide microRNA. Furthermore, some microRNA star sequences have been shown to be phylogenetically conserved and may play a role in gene regulation (Guo and Lu, 2010). From these results, we conclude a positive correlation between induced epigenetic modifications and increased gene expression in a group of microRNAs after three days of induced differentiation. We postulate that these particular microRNAs may play a prominent role in lineage restriction towards maturing neurons. In summary, ChIP sequencing for enriched permissive and repressive marks has uncovered a few select microRNA genes that are epigenetically regulated after valproic acid treatment and may assist in modulating the neuronal phenotype.

Exogenously expressed microRNAs increase neurogenesis

As an initial validation that microRNAs could modulate the phenotype of differentiating neural precursor cells, we transfected strand-specific PremiRs, (Ambion,

Austin,TX) that act as mature microRNA mimics, into the multipotential clone L2.3. This immortalized clone was used because the Fischer clones had not yet been derived and it was necessary to test the hypothesis that a select group of neurogenic microRNAs could induce a phenotypic response. Since their discovery, the search for functional microRNAs and their expression patterns has been a highly researched topic due to their roles in neural development and maturation (Bartel, 2004; Krichevsky et al., 2003;

Gao, 2010). We hypothesized that the expression of specific combinations of microRNAs that are expressed throughout neural development play a role in determining the final phenotypic state. We were particularly interested in identifying specific groups of microRNAs involved in neuronal differentiation. Using the neuronal clone L2.2 and multipotential clone L2.3, a potential set of neurogenic microRNAs (mir-9, mir-124, mir-

56

153, mir-182) was identified by comparing the cross-correlated the expression patterns of microRNAs and a subset of transcription factor mRNAs (Goff et al., 2008). Among those identified as brain-enriched microRNAs by previous reports, mir-9 and mir-153 are expressed in proliferating and differentiating neural cells (Mortazavi et al., 2006;

Kapsimali et al., 2007) while mir-124 expression is restricted to differentiating neurons

(Kapsimali et al., 2007; Yu et al., 2008).

To test whether these microRNAs could modulate the phenotype of differentiating neural precursor cells, we transfected strand-specific PremiRs (Ambion,

Austin,TX) into the multipotential clone L2.3. Each of these small RNAs were overexpressed separately and in combination to identify any microRNA synergies. We also overexpressed a non sequence specific RNA and a no RNA template as negative controls. PremiRs are double stranded molecules that are appropriately loaded into the microRNA machinery complexes. This preferential loading then allows for the over- expression of the strand-specific microRNA sequence. We hypothesized that these microRNAs were capable of increasing the percentage of neuronal cells by increasing

TuJ1 positive cells after induced differentiation. Transfected cells (n=3) were differentiated for three days and quantified for TuJ1 expression using flow cytometry

(Figure 23A). TuJ1 positive cells were significantly increased compared to the scrambled negative control having a p-value of <.0.05 using Students t-test. Next, we asked if it were necessary that all four microRNAs be over-expressed together or would we observe a similar effect using individual microRNAs? In three separate experiments, we transfected the combination of all four neurogenic microRNAs (mir-9, mir-124, mir-153, mir-182), in addition to each microRNA individually (Figure 23C). Results suggest that two of the four microRNAs, mir-9 and mir-153 were sufficient by themselves to increase levels of TuJ1 positive cells. Interestingly, both of these microRNAs increase levels

57

comparable to the combination suggesting there is not a synergistic increase in TuJ1 production using all four neurogenic microRNAs.

We then investigated whether knocking down microRNA expression would produce the opposite effect by dampening neuronal progeny (Figure 23B). We observed no effects on the numbers of Tuj1+ cells when AntimiRs targeting each of the neurogeneic microRNAs described above were transfected individually (data not shown). Next, a combination of all four Antimirs was transfected will the goal of inhibiting the endogenous microRNAs simultaneously. Although we did not observe significant differences between treatments, we did notice a downward trend of less neurons in the

Antimir samples. These results suggest that exogenous expression of some of these microRNAs both individually and in combination have a neurogenic effect and are sufficient to enhance a pro-neuronal effect on multipotential neural precursor cells.

Furthermore, the inability to fully reverse this effect also suggests that there are potentially other microRNAs that have pro-neuronal roles and may function in parallel pathways. By not successfully targeting all neurogenic microRNAs we were still unable to completely diminish neurogenesis.

Summary of Section B: Epigenetic regulation of neural precursors

Our preliminary comparison of all three Fischer clones showed clear epigenetic differences with key neuronal and glial transcription factors and other regulators involved in neural fate decisions. Specifically, our initial focus was on well-known histone modifications that were previously linked to gene activation or repression. Combinations of the active mark H3K4me3 and repressive mark H3K27me3 provided an interesting snapshot of chromatin state that preceded cell differentiation. We learned that these

58

particular neuronal and glial regulatory regions had epigenetic signatures prior to differentiation and, in a sense, prepared the cell for future gene expression. Taking a more global view of the epigenetic state of the clones, we surprisingly did not find any significant differences between the clones in their undifferentiated states using peak annotation and gene ontology analysis. However, after differentiation of the multipotential clone CTX8 with neuronal promoting factors BMP2 and valproic acid, we observed acetylation changes in neuronal development transcription factors and microRNAs. These epigenetic changes suggest a coordinated regulation of neural genes that assist in modulating cell fate.

59

Section C.

Enhanced reprogramming of neural precursor cells towards interneuronal subtypes

The production of stable, expandable populations of neural precursors that differentiate consistently into neurons with specific neurochemical properties is a goal for both regenerative medicine and the study of neuronal mechanisms of disease.

Interneuronal development progresses through a series of regulatory steps starting with an initial commitment towards the interneuronal class, and then later proceeds into specific subtypes (Vitalis and Jean Rossier, 2010; Butt et al., 2005).

Through the isolation and reprogramming of forebrain cells, we have learned that the immortalized set of neural clones (CTX8, L2.3), generates some broad phenotypes than those with more restricted capabilities (GE2, GE6, L2.2) (Li et al., 2008; Li et al.,

2011). Of the three reprogrammed Fisher clones, GE6 was shown to differentiate into primarily GABAergic functional interneurons. Three transcription factors; Ascl1, Dlx1

Dlx5 (to be referred to here as ADD) were all confirmed to be preferentially expressed in this neuronal clone during extended differentiation (Figure 8). Similar to L2.2, GE6 consistently showed positive expression for general interneuronal markers such as

GABA and Gad 65/67. Both the microarray and qPCR results indicate a temporally induced network of genes leading to consistent cell fate restriction. Furthermore, within the restricted neuronal clones, distinct subtype identities are apparent when the clones were differentiated. Immunostaining and western blots for additional interneuronal subtypes revealed that the differentiated GE6 clones expressed interneuron markers

NPY and SST and just a small set were positive for Calretinin (Li et al, 2011). In contrast, L2.2 clones gave rise to mostly Calretinin positive cells but showed very little

60

NPY and SST positive progeny (Li et al., 2008). Moreover, both expressed broad interneuronal markers such as VGAT and Gephyrin (Figure 10) (Li et al., 2011). In addition, these primarily neuronal restricted clones were negative for glutamatergic markers, confirming subtype specificity.

In electrophysiology assays, both L2.2 and GE6 clone were synaptically active, however, differentiated L2.2 clones could only form synapses when co-cultured with hippocampal primary neurons (Li et al, 2011). In contrast, GE6 differentiated clones were synaptically active both alone and in co-cultures. In fact the GE6 action potential traces displayed an even more robust electrically active signature and formed more mature interneurons compared to the L.2.2 clone. Once again, this suggests that given similar differentiation conditions, GE6 is pre-programmed to develop into a specific subtype of interneuron. All of these distinct differences suggest the successful isolation and maintenance of interneuronal subtypes. We also predict that these differences have downstream consequences, affecting interneuron maturity and function.

The goal was to learn how to program cells to both enhance and direct neurogenesis towards specific neuronal subtypes. We approached this by combining what was learned in the initial characterization assays (Figure 2C), in addition to the epigenetic and gene expression profiles described in the sections A and B. For example, in addition to the genes that are essential for neuronal development in GE6, we also confirmed that Ascl1 could be epigenetically regulated in the multipotential clone CTX8

(Figure 21). Treatment with the HDAC inhibitor valproic acid increased acetylation enrichment surrounding the Ascl1 gene and also enhanced neurogenesis in these multipotential clones (Figure 18, Figure 19). These results, along with the GE6 observations, suggest that all three of these transcription factors (Ascl1, Dlx1, Dlx5) are

integral to the formation of the interneuronal phenotype. Since the multipotential clone

61

CTX8 has been phenotypically responsive to factor treatments (CNTF, SHH, BMP2) and chromatin modifications (VPA), this clone was chosen as a relatively uncommitted neural precursor with which to reprogram the interneuronal phenotype observed in GE6. We predicted that exogenous expression of all three transcription factors (ADD) would activate the necessary gene networks to enhance CTX8’s differentiation towards a

GABAergic phenotype. We combined a standard neuronal differentiation protocol with transient co-overexpression of the three ADD transcription factors. In addition, a co- culture environment was used to optimize and provide additional survival and growth factors for neuronal maturation, as used previously for GE6 (Li et al., 2011). By learning both the regulatory factors responsible and where they initiate their downstream pathways, we can better understand the steps leading to neuronal identity and development.

Ascl1 expression is increased in neuronal and multipotential clones

Our goal was to obtain an expandable population of precursor cells for rapid production of selected neuronal subtypes. Ascl1 was one of the first choices due to the

gene expression patterns and epigenetic state observed in all three Fisher clones. Ascl1 gene expression profiles in the pre and post differentiation conditions were both expressed at relatively higher levels in the multipotential (CTX8) and neuronal clone

(GE6) (Figures 8, Figure 11). Also, the undifferentiated chromatin states were enriched in permissive acetyl histone marks and appeared conducive to future gene expression.

In addition, by globally altering chromatin patterns by the addition of valproic acid, a well- known chromatin modifier, we observed specific increases of histone acetylation within the regulatory regions of Ascl1. In addition Ascl1 historically is a well-known transcription

factor that has been linked to a variety of neurogenic pathways (Casarosa et al., 1999,

62

Hortona et al., 1999, Carlos et al., 2002). Most recently, Ascl1 was also found to be the most critical gene responsible for the direct conversion of mouse fibroblasts into induced neurons (Vierbuchen et al., 2010).

The correlation between the gene expression and chromatin modifications during the early stages of differentiation suggests that Ascl1 contributes to the initial production of neurons. At this time it is still unclear if Ascl1’s expression is responsible for generating certain neuronal subtypes or is just a pan neuronal transcription factor. It has been demonstrated that Ascl1 does play a role in both neurite outgrowth and the

generation of interneurons (Poitras et al., 2007, Battiste et al., 2007). We predicted that

the overexpression of Ascl1 would increase both the percentage of neurons generated

and also contribute to morphology enhancements such as increased neurite outgrowth

or dendritic branching. We chose to overexpress this transcription factor into the

multipotential clone (CTX8) for multiple reasons; first, it closely resembles a radial glial

cell and second, it has previously been shown to rapidly respond to a variety of growth

factor treatments by altering its phenotype percentages, and lastly, CTX8 over time

generates primarily glial cells as a default cell fate. Therefore, it is a good model cell that

is both reactive to external factors and will be relatively easy to quantitate subtle shifts in

neuronal/glial progeny.

Reprogramming through transient transfection is rapid and efficient

CTX8 precursor cultures (n=3) were transfected with a Chicken /Ascl1

plasmid or the uninserted vector plasmid pUC19 used as an empty vector control. To

monitor the transfection efficiencies and track transfected cells, a pCAG-DsRed

construct (Matsuda and Cepko, 2004), producing a red fluorescent protein (RFP) was

63

co-transfected into both conditions that was visible within 12-16 hours using epifluorescence microscopy. Differentiation was induced approximately 12 to 16 hours post transfection by bFGF withdrawal. The range of RFP intensity was variable, suggesting that plasmid copy numbers were not always equal. To address the need for over-expressing multiple plasmids and to verify transfection consistencies, we completed a pilot study in one of the earlier immortalized rat clones, L2.3 (Li et al., 2008). Since this precursor was not derived from a GFP expressing rat, were able to co-transfect both

GFP and Ds-Red producing plasmids and then monitor their co-expression (Figure 24A).

An average co-expression of at least 85% gave us confidence that each successfully transfected cell most likely received both plasmid constructs and that co-transfecting two or more plasmids should be adequately expressed in a successfully transfected cell

(Figure 24B). In all described experiments going forward, we assumed that each successfully co-transfected cell contained a combination of all electroporated constructs.

Exogenous expression of Ascl1 increases neuronal progeny and enhances morphology

Within three days of the transfections followed by induced differentiation by bFGF withdrawal, we observed drastic morphology differences between the Ascl1 transfected clones and the empty vector controls. By viewing only successfully-transfected, Ds-Red positive cells, we observed that Ascl1 transfected cells exhibited a highly branched morphology that is reminiscent of a more mature neuronal phenotype (Figure 25). This branched morphology was evident early on and it persisted and increased in complexity up to two weeks post differentiation.

64

Next, we tested our prediction that the introduction of Ascl1 into the multipotential cells would influence cell fate by generating more neuronal cells. Immunocytochemistry was completed on cross-linked cells using antibodies against the neuronal marker TuJ1

(Figure 26A). To confirm that Ascl1 had an influence on directing the phenotype, only the

Ds-Red positive cells (successfully transfected cells) in each condition were quantified and scored by a blind observer as either TuJ1 positive or negative. As predicted, the exogenous expression of Ascl1 significantly increased the percentage of TuJ1 positive cells by approximately 50% and a p-value of <0.01 using Student’s t-test (Figure 26B).

Longer term transfected cultures were tested for neuronal function. Since we observed such striking morphology differences compared to the controls, we wanted to see if these were truly maturing into functional neurons. In collaboration with Dr. Mark

Plummer’s lab, 12 to 14 day differentiated cultures were recorded for induced action potentials and synaptic activity. While the Ascl1 overexpressed cultures could fire occasionally, no synaptic activities were ever observed or recorded (data not shown).

Therefore, Ascl1 overexpression was a good first step towards generating an increased population of neuronal cells from a primarily non-neuronal clone. However, the goal is to learn the programming rules to produce increased populations of interneuronal subtypes. Ascl1 overexpression was a start, but we needed additional components to signal and activate interneuronal pathways.

Dlx family members activate interneuronal networks

To continue in the pursuit to develop an expandable population of interneuronal subtypes, we next focused our attention on the distal-less family (Dlx). Dlx family members are ventrally derived and have been suggested to play key roles in regulating interneuron development (Butt et al., 2007; Le et al., 2007; Yu et al., 2011).

65

As described previously, upon differentiation the gene expression profiles of multiple members of this family (Dlx1, Dlx2, Dlx5, Dlx6) were up-regulated in the neuronal clone

and only slightly in the multipotential clone CTX8. Based on the gene expression, protein

results and recent studies, we predicted that the expression of some members of the Dlx

family is critical in generating GE6 functional interneurons. Therefore, we hypothesized

that they would be good candidates for reprogramming the multipotential clone CTX8

towards an interneuronal subtype.

Dlx genes are organized as bigene clusters in the rodent genome: Dlx1/2, Dlx3/4

and Dlx5/6 (Poitras et al., 2007). Although we confirmed by qPCR that four members of

the Dlx family were upregulated upon induced differentiation in GE6 cells, we chose Dlx1

and Dlx5 as initial candidates to represent both clusters. Similar to the Ascl1

overexpression in our multipotential cells, we cloned Dlx1 and Dlx5 into the same CAG

vector as discussed previously. Co-transfections were performed by combining Dlx1 and

Dlx5 in addition to Ascl1 and the Ds-Red plasmids.

Interneuronal subtypes between neuronal restricted clones L2.2 and GE6 displays

differences in maturation and function

Since the GE6 clone clearly represents an interneuronal precursor consistently

expressing Dlx family members, we hypothesized that we could use the Dlx

transcriptional cascade to influence and drive the multipotential clones towards a similar

neuronal path. The rationale was that the introduction of all three transcription factors;

Ascl1, Dlx1, Dlx5 (ADD), into the multipotential clone would serve to mimic the GE6

phenotype and enhance general neuron production, thereby programming a neural

precursor towards an interneuronal phenotype. Following over-expression and induced

66

differentiation for at least four days, we examined whether the combination treatment enhanced neurogenesis similar to the Ascl1 assays. We observed robust increases in the morphology and intense TuJ1 positive staining within the combination ADD treatments compared to controls (Figure 27).

In collaboration with Dr. Mark Plummer’s lab, electrophysiology recordings were completed on longer term cultures (12 days) to analyze the transfected cells for functionality. Whole-cell patch clamp recordings were performed on the controls and

ADD transfected cells that maintained red fluorescence and had a multi-polar appearance. While some action potentials could be induced, no synaptic activities were observed when recording in the voltage clamp mode (data not shown). Therefore, we conclude that the ADD combination treatment enhances the morphology and neuronal markers of CTX8, yet is not sufficient to make them functional within the current culture environment.

We next revisited the original gene expression profile (Figure 6) and immunostaining results (Figure 9) of multipotential clone CTX8. It was evident that the clones began with the minimum expression needed to generate immature neurons.

However, with extended differentiation assays in culture, the neurons that had initially developed still did not progress towards mature neurons compared to their neuronal restricted counterparts GE6. Surprisingly, the quantity and quality of neurons decreased over time and their gene and protein expression profiles suggested a lack of neuronal maturation. Since we had been unable to produce fully functional mature interneurons from CTX8 either by induced differentiation protocols or by the overexpression of the

ADD transcription factor cocktail, they may be developing in an inappropriate cellular milieu. Therefore, we proposed that the precursors may further develop within a more robust neuronal environment.

67

Co-cultures provide ADD treated precursors with an environment for neuronal maturation

Co-culture experiments with either hippocampal or cortical neurons can provide a rich environment for developing young neurons by supplying the nutrients needed for proper maturation. Because we believe that the multipotential cells are lacking in survival and/or maturation signals, the intent was to introduce them into an environment that would deliver the proper survival and guidance signals towards neuronal differentiation and maturation. In collaboration with Dr. Mark Plummer’s laboratory, dissociated rat hippocampal and cortical neurons from embryonic day 18 were plated and maintained on either 35 mm tissue culture plates for electrophysiology or on laminin coated glass coverslips for immunocytochemistry. The hippocampal and cortical neurons had already been previously plated for 14 and 7 days respectively and cultured in standard neuronal culture media. CTX8 neurospheres were dissociated and transfected with the ADD combination or empty vector controls. Electroporated cells were then plated onto the hippocampal or cortical cultures. Approximately equal amounts of cells were added to each co-culture with the goal of keeping the ratios of primary to immortalized clones equal.

CTX8 cells were easily recognizable by their GFP expression and successfully transfected cells were visualized by RFP expression within 12-16 hours. Cells attached adequately and based on morphology, seemed to integrate and differentiate with the primary cells. Strikingly, within two days the ADD treatment displayed clear morphological differences compared to the empty vector controls. Not surprisingly, the

ADD transfected cells displayed a more robust neuronal morphology with longer and more complex neuronal processes compared to the control samples. These differences were maintained and progressed throughout the length of the experiment (Figure 28).

68

The neurite lengths and branch points were traced and quantified using the Bonfire program (Figure 29) (Langhammer et al., 2010). Bonfire analysis confirms the ADD treated samples had strikingly more branch points, terminal points, number of processes and total cable length. Interestingly, the average lengths of the neurite processes were longer in the control samples. We reason that this is due to the more complex branching architecture of the ADD differentiated neurons.

We next addressed the question of whether the combination of transfections within a richer co-culture environment would assist and drive the development of interneurons. The cortical co-cultures plated on glass coverslips were maintained for an additional 14 days after the transfections. Unfortunately, the fluorescence levels of Ds-

Red diminished over the course of the co-culture experiment, which made it difficult to observe all successfully transfected cells. However, we can conclude that the addition of the three transcription factors had a striking effect on the Ds-Red positive cell’s process length and branching over the course of the experiment before the intensities faded.

Cells were cross-linked and immunostained for GAD 65/67, a known marker of

GABAergic interneurons (Scheffler et al., 2005; Lévesque and Parent, 2005). After two weeks of differentiation, the ADD cultures showed slightly increased GAD 65/67 staining indicating that they are on the path towards becoming interneurons (Figure 30A).

Although the GAD intensity in the CTX8 cells were not as strong as in the primary cortical cells, it is not surprising due to the heterogeneous mix of cortical cells. At this stage of development, the forebrain contains varying levels of neuronal subtypes. In addition, we observed increases in synaptophysin suggesting that either the CTX8 cells or the endogenous hippocampal cells were increasing their synaptic activity in culture.

Next, we stained for the more mature interneuronal marker VGAT and excitatory marker

Vglut-1 (Craig et al., 1996; Nunzi et al., 2003; Nakamura et al., 2005). As expected in a

69

hippocampal co-culture environment, there were cells expressing both markers both the controls and ADD co-cultures. Interestingly, because we can track the successfully transfected cells by their Ds-Red expression, we only observed some CTX8 transfected cells with neuron morphology expressing VGAT in the ADD transfections (Figure 31).

The Vglut-1 staining assays showed positive in both non Ds-Red and transfected (Figure

32). Again, only the ADD transfected cultures appeared to co-localize with Vglut-1 in a minority of cells. It was difficult in both immunostaining assays to deduce if the rest of the positive staining that was not attributed to the ADD transfections were from unsuccessfully transfected CTX8 cells or from the hippocampal cell culture.

Nevertheless, we conclude that the ADD transfections enhance neuronal morphology and generate both VGAT and Vglut-1 positive cells environment during extended differentiation, suggesting that the transcription factor combination is driving the cells towards a more mature fate and the co-culture environment is assisting this process. We postulate that this was achieved by either direct activation of the downstream neuronal network in the transfected cells and/or by signaling of endogenous cells in order to generate additional neurons.

ADD treated co-cultures enhance neuronal function and increase synaptic activity

Based on the slight increases in synaptophysin and GAD 65/67 staining in the

ADD treated condition, we next asked if the co-culture environment is also stimulating functional neurons and if the additional ADD factors are enhancing neuronal maturity. In collaboration with Dr. Mark Plummer’s laboratory, we performed electrophysiology on both the CTX8 co-cultures (hippocampal and cortical). Transfected CTX8 cells were fairly easy to distinguish since RFP was still visible in the most highly expressed electroporated clones.

70

After 12 days in vitro, whole-cell patch clamp recordings were performed on transfected cells that had a multi-polar appearance and fluorescing both green and red.

Comparing the control to the ADD combination, the ADD transfected cells were observed to have more mature inward currents and action potentials compared with the controls (Figure 30B). The presence of synaptic currents was also investigated in voltage clamp. The post-synaptic currents (PSCs) observed from the ADD cells were more frequent and had larger amplitudes than in the control cells (Figure 30C). The ADD treatment also displayed a mixture of both excitatory post-synaptic currents (EPSCs) as well as inhibitory post-synaptic currents (IPSCs), whereas the controls showed only

EPSCs. These results suggest that the combination of the ADD combination treatment, immersed in a co-culture environment, enhanced both neurogenesis and the beginnings of an interneuronal phenotype through immunostaining and electrophysiology analysis.

Although the CTX8 transfected clones did not display its neuronal phenotype as robustly as GE6, we can conclude that neural reprogramming, epigenetic and gene expression profiling methods can be beneficial in the identification of essential regulatory components that for driving cell lineages.

ADD transcription factors bind to neural component regulatory regions

Exogenously expressed transcription factors are expected to interact with genomic regulatory sequences where they alter patterns of expressed genes, causing a stable change in phenotype. We believe that the transient expression of the ADD transcription factors bind to certain neuronal regulatory regions increasing or maintaining their gene expression. In turn, the alterations in expression enhance the production or maturation of neuronal phenotypes. Therefore, we mapped genomic binding sites of each of the transcription factors in the ADD mixture as well as the ADD mixture itself.

71

Using ChIP, we tested the hypothesis that the overexpressed ADD constructs enhanced neuronal maturation by binding near neuronal genes. Each individual plasmid was modified to express a triple FLAG tag coding sequence (Wang et al., 2008). This allowed for more efficient chromatin immunoprecipitation using antibodies directed against the produced epitope since the affinity for the triple FLAG tag is greater than a single tag alone (Wang et al., 2008). CTX8 cells were harvested for ChIP one day after transfection of the ADD transcription factors. Mapping of epitope-tagged transcription factors to genomic promoters by chromatin immunoprecipitation sequencing identified potential target sites of the combination treatment. Peaks were annotated based on the nearest transcription start site. GO analysis on these enriched genes was performed and highlighted numerous cellular component genes involved with neuronal formation (Table

3). Some of these GO terms included: cell projection, neuron projection, axon, synapse and ion channel complex. These results suggest that the exogenously expressed transcription factor combination is binding near neuronal component regulatory regions and may be actively playing a role in the observed neuronal maturation and modulating their expression.

Overall, the increases in pan neuronal and interneuronal markers, action potentials and synaptic activity lead to the conclusion that the ADD combination treatment assists in the enhancement of interneuronal development within these multipotential precursors. Although the efficiencies of reprogramming towards interneurons are still low at this time, additional transcription factors or growth factor conditions may be needed for further optimization.

72

III. DISCUSSION

Stem cell therapeutics has recently been touted as the next game changer in the field of personalized medicine. Countless claims have been made, ranging from the curing of a wide array of neurodegenerative diseases and traumatic injuries, to the entering of a new era of in vitro disease modeling using reprogrammed cells. Indeed, within the past five years, there has been a resurgence of interest due to the discovery and application of induced pluripotent stem cells (iPSc). The ability to reprogram one’s own tissues, whether it is fibroblasts, hair or blood, holds great promise for healing and uncovering previously unknown neurodegenerative pathways. Human embryonic stem cells that have been derived from blastocysts are currently a benchmark to which all adult and iPS derived cells are compared. Multiple studies have recently highlighted contrasting results between the two, and ask whether iPS cells are truly pluripotent and have the same flexibility to differentiate toward all cell types (Bar-Nur et al., 2011, Ohi et al., 2011, Jandial et al., 2011). Epigenetic memory may be playing a significant role, thereby limiting certain phenotypes. Therefore, it would be beneficial to identify the circumstances where a stem cell has the intrinsic capabilities to be directed towards the reprogrammer’s desired lineage. To administer stem cell clinical treatments in the future, it will be imperative to manufacture both sufficient quantity and the desired phenotype.

Learning the rules of lineage regulation is necessary for the full realization of stem cell cures

The in vitro usage of rodent stem cells has historically been the choice model

system and has pioneered the path towards human stem cell applications. In addition,

the easing of governmental restrictions and the relative simplicity of iPS production have

also been catalysts for human stem cell progress. However, human production of neural

73

stem cells is still very prone to heterogeneity. Researchers have been consistently improving their techniques in obtaining large quantities of neural stem cells from the pluripotent state. However, due to their heterogeneous nature, manufacturing large quantities of homogenous neural subtypes still remains a challenge (Dhara and Stice,

2008; Elkabetz and Studer, 2008; Placantonakis et al., 2009; Peljto and Wichterle,

2011). Since we were interested in the regulation of neural subtype specificity, the development of reprogrammed rat neural precursors by our colleagues in the Grumet lab met our requirements and produced clones that were less heterogeneous than their primary cell, ancestors.

In this thesis, we questioned how neuronal and glial phenotypes become specialized and what factors are responsible for shaping their cell fate. Our approach to this was to investigate regulatory mechanisms such as epigenetic chromatin modifiers and transcription factor programming, to confirm our predictions that these play a role in driving and maintaining neural lineage restriction. What accounts for the phenotypic differences in these precursors? Extrinsic factors such as neural orientation growth factors are likely to shift precursor cells towards different fates (Panchinson and McKay,

2002; Song and Ghosh, 2004; Guillemot, 2007; Li and Grumet, 2007). Both independently and in response to growth factors, intrinsic factors such as patterns of transcription factor expression and epigenetic marking may explain the cellular diversity.

We examined each of these extrinsic and intrinsic mechanisms in a relatively homogeneous model system to understand the mechanisms driving cellular fate specification.

First, we predicted that that during the height of rat neurogenesis, embryonic stage (E14.5), there would be multiple precursor cells with varying phenotypic potential and that these could be clonally derived yielding a more homogeneous population of cell

74

fate potential. Our results confirmed that specific areas of the brain contain precursors with the intrinsic capability to generate distinct neuronal subtypes. Furthermore, we obtained two additional precursors representing different stages of neural development; a glial restricted and a multipotential neural stem cell like precursor. This was first accomplished by using rat immortalized embryonic forebrain clones as contrasting representative models for neural development and lineage restriction.

We next confirmed our predictions that in addition to gene expression differences, there are intrinsic epigenetic differences between these precursors. For example, by investigating both permissive and repressive histone modifications in each clone’s undifferentiated state, we observed some chromatin signatures that predicted future gene expression in the neuronal and glial clones. Furthermore, we learned that there is also epigenetic regulation taking place throughout cellular differentiation.

During standard differentiation of the multipotential clone CTX8, the neurogenic transcription factor Ascl1 was expressed. Furthermore, in response to treatment with a chromatin modifier, Ascl1's gene expression was further increased and correlated with enhanced neurogenesis. Transcription factors induce multiple downstream networks, with the end result of directing and stabilizing cell fate. Furthermore, we uncovered a group of microRNAs that are epigenetically regulated in response to chromatin modifications. This additional epigenetic layer of regulation contributes to the modulation of lineage restriction occurring during neural development. We conclude that combination of epigenetic modifiers, transcription factors and growth factor treatment may be an approach to achieve enhanced neuron generation and specificity.

In this thesis we utilized immortalized, clonal precursor cells from rat neural forebrain development to investigate the interplay between these intrinsic and extrinsic

75

mechanisms. As one example of a desired cellular fate, we ultimately focused on a cell clone having the capability of producing inhibitory GABAergic cells with the ability to form synapses (GE6; Li et al., 2011). By learning the rules of cell type specification from this cell we attempted to reprogram a less restricted cell (CTX8) to acquire a similar phenotype. While this strategy was not entirely successful, the progress made demonstrates that we now understand enough about the system to continue to explore cellular reprogramming methods as a potential for engineering desired therapeutic populations.

Homogeneous models of neural forebrain development using cell clones

Following the progression of the developing forebrain is a daunting task. The inherent complexity that accompanies a surprising low number of cell divisions before its completion is a testament to the strict regulation that is required (Nowakowski and

Hayes, 1999). Dynamic crosstalk between intrinsic and extrinsic mechanisms coordinate to direct cell migration and fate. The central dogma of neural development is the sequential generation of neurons first followed by glial generation (Temple, 2001;

Guillemot, 2005; Miller and Gauthier, 2007). Although this has been confirmed in countless studies, it does not strictly state that all neural precursors present in the early stages of neural development have the capacity to generate neurons at all times. In other words, there are always levels of lineage restriction occurring even during the early stages of neural development, and in many instances before protein or gene expression

(Delaunay et al., 2008). To properly uncover the rules of regulation, we predicted that studying neural stem cells or precursors with distinct intrinsic properties would be pertinent in learning the mechanisms behind cell fate programming.

76

One possible approach was to dissect and dissociate rat forebrains at different embryonic stages. Although this may be a reasonable tactic, we did not go this route for a few reasons. First, the earliest stages of neural development after the neural plate folds and before substantial neurons are generated, is difficult to dissect and obtain large quantities of neuroepithelial cells for culturing. Second, we preferred cells that were actively dividing but intrinsically have progressed to where their cell fate was partially or fully predetermined. Therefore, we hypothesized that during the neurogenesis phase of rat development (E14.5), the forebrain would contain neural precursors with the ability to generate all three neural categories: neurons, astrocytes and oligodendrocytes.

Although, some of these precursors may not yet express these subtype specific markers or genes, previous groups have postulated that neural restriction may occur well before a noticeable phenotype (Delaunay et al., 2008). Since E14.5 precedes gliogenesis, we were confident that this particular stage of development could yield the contrasting precursors. To achieve this goal, immortalized cell clones using v-myc were derived from the forebrains of E14.5 Fischer rats, that were previously subdivided into dorsal (cortex) and ventral regions (ganglionic eminences) (Figure 2). This method was based on the previous generation of multipotential (L2.3) and neuronal (L2.2) cell clones by the

Grumet group, the latter having the capabilities to generate functional GABA producing interneurons (Li et al., 2004; Li et al., 2008). Although this thesis does not discuss these previous studies in detail, they were the predecessors to the Fischer clone experiments.

Since we predicted L2.2’s lineage started in the ventral area of the forebrain, Dr. Hedong

Li decided to dissociate cells from both the dorsal and ventral regions, predicting that there would be isolated clones with interneuronal capabilities derived from the ventral areas of the forebrain, the ganglionic eminences (GE). Obtaining clones with the inherent capability to generate functional GABAergic interneurons is desired for a few reasons. First, if one isolates a more homogeneous clone with this phenotype, we

77

predict we can learn some essential components responsible for the subtype formation such as transcription factors that activate downstream networks driving cell fate. Next, by applying the essential factors responsible for interneuronal subtype specificity towards human cultures, we may be able to direct a more homogeneous population of cells that could be used for therapeutic purposes.

The benefits of immortalization

The use of primary cells has been notoriously difficult to maintain consistency due to the rapid differentiation progression and passage drift. Therefore, the reprogramming approach using retroviral infection with v-myc allowed the isolated clones to remain mitotically active in the presence of bFGF and to also minimize passage drift. As predicted, we conclude that all three Fischer clones, while in their undifferentiated states, maintain cell division in the presence of bFGF, remained Nestin positive, and do not express any neuronal or glial markers before differentiation is induced (Figure 4, Figure 5, Figure 6). The usage of v-myc as a reprogramming tool was pioneered by Evan Synder while in the Cepco lab, where they stably transformed multipotential NSCs from the mouse cerebellum (Ryder et al., 1990, Synder et al., 1992) for the purpose of unlocking the mechanisms of neural development. These studies were one of the first to develop immortalized models (C17) of neural development, with the goals of obtaining more stable and less heterogeneous clones that represent the stages of neuronal or glial commitment.

Since these initial studies, human immortalized precursors have now been generated from the diencephalic and telencephalic brain regions of a 10.5 weeks

78

gestational age human fetus (De Filippis et al., 2007). This work successfully produced functional neurons and oligodendrocytes from the stably reprogrammed NSCs. Reasons behind this approach were that it had been a notoriously difficult process to maintain and generate large amounts of human NSCs because of their relatively slow growth kinetics.

By introducing v-myc, the resulting clones expanded three to four times faster while staying relatively stable before differentiation induction. In addition, by producing rapid propagating cells, they were now more amenable to high-throughput genetic and drug screens.

The stem cell field in general has not fully embraced human immortalization of fetal neural stem cells and there are a few reasons why this has not become standard practice. First, v-myc is an oncogene that has profound effects on cell division and even though the authors state its expression is essentially silenced during differentiation; no clinical trials would accept these transformed cells unless the gene was excised before transplantation. We also predicted the silencing of v-myc in the Fischer clones and did observe significant decreases in gene expression, but not complete silencing (Figure 9).

Therefore, the use of these clones lies in their intrinsic capabilities and what we can learn about their internal programming, but not for therapeutic applications.

Another reason is that human fetal neural stem cells are not easy to obtain and are also not patient specific. They still need the use of immunosuppressive treatments for transplantation studies and can be harmful by causing tumors (Amariglio et al.,

2009). Lastly, and the most significant reason has been the emergence of iPS technology. Researchers are now able to generate human patient specific pluripotent stem cells and essentially generate unlimited amounts of cells for drug screening, disease modeling and potentially transplantation (Nakagawa et al., 2007; Yu et al.,

79

2007). However, the successful production of neural specific phenotypes, specifically neuronal subtypes is still in its infancy.

In summary, the usage of human ES cells and immortalized human NSCs still has an important role within the field. Both of these should be used as standards or roadmaps for iPS derived neural cells. It is clear that the programming rules from all stages of development are still widely unknown. The usage of multiple cell lines from alternate techniques should be carefully compared in order to optimize neural lineage programming.

Neural clones have unique identities

To test the predictions that immortalized neural clones isolated from the forebrain would yield different levels phenotypic capabilities, thorough protein and gene expression assays were completed to characterize the cells. Confirming our hypothesis,

17 Fischer clones had a range of phenotypes including the three levels of lineage restriction we predicted; neuronal restricted, glial restricted, and multipotential (Figure 3).

Similar to previous studies using immortalized cells as models for neural development

(Ryder et al., 1990, Synder et al., 1992; Villa et al., 2000; De Filippis et al., 2007; Li et al., 2008), obtaining these three contrasting cell fate precursors now allowed us to further investigate the internal mechanisms driving their identities. Three clones were narrowed down by a combination of approaches that included cell morphology, immunostaining and gene expression. Specifically, GE6, the primarily neuronal clone was initially selected based on its gene expression profile and comparing it to a previous

GABAergic, interneuronal-like clone generated by the Grumet lab L2.2 (Li et al., 2008).

80

In addition to producing primarily Tuj1+ progeny, the clone expressed more Dlx family genes compared to the rest of the clones (Figure 8, Figure 9). In addition, GE6 clustered closely with L2.2 in a hierarchical clustering analysis that was completed based on the expression profiles of a subset of genes (Figure 2C). Because the Dlx family is known to play a role in interneuron production (Panganiban and Rubenstein, 2002), this was a signal to us that GE6 was a candidate for generating GABAergic neuronal subtypes.

The confirmation of locating an interneuronal producing clone derived from the ganglionic eminence was expected. This was the main reason that Dr. Li separated the dorsal and ventral regions before clonal immortalization was performed. Interneuronal precursor cells are known to originate in the ventral forebrain before their tangential migration into the cortex during development (Anderson et al., 1999). The gene expression profiling also led us to investigate the functional properties of the Fischer clones. We were intrigued by the fact that multiple neuropeptide and synaptic component genes were preferentially up-regulated in GE6 during the later time points once differentiation was induced. This suggested that this clone may already be capable of generating functional neurons in culture. To confirm that GE6 cells were maturing throughout differentiation, positive immunostaining with interneuronal markers (VGAT and Gephryin) and synaptic marker Synaptophysin were widely observed (Figure 10).

GE6 clones were verified the Grumet group to be functional GABAergic neurons both alone and in co-culture with primary dissociated hippocampal cultures (Li et al., 2011).

The confirmation that GE6 cells synapse with each other is a testament to how restricted and functional some of these clones truly are and how expression profiling can be beneficial for neural stem cell screening. One of the exciting benefits of generating a stable reprogrammed neuronal precursor is to uncover the mechanisms of lineage regulation. We took advantage of both the epigenetic and gene expression profiling in

81

GE6 and applied it towards the more multipotential cell clone CTX8 in order to recapitulate GE6’s phenotype by enhancing and directing neurogenesis towards an interneuronal subtype. These approaches will be discussed in detail with the next few sections.

The isolation of a glial restricted clone, GE2 was a welcome addition as it had been previously elusive in the earlier immortalization experiments. As described earlier, no neuronal markers were observed before or after induced differentiation (Figure 3,

Figure 4). The expression profiles displayed distinct differences between clones showing that GE2 and GE6 differed most out of the three clones (Figure 7). Even though E14.5 in the rat is considered the height of neurogenesis, we were not surprised to isolate a precursor that was limited to the glial lineage at this stage. As mentioned earlier, even though widespread gliogenesis has not yet begun, we believe the lineage restriction of neuronal and glial clones begins before gene or protein expression is apparent.

However, we did not predict glial restricted precursors (GRPs) would necessarily be located in the ganglionic eminence. In fact, we can speculate that since glial cells well outnumber neurons in the entire nervous system, then GRPs can be expected to be found in both dorsal and ventral regions of the forebrain. Perhaps if we had isolated the cells at a later temporal period such as E17, we would have improved our chances of locating GRPs in both regions since neurogenesis is diminishing and gliogenesis is increasing during this developmental period (Das, 1977; Barnabé-Heider et al., 2005).

Although we did not explore GE2 further, this precursor cell should be valuable in future studies to identify mechanisms leading glial cells.

Of course, obtaining a multipotential clone was predicted and verified. This was not surprising as many neural stem or precursor cells are still being generated in the forebrain during this time (Guillemot, 2007). Therefore, to isolate a multipotential cell

82

clone of this nature absolutely represents cortical development during this period. CTX8 cells were able to generate neurons, astrocytes and oligodendrocytes in standard differentiation conditions (Figure 3, Figure 5). In addition, they had more of a radial glial morphology (bi-polar appearance) compared to the other two Fischer clones (Figure 5).

Interestingly, we concluded that growth factor treatments could rapidly and strikingly adjust neuron and glia ratios, showing the inherent plasticity remaining in this clone. However, surprising result was the diminishing neuronal to glial ratios during extended differentiation in its default state without any growth factor treatment (Figure 5).

We unexpectedly saw levels of neurons diminishing after at least 3 days of induced differentiation. In addition, GFAP positive astrocytes increased throughout that time and far outnumbered neurons at 6 and 14 days post differentiation. This was surprising because we had not previously observed this phenomenon with our previous multipotential clone L23. We therefore concluded that CTX8 clones favor primarily the non-neuronal phenotype over extended differentiation. There may be a few reasons why the neuronal cells diminish. First, although the clone has the capability to generate both phenotypes, the astrocytic phenotype may be a more intrinsically favorable outcome. We investigated this through gene expression assays along with epigenetic analysis and conclude that CTX8 cells lose permissive epigenetic modifications mimicking the loss of

Tuj1+ cells over time. We took advantage of these findings by using this as a model to enhance neurogenesis by using both epigenetic and transcription factors critical for regulating neuronal restricted clones. These epigenetic markings and phenotypes can be reversed or maintained through the treatment of BMP2 or valproic acid. Another reason for the loss on neurons may be the lack of transcription factor activity to drive the neuronal lineage. We found that valproic acid treatment also induced Ascl1, a known neuronal transcription factor (Bertrand et al., 2002; Guillemot, 2007), along with

83

enhanced neurogenesis in CTX8 cells. In summary, the isolation and development of these three representative clones are useful for contrasting their inherent capabilities and to study the regulation of neural development.

Do epigenetic marks distinguish cortical neural precursors with distinct fates?

We next wanted to peer into the mechanisms responsible for creating the contrasting lineages that arise during neural development, essentially giving “the cell” its intrinsic identity. It is important to remember that all cells isolated generally contain the same DNA sequence. Essentially, what defines a cell’s phenotype is the combination of all the genes and proteins it expresses. How does the cell remember and also maintain its own identity? We hypothesized that epigenetics must play a role in this process by regulating current and future gene expression. Furthermore, we predicted that the

Fischer clones maintain their own distinct epigenetic signatures and that these differences distinguish neural precursors in the forebrain resulting in distinct fates.

Testing this prediction on the three neural development representative clones, the hypothesis was that chromatin marks form an epigenetic signature that represents the current state of the cell, and these signatures can also predict future gene expression.

One way epigenetic regulation guides cellular fate is to prepare the cell to react to certain extracellular factors such as growth factors, but only at the opportune time. It has previously been shown that the same growth factor can have different effects on cellular growth and differentiation during diverse temporal periods of development

(Takizawa et al., 2001). For example, CNTF is expressed at both early and later periods of neural development. It is a known activator of the JAK/STAT pathway that directly up-

84

regulates GFAP expression. However, LIF treatment (a CNTF analog) does not induce

GFAP expression until later stages. This is because during the earlier stages of development, the regulatory region of GFAP is DNA methylated. This prevents the binding of STAT3 and which results in no GFAP transcription. Not until the methyl group is removed is the cell allowed to react to this particular growth factor. This is just one example of how we believe that epigenetic mechanisms are a component of what some refer to as “cellular context” (Dougherty et al., 2009). Our goal was to uncover other

“cellular contexts” and explain them through epigenetic mechanisms. We chose to focus on histone modifications because of the many combinations that correlate with gene expression (Feinberg, 2008; Mehler, 2008), and also the potential to alter chromatin for future therapeutic strategies.

Human ES cell chromatin signatures were the initial inspiration for us because of their contrasting patterns in cell types once differentiated. One of the earlier genome wide studies completed discovered drastic differences in human fibroblasts, embryonic stem cells and neural stem cells (Bernstein et al., 2006). Based on this and other pioneering studies (Ren et al., 2000; Lieb et al., 2001; Shivaswamy et al, 2001; Johnson et al., 2007; Robertson et al., 2007; Barski et al., 2007), we reasoned that contrasting phenotypic neural precursors developing in the brain would also contain distinct histone modifications, analogous to the earlier periods of development transitioning from ES cells towards NSCs. Furthermore, we hypothesized that forebrain precursors would house epigenetic markings that preceded transcription, in a sense preparing the cell for its eventual gene expression. Uncovering epigenetic signatures that can predict future cell fate would open up a potential screening strategy in the field of directed differentiation. If one could know beforehand that a particular cell line is refractory for certain neural genes, they may choose to use another line or possibly treat the cells with

85

specific chromatin modifiers to assist in the differentiation process. A similar approach has already been applied in the iPS field of reprogramming. The addition of valproic acid, inhibiting HDAC and promoting acetylated chromatin, has been used to increase the efficiency of iPS cell generation (Huangfu et al., 2008).

We investigated the chromatin state from the three immortalized Fischer neural clones in their undifferentiated state. We predicted that certain histone modifications would represent the current expression of lineage genes or would correlate with future expression. Furthermore, we also expected that the chromatin markings were also dynamic and would be altered with either external factors expressed during neural development, or certain chromatin modifiers such as histone deacetylases. This in turn would activate or repress certain lineage genes, ultimately directing the neural precursor towards a particular fate.

To test our predictions, we took advantage of the gene expression differences between clones from the microarray studies (Figure 11). To start, Ascl1 was chosen due to its known role in neuronal regulation as a transcription factor and its expression profile among the three clones. In their undifferentiated states, mRNA expression was relatively higher in both the neuronal restricted and multipotential clones compared to the glial restricted clone. We reasoned this was a model gene to test our hypothesis that these clones contain distinct epigenetic signatures near transcription start sites (TSS). Results from ChIP-qPCR using permissive and repressive histone modifications displayed significant differences, especially between the neuronal and glial restricted clones, GE6 and GE2 (Figure 12). In this context, these results confirm that the chromatin state of

Ascl1 correlates with current and future gene expression.

86

Although this was a good start, we were also interested in discovering epigenetic marks that preceded downstream transcription because this could be used as a potential screening mechanism as described above. We again reviewed the microarray data set for genes that all had similar expression values in the undifferentiated state but showed clear differences in expression between clones when differentiation was induced (Figure

13). Three genes were chosen based on these criteria: Erbb4, Cbs, VGAT. Specifically,

Erbb4 abd VGAT were chosen because they were expressed only in the neuronal and multipotential clones (GE6, CTX8) when the clones were differentiated. In contrast, Cbs was expressed only in the glial and multipotential clones (GE2, CTX8) once differentiated. Therefore, the genes provided a sufficient gene expression contrast between cell clones and were good choices to test the epigenetic signatures of all three clones.

One of these was Erbb4, a gene that codes for a receptor protein for Neuregulin, was found to increase its expression in both days three and seven post differentiation in only the neuronal and multipotential clones. This was an interesting gene to us because it has been previously shown to be integral part of neural development (Birchmeier,

2009). More recently, the connection between Neuregulin1 (Nrg1) and Erbb4 has been linked to important risk genes for schizophrenia. Fazzari et al. described a study where

Nrg1 and Erbb4 signaling controls the development of inhibitory circuitries by regulating the connectivity of specific GABA containing interneurons (Fazzari et al., 2010). The

Erbb4 ChIP results showed an inverse relationship between permissive and repressive marks between the neuronal and glial restricted clones (Figure 13). This finding supports our hypothesis that although transcript levels may be relatively similar in the undifferentiated clones, their chromatin signatures are already in place for future gene expression towards their respective phenotypes.

87

The next candidate gene from the array dataset was Cystathionine Synthase

(Cbs), an important for homocysteine . Deficiencies in the CBS protein are linked to homocystinuria, which in turn has been correlated with mental retardation. In addition, abnormal homocysteine metabolism has been associated with numerous neurological disorders including depression, schizophrenia, and Alzheimer's disease (Enokido et al., 2005). There has been a debate regarding the location and subtype of CBS expressing cells in the nervous system. The group observed CBS protein expression in various stages of mouse development ranging from E13 to P8

(Enokido et al., 2005). They concluded that CBS expression increased during late embryonic stages and were found in radial glia and astrocytes, but were not found in neuronal populations. These findings are very similar to our gene expression results in glial restricted GE2 cells and made this a possible candidate for epigenetic regulation.

Similar to Ascl1 and Erbb4, we performed ChIP with H3K4me3 and H3K27me3 and designed primers near the TSS of Cbs. Confirming our predictions, we observed contrasting results compared to Erbb4 enrichment pattern. Within GE2 chromatin, the

Cbs regulatory region was relatively more enriched for permissive marks and less for repressive, while the neuronal clone GE6 displayed the opposite enrichment pattern.

Once again, one can speculate that these epigenetic chromatin modifications are actively preparing these neural precursors towards their eventual phenotypes, before their downstream gene expression patterns are realized. These initial ChIP qPCR experiments confirmed our hypothesis that undifferentiated neural precursors contain distinct epigenetic signatures and these chemical modifications play key roles in their lineage restriction.

Choosing to expand on the directed ChIP qPCR results on the undifferentiated

ChIP signatures, ChIP sequencing was performed to get a better overall perspective of

88

where histone modifications are enriched and to potentially discover epigenetically regulated genes throughout the Fischer clone genomes. As mentioned earlier, although these histone modifications have been documented to be deposited near transcription start sites, the ChIP qPCR method is at best an estimated guess for discovering enrichment patterns. We hypothesized that the epigenetic signatures would be distinct at this stage of development and would highlight additional epigenetically regulated neuronal or glial networks. Confirming the previous ChIP qPCR data, we also observed permissive enrichment near the TSS in both Erbb4 and Olig2, while VGAT was enriched only in the repressive histone modification H3K27me3 (Figure 14).

To get an overall view of each clone’s global chromatin state, we performed a gene ontology (GO) analysis of all sequenced peaks and analyzed the gene networks that were enriched in either the permissive H3K9/14ac or the repressive H3K27me3 modifications (Figure 15). All peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a Benjamini &

Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset. We compared the highest ranked GO terms and then grouped them into broader categories to highlight the most enriched categories per sample. Supporting our hypothesis that there are inherent chromatin differences between clones, we observed several GO category differences. A few interesting points to consider was that the multipotential clone CTX8 was relatively enriched in more General Development and

General Differentiation categories compared to the other more restricted clones. This could be an epigenetic representation to its multipotential nature in that it is not yet fully committed to a particular lineage.

However, not all results were expected. For example, the glial restricted clone,

GE2 was slightly more enriched in the Neuronal category that the neuronal clone GE6.

89

This was surprising since both gene expression and protein assays confirm its non- neuronal phenotype. To explain why this result contradicted our original hypothesis that there would be global chromatin signatures predictive of downstream gene expression, we should consider multiple points. First and foremost, the Fisher clones may be more similar in their epigenetic state then we originally thought. Since most of the mRNA expression differences between clones occurred after differentiation was induced, it is possible that they are more similar in their epigenetic state prior to differentiation. There are an increasing amount of histone modifications being correlated with gene expression. We foresee that additional chromatin maps in combination might highlight the more subtle changes within these clones. In addition, it is possible that the Neuronal category and GO terms that comprise it may be overly broad and may include some non-neuronal genes. Lastly, differentiation may be needed to both induce and track dynamic histone changes over time to observe additional epigenetic regulation of these precursors. We confirmed this last reason by analyzing temporal chromatin maps throughout differentiation, with the addition of growth factor BMP2 and chromatin modifier, valproic acid. We chose to only use the multipotential clone CTX8 for these experiments due to its flexible response to external growth factors (Figure 16). Overall, we conclude that there are intrinsic epigenetic signatures within the Fischer clones, suggesting that these histone modifications play a role as regulatory components during nervous system development.

Growth factors affecting cell fate regulate epigenetic marks

Growth factors are well known to cause several phenotypic responses during neural development, up-regulating or diminishing certain neuronal and glial pathways (Li

90

and Grumet, 2007). Since we observed a dramatic response to three growth factors; a

Sonic Hedgehog Agonist, CNTF, and BMP2 (Figure 16), we hypothesized that these factors may be generating their cell fate responses in part through histone modifications.

To test this, we performed a differentiation time course using BMP2 treatment and tracked genome wide acetylation marks using ChIP-Seq. BMP2 was the factor of choice because it generated both Tuj1+ neurons and GFAP+ astrocytes, and the neuronal morphological responses produced a more mature looking neuronal cell compared to the control. The global ChIP-seq analyses for the differentiated treatment were completed similarly as the undifferentiated ChIP-seq analysis. We ran a GO analysis on all acetylated enriched peaks. Our initial interests were in the epigenetically regulated permissive marks and predicted we would find upregulated neural pathways responsible for affecting cell fate. By comparing the top affected categories during each time point, an interesting trend was found. There was maintenance of both nervous system related and neuronal genes in the BMP2 treated samples compared to the controls during three and seven days of differentiation (Figure 17). The control group had decreases in both of these categories by day seven. Since we observed similar phenotypic responses in immunostaining assays where TuJ1 positive cells diminish over time but are maintained or promoted with treatment, these epigenetic changes suggest a coordinated regulation of neural genes that may be assisting in modulating neuronal cell fate throughout differentiation.

Changing epigenetic marks alters cell fate

At this stage, we have observed several histone modification markings present before neural cells develop into their mature phenotypes, and observed that these

91

epigenetic signatures can represent both current and future gene expression patterns

(Figure 12, Figure 15). Furthermore, we have also confirmed that growth factors can affect epigenetic marks in multiple neural gene networks throughout differentiation, suggesting that these chromatin alterations mold ongoing lineage restriction (Figure 17).

Next, we wanted to take these epigenetic/phenotype correlations a step further and hypothesized that directly altering chromatin structure would lead to downstream gene expression changes altering the phenotypic outcome. The approach used was the treatment of CTX8 cells with valproic acid (VPA), a known histone deacetylase (HDAC) inhibitor. Histone deacetylase inhibitors alter chromatin structure by interfering with endogenous histone deacetylases, resulting in a net increase of acetylated histones.

This in turn, has a direct effect on the chromatin structure by neutralizing the positive charge of the histone tails and decreasing their affinity for DNA. This modifies the nucleosomal conformation into a looser configuration, making it more accessible for the binding of transcriptional regulatory proteins (Norton et al. 1989; Hong et al. 1993;

Struhl, 1998) and loosens the underlying DNA making it more viable for transcription.

In recent years, there have been several studies promoting the wide ranging effects of HDAC inhibitors and the potential therapeutic benefits of applying them towards diseases areas such as cancer and neurodegenerative diseases. Some recent nervous system related diseases include: Rett syndrome, Friedreich's ataxia,

Huntington's disease and multiple sclerosis (Krämera et al., 2001; Kazantsev and

Thompson, 2006). In addition, neurogenesis has been shown to be stimulated and enhanced after treatment with these chromatin modifiers (Kim et al., 2009; Yu et al.,

2009). For example, VPA specifically has been shown to induce neuronal differentiation in multipotent progenitor cells by up regulating certain neuron-specific genes (Hsieh et al., 2004). Most recently, the Hobert group from Columbia University, has discovered

92

that the removal of a specific chromatin remodeling component, LIN-53, removes a barrier to reprogramming and can directly convert Caenorhabditis elegans germ cells into neurons. However, most excitingly, this process can be mimicked by chemical inhibition of HDACs using two inhibitors, VPA and trichostatin A. The authors further suggest that "these results nonetheless provide a strong indication that histone modifications are key players in restricting the ability of a transcription factor to reprogram cellular identity” (Tursun et al., 2011).

We hypothesized that directly altering chromatin structure, specifically through

HDAC inhibitors, would globally affect histone acetylation levels leading to downstream gene expression changes, and finally altering cell fate. CTX8 was chosen for testing for a few reasons. This clone was chosen due to its inherent multipotential capabilities and its recent flexible phenotypic response when subjected with certain growth factors. Slight alterations in the generation of neurons or astrocytes would be relatively easy to quantitate, compared to the more restricted clones.

We needed to first verify that administration of VPA to CTX8 cells would globally increase histone acetylation levels. Cells were differentiated for three days with our without the HDAC inhibitor. Striking increases in H3K9/14ac were observed compared to both the control sample (Figure 18A). Now that we had confirmation that VPA altered chromatin structure in the multipotential clone, CTX8, we tested the hypothesis that these acetylation changes would lead to cell fate changes over time. Both short and long term differentiation assays were completed and then assayed for both neuronal and glial markers. Using a characterization panel comprised of immunostaining, flow cytometry and analysis of neurite morphology, we confirmed our predictions that VPA treatment affects cell fate by both increasing the quantity of neuronal cells and increasing neurite process lengths (Figure 18B, Figure 19). This type of enhanced neurogenesis has been

93

observed previously by other groups (Hsieh et al., 2004), but it raises the question as to why only neuronal markers were increased and not glia? It is still currently not known why there are more reports of neuronal increases since this particular HDAC inhibitor is known to affect acetylation globally. There may be some currently unknown targeting mechanism towards neuronal genes but this is just speculation at this time. However, since the applications of VPA have also been recently extended into the areas of iPS reprogramming (Huangfu et al., 2008), a more contextual explanation is more likely. We speculate that the addition of VPA in very early neuroepithelial cells or in much later stages of gliogenesis may have additional phenotypic effects that include other non- neuronal phenotypes.

The next question we asked was if we could find a link between the global acetylation increases and the increased neuronal cell phenotypes that were observed.

To address this, we took a genome-wide view of all acetylation enrichment changes using ChIP-seq and compared the VPA-treated samples and controls. All enriched chromatin fragments were sequenced and aligned to the rat genome. Once again we employed a GO analysis where all acetylated peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a Benjamini & Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset. We compared the highest ranked GO terms and then grouped them into broader categories to highlight the most enriched categories per sample. Many of the most significantly enriched GO terms included neural-related categories such as: neural tube formation, neural tube development, neurite outgrowth and morphogenesis.

This was satisfying to us as it confirmed that the acetylated epigenetic signature of CTX8 cells during this developmental period identifies with neural development. Interestingly, confirming our prediction, while there was a diminishing number of both nervous system

94

related (Figure 20A) and neuron specific gene (Figure 20B) categories in the control group, there appeared to be retainment or maintenance of these neural categories within the VPA treated samples. These results supported our hypothesis that the global increase due to VPA treatment is targeting neuronal gene networks on some level. We wanted to take this a step further and identify some specific neuronal genes that may be epigenetically regulated through the treatment of VPA. We postulate that some of the specific genes detected throughout these treatments could add to the general understanding of cellular plasticity during development and how a neural precursor's fate might be directed with certain chromatin modifiers. The goal was to observe changing histone modifications that are regulated upon induced differentiation, locate the specific regulated genes and then deduce their importance or lack thereof in follow up lineage progression studies.

To strengthen the quality of the results and to reduce the number of genes to consider, we selected a more stringent list of histone mark-associated genes for further analysis. Therefore, we filtered out all peaks that were not within 3 kb of each gene’s transcription start site. The 3 kb filter was arbitrarily chosen based on previous histone modification signatures that are usually enriched near promoter regions close to the TSS of each gene (Bernstein et al., 2006; Kouzarides, 2007). Although we acknowledge that other regulatory regions a further distance away may play significant roles in directing transcription, such as enhancer regions, we felt that limiting enrichment near the TSS was beneficial to quickly locating the most essential neural development-associated genes.

Although we have yet to follow up on most of the genes that have been associated with the VPA maintained neuronal pathways, a well-known neuronal transcription factor was included after the filtering. Interestingly, Ascl1 was found to be

95

strongly enriched in acetylation after VPA treatment, spanning approximately 2 kb throughout the coding sequence of the gene (Figure 21). In addition, its mRNA was also confirmed to increase compared to the control, supporting the dogma that increases in histone acetylation positively correlate to increases in gene expression. This finding was of interest to us, because although it is known that Ascl1 plays a role in enhancing neurogenesis, it has also been shown to preferentially regulate the development of interneurons (Poitras et al., 2007, Battiste et al., 2007, Zhang et al., 2010). Because we have shown that enhancing the generation of interneuronal subtypes can occur by overexpressing Ascl1 in combination with members of the Dlx family, we have now learned that Ascl1's expression can also be regulated by valproic acid. Therapeutic strategies using drug treatments such as HDAC inhibitors have already been tested

(Johnstone, 2002; Egger et al., 2004; Glaser, 2007). Expanding on these studies by learning the downstream targets that they activate may assist in future strategies to modulate specific gene networks. We conclude from this, that many TFs are epigenetically regulated and that numerous neuronal pathways may be activated, leading to increases in neurogenesis.

One class of epigenetic regulators likely to be affected by global chromatin acetylation are microRNAs. Their role in nervous system development has been an interest since we observed a cluster of neuronal genes that enhance neurogenesis (mir-

9, mir-124, mir-153, mir-182) (Figure 23). Numerous reports on how microRNAs assist in guiding neural precursors towards neuronal or glial fates have been reported

(Krichevsky et al., 2003; Miska et al., 2004). However, the epigenetic regulation of neural stem or precursor cells is still largely unknown. To the best of our knowledge, this is one of the first studies to probe the epigenetic regulation of microRNAs in immortalized neural precursors. New insights into the transcriptional regulation of microRNAs

96

discovered in this manner may be useful for future therapeutic strategies where specific neural phenotypes are required.

After filtering the data set to include peaks within 3 kb of each TSS, we uncovered multiple microRNAs with increased acetylation following VPA treatment

(Table 2). qPCR on many of these microRNAs confirmed the gene expression increases in response to VPA treatment (Figure 22). Both mir-9 and mir-30b had increases in

H3K9/14ac within their regulatory and coding regions. It is very interesting that both of these known neural genes are epigenetically regulated when treated with an HDAC inhibitor. We can speculate on their importance since they that have been previously identified with brain development and specifically neurogenesis (Krichevsky et al., 2003;

Gao, 2010; Song et al., 2011). Another interesting microRNA that was discovered was mir-125b. In contrast to mir-9 and mir-124, the regulatory regions surrounding mir-125b were enriched in acetyl marks in both the control and VPA treated samples. Although we observed slight increases in acetylation with the addition of VPA, we can speculate that mir-125b may be essential for both neurogenesis and preparing the cells to react to future extrinsic factors. mir-125b has been implicated as having a role in neuronal development (Minh et al., 2010), but has been relatively overshadowed by mir-9 and mir-

124 as the most enriched microRNAs in the brain. In summary, we have evidence to support our prediction that microRNA expression can also be regulated by the increased acetylation of histones due to VPA treatment. We suspect that the increases in microRNA expression in conjunction with other de-repressed transcription factors due to

HDAC inhibition are responsible for the increases in both neurons and neuronal process lengths. Therefore, by filtering epigenetic enrichment patterns in these multipotential cells, we were able to uncover specific epigenetically regulated microRNAs that may have may play a prominent role in lineage restriction towards maturing neurons.

97

Transcription factors regulated during GABAergic differentiation can exogenously alter cell fate by regulating neurogenic genes

A primary role for GABAergic inhibitory interneurons is to synapse on glutamatergic projection neurons. This activity effectively modulates the excitatory functions in the forebrain. Loss of interneurons due to brain trauma can have a range of devastating effects, including hyperexcitability that may cause seizures (Fritsch et al.,

2009). Furthermore, some GABAergic interneuron subtypes have been linked to neurological diseases such as Schizophrenia (Woo and Lu, 2006). Thus, the generation of expandable stable GABAergic interneurons can be useful for transplantation purposes to alleviate some of these nervous system malfunctions. For example, treatment with functional interneurons can be beneficial for treating epilepsy and allodynia (Lowenstein et al., 1992; Mukhida et al., 2007; Eaton and Wolfe, 2009). Recent advances in reprogramming have designed approaches to directly convert non-neuronal cells into functional neurons, however creating specific neuronal subtypes using these methods is still in its infancy (Vierbuchen et al., 2010; Tursun et al., 2011).

Briefly, the approaches taken to uncover some of the programming rules during interneuronal development were to first isolate and immortalize a ventral forebrain precursor that is capable of generating a relatively homogenous population of functional interneurons. Next, we investigated the gene expression and protein profiles of this clone (GE6), comparing it to two other contrasting non-interneuronal phenotypes. Lastly, by identifying three transcription factors believed to be essential for interneuron development and function in this clone, we then exogenously expressed these TFs into a multipotential clone (CTX8) in an attempt to program its cell fate.

98

We have isolated and reprogrammed three neural clones with contrasting intrinsic abilities (Figure 2, Figure 3). Within these precursors, specific neural pathways are activated leading to the eventual maturation of neurons and glia. Of the three reprogrammed Fisher clones, GE6 was shown to differentiate into primarily GABAergic functional interneurons (Figure 9, Figure 10). Three transcription factors; Ascl1, Dlx1

Dlx5 (ADD) were all confirmed to be preferentially expressed in this neuronal clone during extended differentiation (Figure 8). In addition, within the multipotential clone

CTX8, Ascl1 was shown to be epigenetically regulated by VPA (Figure 21). The increased acetylation and gene expression is thought to be partially responsible for the observed enhanced neurogenesis in these multipotential clones. These results confirmed our predictions that we could isolate and stably transform homogeneous precursors and also suggests that all three of these transcription factors are integral to the formation of the interneuronal phenotype. Since the multipotential clone CTX8 has been phenotypically responsive to growth factor treatments (CNTF, SHH, BMP2) (Figure

16) and chromatin modifications (VPA) (Figure 18), this multipotential clone was chosen to recapitulate the interneuronal phenotype observed in GE6. We hypothesized that exogenous expression of all three transcription factors (ADD) would activate the necessary gene networks to enhance CTX8’s differentiation towards a more interneuronal phenotype.

Ascl1 is a well-known transcription factor that has been linked to a variety of neurogenic pathways (Casarosa et al., 1999, Horton et al., 1999, Parras et al., 2002). It has also been shown to be involved in the specification of interneuronal subtypes

(Jakovcevski et al., 2011). There were multiple reasons we were initially intrigued with

Ascl1 as our first reprogramming candidate. First, in the neural representative clones,

Ascl1's expression patterns in the pre and post differentiation conditions were both

99

expressed at relatively higher levels in the multipotential (CTX8) and neuronal clones

(GE6) (Figures 8, Figure 11). Furthermore, the undifferentiated chromatin states were enriched in permissive acetyl histone marks and appeared conducive to future gene expression (Figure 12). In addition, by globally altering chromatin patterns by the addition of valproic acid, a well-known chromatin modifier, we observed specific increases of histone acetylation within the regulatory regions of Ascl1 (Figure 21). Lastly and most recently, Ascl1 was also found to be the most critical gene responsible for the direct conversion of mouse fibroblasts into induced neurons (Vierbuchen et al., 2010).

Based on these results, we predicted that the exogenous expression of Ascl1 would have a profound effect on the cell fate of CTX8, by either increasing its neuron production or by developing more mature neurons.

To begin, the overexpression of Ascl1 alone had rapid and striking results easily viewed with the co-expression of fluorescent protein RFP. Within three days, noticeable increases in neurite process lengths and branching were apparent suggesting a neuronal phenotype (Figure 25). Interestingly we were surprised that when quantified, the total amount of neurons did not increase significantly. However, by quantifying only the transfected cells using RFP, the neurons counted outnumbered the control treatments (Figure 26C). One possible explanation for these somewhat conflicting results may be that Ascl1 overexpressed cells become post-mitotic well before the rest of the culture and get overrun by glial cells. One should remember that most of CTX8 sells will eventually proceed down the glial lineage (Figure 5) and this particular phenotype will still multiply even after bFGF withdrawal, albeit at a slower pace. The other explanation is that the transfection efficiencies were lower than expected, so transfected cells were a minority compared to the overall TuJ1 cells in culture. In collaboration with Dr. Mark Plummer’s laboratory, Ascl1 overexpressed cultures were

100

extended to approximately 10 to 12 days in culture and tested for electrical activity, specifically for induced action potentials and synaptic activity. While the overexpressed cells could fire the occasional action potential, no synaptic activity was ever observed.

Additionally, we also attempted to test BMP2 growth factor-treated cultures for electrical activity due to their rapid responses and mature morphologies after treatment. Although the BMP2-treated cultures did seem to have increased action potential frequencies, again no synaptic activity occurred (Plummer group, data not shown).

At this stage we concluded that both external growth factors and Ascl1 overexpression were sufficient for pushing the multipotential clone towards a neuronal phenotype but were insufficient for maturing the cell to the point of synaptic activity.

There are a few reasons why these approaches were not enough to drive the cell towards a fully functioning neuron. First, although Ascl1 has been previously shown to increase neurogenesis and to even directly reprogram fibroblasts into functional neurons

(Vierbuchen et al., 2010), our expression system is transient, so it may not be powerful enough to direct mature neuronal subtypes in all cellular contexts, especially in a primarily glial producing multipotential cell. Second, the amount of glia generated in the

CTX8 cultures may be inhibitory for proper maturation. Lastly, certain essential factors that are normally present in the neural milieu during development may be absent. Some of these may be responsible for providing the nutrients needed for proper maturation.

Taking all of these scenarios into account, we decided to combine some components that were previously lacking in our long term cultures. A combined approach has worked in the past, with such recent successes in iPS production. The Yamanaka group originally started with approximately 25 genes before this team optimized and narrowed the essential pluripotency factors to just 4 TFs (Takahashi and Yamanaka, 2006).

101

Dlx family members originate in the ventral areas of the forebrain and have been suggested to play key roles in regulating interneuron development (Butt et al., 2007; Le et al., 2007; Yu et al., 2011). For example, one of the functions of these transcription factors is to promote the tangential migration from ventral to dorsal regions of the telencephalon (Anderson et al., 1999; Eisenstat et al., 1999; Marin and Rubenstein,

2003). Therefore, when four Dlx members were confirmed to be expressed throughout differentiation in the interneuronal like clone GE6 (Figure 9), they were reasonable candidates to join Ascl1 in the attempted reprogramming of the CTX8 clone towards a more interneuronal subtype.

For our combinatorial approach, all three TFs: Ascl1, Dlx1 and Dlx5, were transfected in order to enhance the generation of interneurons. In addition, once overexpressed, the transfected cells were then immersed into a hippocampal cell co- culture environment to receive any additional factor components that may assist with neuronal maturation. Although the Dlx genes did not appear to be epigenetically regulated like Ascl1, they were chosen based on the expression profile of the interneuronal clone GE6. We postulate that the increased expression of this transcription factor family is partially responsible for the interneuronal markers expressed and successful synaptic activity observed within these cultures. Therefore, Ascl1’s purpose was to increase both neuron output and to push the cells towards an interneuronal fate.

Overexpression with all three factors plus the RPF plasmid Ds-Red were completed and plated onto E18 primary rat hippocampal cultures. Once again, the RFP expression displayed striking differences; such as increased process lengths, branching and overall neuronal morphology, within a few days compared to the control plasmids (Figure 19).

After 12 days, transfected cells were recorded for action potentials and synaptic activity.

102

Some important findings were gained from the co-culture experiments. First, the control transfected cells displayed a more branched morphology compared to previous non co-cultured experiments. Next, both action potentials and low level synaptic activity was observed. This supported our prediction that this enriched environment would assist the cells in their neuronal development. Perhaps the hippocampal cultures released growth factors to enhance both survival and development. In addition, as mentioned above, the ADD co-cultures showed increased branching and neurite lengths and displayed neuronal morphologies throughout the experiment. Also confirming our predictions, the ADD transfections produced action potentials when induced and also showed a more enhanced synaptic profile (Figure 30B,C). While the control cultures displayed low level synaptic activity reminiscent of excitatory peaks, the ADD cells showed much larger synaptic peaks with a hint of inhibitory synaptic activity. Because

CTX8 cells were co-cultured with hippocampal cells, we cannot be certain that the enhanced synaptic profile is due to the CTX8 cells generating the increased synaptic activity. To be sure, CTX8 transfected cells would need to be cultured and recorded alone. One possibility to take advantage of the co-culture environment would be to use the conditioned media from either the co-culture or hippocampal culture onto isolated

CTX8 cells. We have not yet completed this experiment but it is part of our future plans.

It is also intriguing to suggest that the ADD overexpressed CTX8 are assisting the cells or the endogenous hippocampal neurons to increase their synaptic output.

In addition to the functional characterization of cells by electrophysiology, we wanted to know if the ADD cells were progressing to more mature neuronal phenotypes compared to the controls. Both synaptophysin and GAD 65/67 immunostaining results pointed to the slightly increased expression in ADD neurons because they appeared to co-localize with Ds-Red positive cells (Figure 30A). We were not surprised that the GAD

103

65/67 intensity in the CTX8 cells was not as strong as in the primary cortical cells due to the heterogeneous mix of cortical cells. At this stage of development, the forebrain contains varying levels of neuronal subtypes. To further investigate the nature of these developing neurons, immunostains for the more mature interneuronal marker VGAT and excitatory marker VGlut-1 (Craig et al., 1996; Nunzi et al., 2003; Nakamura et al., 2005) were performed. We observed cells expressing VGAT in both the controls and ADD co- cultures. Interestingly, because we can track the successfully transfected cells by their

Ds-Red expression and supporting our hypothesis that ADD overexpression would enhance interneuron like cells, we only observed very few CTX8 transfected cells with neuron morphology expressing VGAT in the ADD transfections (Figure 31). However, this co-localization was not seen in the control samples. The Vglut-1 staining assays provided similar results showing cells expressing Vglut-1 in both the controls and ADD co-cultures (Figure 32). However, what was not predicted was that we also observed that the ADD transfected cultures appeared to co-localize with Vglut-1. Although it was difficult in both immunostaining assays to deduce if there was definitely co-localization, we must say that the addition of VGlut-1 cells was not predicted.

Why is the ADD combination not producing only interneuronal like cells? It is possible that the inherent capability of CTX8 cells is still intact and even though the ADD factors assist in neuronal production, they may also induce glutamatergic pathways.

Another reason may be that the Dlx factors are not enough to fully trigger the expression of VGAT because of CTX8’s chromatin profile. In the original histone modification ChIP assays, the VGAT regulatory region was highly enriched in repressive epigenetic marks

(Figure 12, Figure 14), suggesting a refractory response to VGAT expression. Perhaps a combinatorial approach using VPA with the ADD factors would break down any refractory barriers and achieve a more specific interneuronal phenotype. Therefore, the

104

data suggests that the ADD transcription factor combination and co-culture environment enhances neuronal morphology and generates few VGAT and VGlut-1 positive cells during extended differentiation. While the phenotypes were not as fully mature compared to the original restricted clones GE6, we did show that neural reprogramming, epigenetic and gene expression profiling methods can discover regulatory components responsible for driving cell lineages. Future optimization is recommended because it will be necessary to confirm which items may be dominant or dispensable for interneuronal commitment.

In summary, using immortalized clonal precursor cells derived from rat forebrain development enabled us to investigate cross-talk between intrinsic/extrinsic mechanisms, and how they coordinate in the molding of distinct cellular phenotypes. We questioned how neuronal and glial phenotypes become specialized and what factors may be responsible for directing cell fate. The isolation of three relatively homogenous contrasting phenotypes assisted in confirming our predictions that each clone maintains a distinct epigenetic signature. Furthermore, these chromatin states in some instances can predict downstream gene expression and may be useful as a screening technique.

In addition, we confirmed our predictions that the altering of chromatin structure regulates certain neuronal genes, resulting in enhanced neurogenesis. Finally, we successfully isolated a clone with the distinct ability to produce inhibitory GABAergic cells with the ability to form synapses. Using this as a model for neuronal restriction, we attempted to reprogram a more multipotential cell towards a more restricted phenotype using a combination of transcription factors thought to play an essential role in interneuron development. Interestingly, this approach increased both neurons and synaptic activity, but did not distinctly produce fully mature interneurons. Although our strategy was not entirely successful in this regard, the progress and lessons learned will

105

shape further reprogramming methods with the goal of engineering desired cellular subtypes for therapeutic applications.

106

IV. FIGURES

Figure 1: L2.2 and L2.3 NSC clones - An early in vitro model for neural development. Two v-myc transduced NSC clones were derived from E14.5 dissociated Sprague-Dawley rat cortex. Both immortalized clones can be maintained as

“undifferentiated” neural precursors in the presence of bFGF. Immunostaining results illustrate that once bFGF is withdrawn, one clone, L2.2, differentiates predominantly into

TuJ1+ GABAergic interneurons. The other, L2.3, exhibits a mixed phenotype expressing both neuronal and glial markers. (Clonal selection, immunostaining, and figure courtesy of Dr. Hedong Li)

107

A. B.

C.

Figure 2: Isolation and immortalization of GFP positive neural precursor clones

from the dorsal and ventral regions of the forebrain. (A) Coronal section of the

forebrain displaying that GFP expressing Fischer rats have been generated (Marano et

al., 2008). (B) E14.5 forebrains were dissected into dorsal and ventral sections

separating the cortex from the ganglionic eminences. Sections were dissociated and

fetal cells were infected with v-myc expressing retrovirus, immortalizing them (Li et al.,

2011). (C) Hierarchical clustering analysis based on the expression profiles of the set of genes listed at left divided the clones into three major clusters. The smallest cluster contained two clones indicating that GE6 is most closely related to L2.2, which has previously been identified as an interneuronal progenitor clone.

108

Figure 3: Differentiated Fischer clones display distinct markers of neuronal and/or glial cell lineage. Cultured cells were harvested and assayed by western blot with antibodies to TuJ1 and GFAP. Anti-GAPDH was used to normalize the sample loading.

V-myc transduced Fischer clones were maintained as Nestin+ in the presence of bFGF.

Western blot results show clones when cultured in the absence of bFGF. GE6 differentiates predominantly into a TuJ1+ phenotype and GE2 into GFAP+ phenotype.

CTX8 expressed both neuronal and glial markers and is considered multipotential

(Assay performed by Sasha B. Godfrey and figure courtesy of Dr. Hedong Li).

109

Figure 4: GE2 represents a glial restricted precursor. A time series displays the differentiation of GE2. Pre-differentiated cells were all Nestin positive. Upon bFGF withdrawal, there is a down regulation of Nestin, a neural precursor marker. In addition, the differentiation assay shows the progressive increase in GFAP+ cells without any expression of the neuronal marker TuJ1. Morphology was assessed by using DIC optics.

When initially plated, GE2 cells clone (GE2) displayed a flat morphology with short process lengths compared to the other two precursors. Upon differentiation, these cells became progressively flatter and exhibited a cobblestone- like morphology.

110

Figure 5: CTX8 is multipotential, but produces primarily non-neuronal cells. A time series displays the differentiation of CTX8. Upon bFGF withdrawal, there is a down regulation of Nestin, a neural precursor marker. In addition, the differentiation assay shows the progressive increase in GFAP+ similar to GE2. TuJ1+ cells are prevalent early following differentiation however; they continually decrease throughout the differentiation time course.

111

Figure 6: GE6 represents a neuronal restricted precursor. A time series displays the differentiation of GE6. Upon bFGF withdrawal, expression of Nestin is maintained, unlike the GE2 and CTX8 clones. In addition, the differentiation assay shows the progressive increase in TuJ1+ cells and minimal GFAP in days three and six. There is a strong co-localization of TuJ1 and GFAP in the longer two week time point. One explanation is that this is a result of de-differentiation of the clones. Another possibility is that the cells were not healthy as there was substantial cell death and short process lengths when observed with the DIC optics.

112

A. GE6

L2.2 CTX8

L2.3

GE2

Li et al., 2011

B.

Figure 7: Microarray gene expression profiles display clear differences between Fischer clones. (A) Affymetrix Rat Focus Microarrays were used to analyze temporal gene expression profiles for all 3 clones. A principal component analysis (PCA) of all expressed genes for GE6, GE2 and CTX8 compared to the interneuronal progenitor L2.2 and multipotential clone L2.3. Cell lines are identified by color and time of differentiation (0, 3, or 7 days) is identified by circle size. Note the low variance between replicate samples and a clear separation among the neuronal, glial and multipotential clones, mirroring initial characterization assays. (analysis provided by Dr.

John Corradi, Bristol Myers Squibb). (B) A hierarchical clustering analysis using correlation as the metric was performed and shows a dendrogram displaying the relationships between all samples. Results demonstrate tight replicate distribution with the neuronal and glial phenotypes most dissimilar. (Analysis provided by Dr. John

Corradi, Bristol Myers Squibb).

113

Li et al., 2011

Figure 8: Heat map of expression profiles identifies a cascade of regulated transcription factors during critical stages of cell fate maturation. Z-scored data are indicated by colors, with red being larger positive values and violet being smaller negative values, relative to the overall mean. Expression profiling shows robust changes in GE6 during differentiation and show initial induction of Ascl1, followed by expression of Dlx family members, Dlx1 and Dlx5. Also during the later stages of differentiation, multiple transcripts involved in neurotransmission (Npy, Sst) and presynaptic proteins

(Snap25, Stxbp1 and Syt1, 4 ,5 ,6) were expressed. The up-regulation of later stage genes indicates the neuronal maturation of this clone and sets it apart from glial clone

GE2 and CTX8, the multipotential clone that does not express any of these mature genes during the later stages of differentiation. (Analysis provided by Dr. John Corradi,

Bristol Myers Squibb.)

114

Figure 9: qPCR of additional Dlx family members, GAD-2 and v-myc mRNAs.

Aliquots of the same preparations of total cellular RNA used in the microarray study were assayed by SYBR green qPCR. Results are expressed relative to the mean of the

Day0 CTX8 group for comparison (plotted as mean +/- SEM; n=3). A two-way ANOVA

(Days, Cell) was run for each assay. Tukey’s post-hoc tests found that the differentiation time point indicated by asterisks were significantly different from Day 0 (*p<0.05;

**p<0.001). These qPCR results also confirmed the microarray expression profiles of

Dlx1 and Dlx5. (Assay completed in collaboration with Mavis Swerdel.)

115

Figure 10: Differentiated GE6 cells generate primarily GABAergic phenotypes.

GE6 cells were pre-differentiated for 3 days without FGF2 and then differentiated with

0.5% FCS for 7 days. Neurons were identified by immunostaining with chick anti-βIII tubulin, recognized by the monoclonal antibody TuJ1, rabbit anti-Synaptophysin, mouse anti-Gephyrin, and anti-Vesicular GABA Transporter (VGAT) but not with anti-Vesicular

Glutamate 1 (VGlut-1) indicating the presence of GABAergic but not glutamatergic neurons (Immunostaining completed by Dr. Hedong Li).

116

Figure 11: Gene expression profiles display lineage differences between Fischer neural clones. All three clones were harvested for total RNA at days 0, 3 and 7 days and ran on Affymetrix Rat Focus Microarrays. Ascl1, a known neuronal transcription factor is already relatively expressed more already relatively expressed more in the multipotential (CTX8) and neuronal (GE6) clones in their undifferentiated states. Erbb4, a receptor for Neuregulin, is induced once bFGF is withdrawn in both GE6 and CTX8.

Vgat, a vesicular transporter for GABA in inhibitory neurons increased dramatically in the

GE 6 clone. Olig 2 was expressed in bothe the multipotential and neuronal clones. In contrast, Cystathionine synthase (Cbs) is expressed only in the multipotential and glial restricted precursors. Microarray results were provided by BMS and are displayed on a log scale.

117

A.

B.

C.

Figure 12: Chromatin immunoprecipitations of distinct neural precursor clones, in their undifferentiated states, identify fate-specific marks. (A) A listing of neuronal and glial genes and their known functions in neural development. These genes were relatively expressed at different levels between Fischer clones. (B) Undifferentiated neuronal (GE6), glial (GE2) and multipotential (CTX8) clones were immunoprecipitated for histone modifications that were known to correlate with either gene activation

118

(H3K4me3) or repression (H3K27me3). qPCR targeting the promoter regions of each

gene detected the relative enrichment of each sample. As predicted, there was an

inverse relationship of permissive to repressive histone marks at key neuronal and glial

genes. *represents the differences between clones (p ≤ 0.05) using Students t-test. (C)

An illustration describing the hypothesis that the promoter regions on certain neuronal genes will be in a more permissive chromatin state in the neuronal restricted precursors relatively compared to glial restricted precursors.

119

Figure 13: ChIP-Seq generates widespread genomic coverage across all chromosomes. Enriched fragments were sequenced and aligned to the rat genome. A genomic coverage map of each undifferentiated Fischer clone is represented by color.

Displayed is the H3K9/14ac peak density map that was scaled to the total number of peaks per Mb per sample. Genomic coverage was as follows: GE6-Mean coverage: 1.1,

Coverage range: 1 - 49. GE2-Mean coverage: 1.1,Coverage range: 1 - 38. CTX8-Mean coverage: 1.4, Coverage range: 1 - 58. regions p-value <=0.1 were converted into peaks

(red) and can be easily visualized using the UCSC genome browser.

120

A. B. Erbb4 Olig2

C. VGAT

Figure 14: ChIP sequencing correlates with previous ChIP-qPCR results, highlighting permissive and repressive histone enrichment. Enrichment peaks are shown as colors representing permissive histone mark H3K9/14ac (green) and repressive histone mark H3K27me3 (red), surrounding the transcription start sites (TSS) of each gene. Erbb4 (A) and Olig2 (B) were both enriched in permissive histone modifications, correlating with the previous Chip-qPCR results (Figure 12B). In addition, the regulatory region of Slc32a1 (C), also known as VGAT was enriched in the repressive marks, also correlating with previous ChIP-qPCR results (Figure 12B). The black bars (____) upstream of each TSS represent the location of the ChIP-qPCR amplicons described in Figure 12B.

121

A.

B.

C.

Figure 15: ChIP sequencing of epigenetic acetyl marks highlights differences between clones in their undifferentiated states. Gene Ontology (GO) analysis of

ChIP sequencing of histone acetylation modifications (H3K9/14 ac) displays the most significant enrichment categories during each time point. There were differences between clones in multiple categories such as General Differentiation and Neuron

Specific. These results suggest that the Fischer clones in the undifferentiated states

122

have chromatin state differences that may affect downstream gene expression. All peaks were annotated to their nearest gene TSS and then assigned counted within a gene ontology biological process. Each GO term was given a Benjamini & Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset compared with the number of all genes for that gene ontology group. We compared the highest ranked GO terms and then grouped them into seven broad categories to highlight the most enriched categories per sample and temporal period.

123

Figure 16: CTX8 fate can be affected by factors present as gradients in developing forebrain. (A) The developing neural forebrain produces growth factor gradients that can have profound effects on cell lineage by inducing neuronal and glial networks (Li et al., 2008). CTX8 clones treated with growth factors showed rapid induction of neuronal or glial markers. Cells were differentiated by bFGF withdrawal and treated with the listed growth factor. After 3 days, cells were cross-linked and immunostained with neuronal marker TuJ1 and glial marker GFAP. CNTF rapidly up-regulated GFAP whereas the

SHH agonist (SHH Ag) seemed to reduce the glial phenotype and induce TuJ1 positive progeny. BMP2 increased both neuronal and glial markers along with a more mature morphology.

124

A.

B.

C.

125

Figure 17: BMP2 treatment promotes the progression of both astrocytes and neurons. (A) CTX8 cells were differentiated by bFGF withdrawal and treated with

BMP2. Cells were cross-linked and immunostained with neuronal marker TuJ1 and glial marker GFAP. By day three of differentiation, BMP2 increased both neuronal and glial markers along with a more mature morphology (long process lengths and complex branching - as indicated by white arrows) compared with controls. (B) Gene Ontology

(GO) analysis of ChIP sequencing of histone acetylation modifications (HeK9/14 ac), displays the most significant enrichment categories during each time point. All peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a Benjamini & Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset. We compared the highest ranked

GO terms and then grouped them into broader categories to highlight the most enriched categories per sample and temporal period. (C) Two of the GO categories from Figure

16B show the H3K9/14ac acetylation enrichment near nervous system related and neuron specific transcription start sites. In the BMP treated samples at three and seven days, results show the maintenance of histone acetylation in these categories during differentiation compared to the controls. These trends mimic the immunostaining results and suggest that BMP2 treatment promotes the acetylation of neural and neuronal development genes, leading to downstream gene activation.

126

A.

B.

Figure 18: Valproic acid alters global chromatin and enhances neurogenesis. (A)

Western blot depicting the global increase of permissive acetylation marker H3K9/14 ac in 1mM VPA treated cells. Total Histone H3 was used as a control. (B) CTX8 clones were differentiated by bFGF withdrawal for 3 days with or without treatment of VPA.

Treated VPA cells increased the number of TuJ1+ and MAP2+ cells at 3 and 14 days

respectively. The Tuj1/GFAP immunostain images display one 20x objective field of

view. The MAP2 immunostains each show a mosaic image comprised of 16 - 20x

individual tile images.

127

A. B.

60 +Valproic Acid

40 Positive

20 TuJ1

of

% 0 Control Dy7+VPA Dy7

Figure 19: Valproic acid increases neurogenesis and neurite process length. (A)

CTX8 cells were differentiated via bFGF withdrawal for 7 days with or without treatment of VPA (1mM). Due to the low quantity of differentiated cells, biological replicates (n=3) were combined to obtain sufficient amounts for flow cytometry analysis. Differentiated cells were dissociated, cross-linked and immunostained for TuJ1 and a mouse IgG as an isotype control. Percentage of TuJ1 positive cells are represented after subtraction of background fluorescence from the isotype control. (B) Control and VPA treated cultures were plated on glass coverslips and cross-linked and immunostained for TuJ1. Positive cells from each condition (1 biological replicate , n=20 cells) were traced using Image J

(Abramoff et al., 2004) and Neuron Studio (Wearne et al., 2005; Rodriguez et al., 2008).

Their lengths and branching were quantified using the Bonfire software package

(Langhammer et al., 2010). VPA treatment led to increased neurite process length

(18%), and total cable length (20%) which is defined as the combined process lengths per cell.

128

A.

B.

Figure 20: Valproic acid alters the chromatin in a network of nervous system and neuron specific genes. GO analysis of ChIP sequencing of histone acetylation modifications (HeK9/14ac), displays the enrichment near neuronal development and neuronal transcription start sites. All peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a

Benjamini & Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset. We compared the highest ranked GO terms and then grouped them into broader categories to highlight the most enriched categories per sample and temporal period. At three and seven days, results show the relative maintenance of histone acetylation in both nervous system related (A) and neuron specific genes (B) in the VPA treated samples, while the control group had decreases in both of these categories. These trends mimic the immunostaining results and suggest that VPA treatment promotes the acetylation of neural and neuronal development genes, leading to downstream gene activation.

129

A. Ascl1

B.

Figure 21: Valproic acid induces epigenetic acetylation markings and gene

expression in neuronal transcription factor Ascl1. (A) Multipotential clones (CTX8)

were treated with VPA (1 mM), differentiated and harvested for ChIP sequencing.

Enriched permissive and repressive histone modifications were sequenced along with a

non-enriched input control. Three days after induced differentiation, the coding region

surrounding known neuronal transcription factor Ascl1 was heavily enriched (spanning

~2kb) with permissive mark H3K9/14ac (green peak) only in the VPA treated samples.

The repressive modification H3K27me3 was also diminished in the VPA samples

compared to the control (red peak). (B) Ascl1 gene expression was assayed using the

total RNA that was harvested after 3 days of treatment. qPCR with primers targeting the

coding region of ASCL1 was run. Results are expressed relative to the mean of the

CTX8 Day 0 sample group (not shown) for comparison (plotted as mean +/- SEM; n=3).

130

Ascl1 expression in the VPA treated samples were found to be significantly different that the Day 3 control sample as indicated by the asterisks (*p<0.05 using Student’s t-test).

131

Figure 22: Select microRNAs are epigenetically regulated by valproic acid. microRNA transcription was hypothesized to positively correlate with acetylated enrichment patterns within the filtered regulatory regions of microRNAs shown in Table

2. Gene expression was assayed using the total RNA that was harvested after 3 days of treatment. qPCR with primers targeting the mature microRNA was run. As predicted, we observed significant increases in microRNA expression in the VPA treated samples relative to controls at Day 3, as indicated by the asterisks (*p<0.05 using Student’s t- test). Results are expressed relative to the mean of the CTX8 Day 0 sample group (not shown) for comparison (plotted as mean ± SEM; n=3).

132

A. B.

C.

Figure 23: Exogenous expression of a select group of microRNAs increases the neurogenic capacity of a multipotential NSC clone L2.3. Gain or loss of function of mir-9, mir-124, mir-153 and mir-182 was assayed in the multipotential clone L2.3.

Individual (A) or a mixture (C) of double stranded microRNA mimics (Ambion Premirs™) were nucleofected into L2.3 clones prior to FGF withdrawal (n=4). After 72 hours of differentiation (-bFGF), cells were stained for TuJ1 and assayed via flow cytometry.

(*p<0.05 using Student’s t-test). Addition of the four predicted neurogenic microRNAs yielded a larger percentage of TuJ1+ (neurogenic) cells compared to the negative control. (B) A mix of Antimirs for all four microRNAs was nucleofected and assayed similarly to the previous assay with the Premirs™.

133

A.

B.

Figure 24: Verifying co-transfection of plasmids using CAG-GFP and CAG-DsRed.

(A) L2.3 cells were co-transfected with 2 μg each of GFP and DS-Red reporter constructs that are a fusion between the CMV enhancer and chicken actin promoter

(Matsuda and Cepko, 2004). Co-localization was observed for GFP and DS-Red 12 to

18 hours post transfection. (B) The percentage of co-transfected cells high and was quantified using Image J (Abramoff et al., 2004). For each image (n=3), intensity thresholds were set for each channel and each cell was scored as single or double positive. Transfections were completed using Lonza’s 96 well nucleofector using the proprietary high efficiency pulse program. Approximately 1x106 cells were transfected in

suspension and were subsequently plated onto laminin coated glass coverslips.

134

Figure 25: Transfection of Ascl1 enhances neurite length and branching. Plasmids expressing Ascl1 and Ds-Red were mixed and electroporated (Lonza) into CTX8 cells.

Cultures were then differentiated for 3 days by bFGF withdrawal. Inverted Ds-Red images show co-transfected cells displaying longer processes and increased branch points overall resembling a more neuronal phenotype.

135

A.

B.

C.

136

Figure 26: Transfection of Ascl1 alone enhances neurogenesis. Plasmids expressing AsclI1 and Ds-Red were mixed and electroporated (Lonza) into CTX8 cells.

Cultures were then differentiated for 3 days by bFGF withdrawal. (A) Mosaic panel of 16 images (20x objective) shows an overview of the widespread increases in TuJ1 intensity in the Ascl1 transfected cultures compared to the controls. (B) A representative tile (20x

objective) from a separate field of view from the image above (A). These images

highlight extensive neurite outgrowth in TuJ1+ cells. (C) Successfully co-transfected

cells were first filtered based on Ds-Red expression and then scored on TuJ1

expression. Co-transfected cells were more likely to express TuJ1 (*p < 0.01 using

Student’s t-test).

137

Figure 27: Transfection of combined ADD transcription factors enhances neurogenesis. Plasmids expressing all three ADD TFs were mixed and electroporated

(Lonza) into CTX8 cells. Cultures were then differentiated for 4 days by bFGF withdrawal. (A) A representative image (20x objective) shows widespread increases in neurite outgrowth in TuJ1+ cells.

138

Figure 28: Co-culture of ADD transfected CTX8 and hippocampal neurons enhances neuronal morphology. CTX8 cells, transfected with Ds-Red, Ascl1, Dlx1, and Dlx5, were co-cultured with E18 hippocampal neurons. Live cell images after 7 days depict a strikingly branched morphology in the ADD treated cells that expressed the Ds-

Red signal. These morphology changes were unique to transfected (DS-Red+) cells as the control’s morphology did not appear as long or branched.

139

Figure 29: Co-culture of ADD transfected Ctx8 and hippocampal neurons enhances total neurite length and branching. CTX8 cells, transfected with Ds-Red,

Ascl1, Dlx1, and Dlx5 (ADD), were co-cultured with hippocampal neurons. Ds-Red positive cells from each condition (1 biological replicate, n=20 control cells, n=17 ADD cells) were traced using Image J and Neuron Studio. Their lengths and branching were quantified using the Bonfire software package (Langhammer et al., 2010). The ADD combination led to increased number of processes, branch points and total cable length, which is defined as the combined process lengths per cell.

140

A.

B.

Control

+ADD

C.

+ADD

Control

141

Figure 30: Transfection of Ascl1, Dlx1, and Dlx5 enhances neurogenesis and synaptic activity. (A) CTX8 cells, transfected with Ds-Red and ADD were co-cultured with hippocampal neurons. After 2 weeks of differentiation, the ADD cultures showed slight increases in synaptophysin and GAD 65/67 staining. (B) Example trace of inward currents (left) and depolarization-induced action potential firing (right) obtained with whole-cell current-clamp recording from control and ADD transfected co-cultures. Both inward currents and action potentials were more mature when transfected with ADD compared with control. (C) Example traces of ongoing synaptic activity. Cells transfected with all three factors had more frequent spikes and more robust mixture of both EPSC

(triangles) and IPSC (circles) compared with the control. Co-cultures and immunostainings were done in collaboration with Dr. Mark Plummer and Joanne

Babiarz.

142

Figure 31: Transfection of Ascl1, Dlx1, and Dlx5 enhances neurogenesis by generating VGAT positive progeny. (A) CTX8 cells, transfected with Ds-Red and ADD were co-cultured with hippocampal neurons. After 2 weeks of differentiation, the ADD cultures showed slight increases in VGAT staining, a marker for GABAergic neurons.

Interestingly, because we can track the successfully transfected cells by the Ds-Red expression, we only observed some CTX8 transfected cells with neuron morphology expressing VGAT in the ADD transfections (arrows). Co-cultures and immunostainings were done in collaboration with Dr. Mark Plummer and Joanne Babiarz.

143

Figure 32: Transfection of Ascl1, Dlx1, and Dlx5 enhances neurogenesis by generating Vglut-1 positive progeny. (A) CTX8 cells, transfected with Ds-Red and

ADD were co-cultured with hippocampal neurons. After 2 weeks of differentiation, the

ADD cultures showed slight increases in Vglut-1 staining, a marker for GABAergic neurons. Interestingly, because we can track the successfully transfected cells by the

Ds-Red expression, we only observed some CTX8 transfected cells with neuron morphology expressing Vglut-1 in the ADD transfections (arrows). Co-cultures and immunostainings were done in collaboration with Dr. Mark Plummer and Joanne

Babiarz.

144

V. TABLES

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000008195 1433Z_RAT ENSRNOG00000011295 Anapc2 ENSRNOG00000020414 Axin1 ENSRNOG00000031424 Ccdc65 ENSRNOG00000036437 5_8S_rRNA ENSRNOG00000004130 Anapc4 ENSRNOG00000020716 Axl ENSRNOG00000025317 Ccdc75 ENSRNOG00000034350 5S_rRNA ENSRNOG00000005854 Angpt1 ENSRNOG00000010096 Azi2 ENSRNOG00000002052 Ccdc80 ENSRNOG00000034406 5S_rRNA ENSRNOG00000023532 Ankfn1 ENSRNOG00000005333 Azin1 ENSRNOG00000012137 Ccdc84 ENSRNOG00000034473 5S_rRNA ENSRNOG00000005502 Ankib1 ENSRNOG00000004316 B0BN98_RAT ENSRNOG00000004057 Ccdc88a ENSRNOG00000034753 5S_rRNA ENSRNOG00000005623 Ankmy2 ENSRNOG00000005641 B1WBR8_RAT ENSRNOG00000007292 Ccdc99 ENSRNOG00000035143 5S_rRNA ENSRNOG00000001204 Ankrd13a ENSRNOG00000008524 B2RYK0_RAT ENSRNOG00000021851 Ccl1 ENSRNOG00000035171 5S_rRNA ENSRNOG00000028168 Ankrd16 ENSRNOG00000002632 B2RYX0_RAT ENSRNOG00000007719 Ccnc ENSRNOG00000035821 5S_rRNA ENSRNOG00000019697 Ankrd28 ENSRNOG00000008205 B2RZ68_RAT ENSRNOG00000020918 Ccnd1 ENSRNOG00000035837 5S_rRNA ENSRNOG00000031335 Ankrd37 ENSRNOG00000003251 B3galt2 ENSRNOG00000019939 CCND2_RAT ENSRNOG00000036038 5S_rRNA ENSRNOG00000023995 Ankrd39 ENSRNOG00000007142 B3gat1 ENSRNOG00000011379 Ccndbp1 ENSRNOG00000036074 5S_rRNA ENSRNOG00000002935 Ankrd40 ENSRNOG00000019804 B3gat3 ENSRNOG00000031656 Ccnh ENSRNOG00000036343 5S_rRNA ENSRNOG00000009664 Ankrd42 ENSRNOG00000021886 B4galt7 ENSRNOG00000002125 Ccni ENSRNOG00000036347 5S_rRNA ENSRNOG00000007110 Ankrd6 ENSRNOG00000036859 B5DF39_RAT ENSRNOG00000014551 Ccnj ENSRNOG00000040545 5S_rRNA ENSRNOG00000019052 Ankzf1 ENSRNOG00000016847 Bace1 ENSRNOG00000003893 Ccnjl ENSRNOG00000040821 5S_rRNA ENSRNOG00000009266 Anp32b ENSRNOG00000001582 Bach1 ENSRNOG00000018691 Ccnl2 ENSRNOG00000041721 5S_rRNA ENSRNOG00000021168 Anp32e ENSRNOG00000008277 Bag1 ENSRNOG00000011512 Ccnt1 ENSRNOG00000035882 7SK ENSRNOG00000014610 Anpep ENSRNOG00000000485 Bak1 ENSRNOG00000017113 Ccnt2 ENSRNOG00000041861 7SK ENSRNOG00000024870 ANS1B_RAT ENSRNOG00000025188 Basp1 ENSRNOG00000019691 Ccs ENSRNOG00000041936 7SK ENSRNOG00000008678 Antxr1 ENSRNOG00000000852 Bat2 ENSRNOG00000009642 Cct4 ENSRNOG00000041938 7SK ENSRNOG00000010668 Anxa6 ENSRNOG00000007884 Bcap29 ENSRNOG00000011632 Cct5 ENSRNOG00000002827 A2bp1 ENSRNOG00000015390 Ap1g1 ENSRNOG00000018783 Bcas2 ENSRNOG00000000923 Cct6a ENSRNOG00000013313 Aadacl1 ENSRNOG00000009988 Ap2b1 ENSRNOG00000020607 Bckdha ENSRNOG00000001592 Cct8 ENSRNOG00000011861 Aadat ENSRNOG00000001709 Ap2m1 ENSRNOG00000009928 Bckdhb ENSRNOG00000019215 Cd151 ENSRNOG00000018317 Aak1 ENSRNOG00000018977 Ap3d1 ENSRNOG00000012394 Bcl2l13 ENSRNOG00000016451 Cd1d1 ENSRNOG00000011182 Aanat ENSRNOG00000011461 Ap3m1 ENSRNOG00000015732 Bcl2l2 ENSRNOG00000002141 Cd200 ENSRNOG00000004708 Aard ENSRNOG00000014928 Apba1 ENSRNOG00000001843 Bcl6 ENSRNOG00000000321 Cd24 ENSRNOG00000002778 Aatf ENSRNOG00000020466 Apba3 ENSRNOG00000012420 Bcl9l ENSRNOG00000033608 Cd276 ENSRNOG00000002636 Abat ENSRNOG00000017849 Apbb3 ENSRNOG00000013052 Bclaf1 ENSRNOG00000015945 Cd3g ENSRNOG00000008557 Abcb8 ENSRNOG00000005542 Apob ENSRNOG00000016754 Bcs1l ENSRNOG00000001964 Cd47 ENSRNOG00000002948 Abcc3 ENSRNOG00000015411 Apobec1 ENSRNOG00000004488 Bdkrb1 ENSRNOG00000001972 Cd47 ENSRNOG00000010064 Abcc4 ENSRNOG00000008174 Appl2 ENSRNOG00000020513 Becn1 ENSRNOG00000001527 Cd80 ENSRNOG00000029178 Abcc5 ENSRNOG00000014405 Aprt ENSRNOG00000008025 Bend5 ENSRNOG00000017536 Cdc16 ENSRNOG00000018345 Abce1 ENSRNOG00000016043 Aqp4 ENSRNOG00000016031 Bicd2 ENSRNOG00000005904 Cdc27 ENSRNOG00000001158 Abcg1 ENSRNOG00000004807 Arf2 ENSRNOG00000012852 Bin1 ENSRNOG00000008348 Cdc34 ENSRNOG00000017120 Abhd2 ENSRNOG00000012623 Arf4 ENSRNOG00000003563 Blmh ENSRNOG00000033426 Cdc37 ENSRNOG00000009244 Abhd4 ENSRNOG00000004791 Arf6 ENSRNOG00000012684 Bloc1s2 ENSRNOG00000002841 Cdc42bpa ENSRNOG00000008167 Abhd6 ENSRNOG00000014429 Arfgap2 ENSRNOG00000007529 Bmf ENSRNOG00000019975 Cdc5l ENSRNOG00000009371 Abl1 ENSRNOG00000007485 Arfgef2 ENSRNOG00000010890 Bmp1 ENSRNOG00000009221 Cdcp2 ENSRNOG00000015762 Abtb1 ENSRNOG00000010533 Arfip1 ENSRNOG00000020717 Bod1 ENSRNOG00000003088 Cdgap ENSRNOG00000014178 Acad9 ENSRNOG00000018440 Arfip2 ENSRNOG00000010133 Bpgm ENSRNOG00000015602 Cdh2 ENSRNOG00000009845 Acadm ENSRNOG00000016610 Arhgap1 ENSRNOG00000020701 Brca1 ENSRNOG00000013503 Cdh24 ENSRNOG00000018114 Acadvl ENSRNOG00000031168 Arhgap15 ENSRNOG00000001111 Brca2 ENSRNOG00000024144 Cdipt ENSRNOG00000007862 Acat1 ENSRNOG00000025624 Arhgap20 ENSRNOG00000012739 Brf2 ENSRNOG00000016088 Cdk10 ENSRNOG00000003185 Acbd3 ENSRNOG00000008659 Arhgap21 ENSRNOG00000000979 Bri3bp ENSRNOG00000006000 CDK12_RAT ENSRNOG00000018789 Accn2 ENSRNOG00000002095 Arhgap24 ENSRNOG00000008249 Brms1l ENSRNOG00000021685 Cdk5r1 ENSRNOG00000013533 Acin1 ENSRNOG00000005809 Arhgdib ENSRNOG00000012415 Brp44l ENSRNOG00000015696 Cdk5rap1 ENSRNOG00000016924 Acly ENSRNOG00000004566 Arhgef15 ENSRNOG00000008414 Bsg ENSRNOG00000018510 Cdk7 ENSRNOG00000011283 Acn9 ENSRNOG00000020027 Arhgef2 ENSRNOG00000019656 Btd ENSRNOG00000022586 Cdk9 ENSRNOG00000005849 Aco1 ENSRNOG00000004831 Arid2 ENSRNOG00000019664 Btd ENSRNOG00000007249 Cdkn1b ENSRNOG00000005260 Acp1 ENSRNOG00000013416 Arid3c ENSRNOG00000016912 Btf3 ENSRNOG00000005024 Cdkn2aipnl ENSRNOG00000013594 Acp2 ENSRNOG00000014653 Arl11 ENSRNOG00000004284 Btg1 ENSRNOG00000010918 Cebpa ENSRNOG00000017494 Acp6 ENSRNOG00000019313 Arl4c ENSRNOG00000003300 Btg2 ENSRNOG00000001293 Cee ENSRNOG00000034254 Actb ENSRNOG00000001080 Arl6ip4 ENSRNOG00000016280 Btrc ENSRNOG00000013628 Cela2a ENSRNOG00000036701 Actg1 ENSRNOG00000007312 Arl8b ENSRNOG00000010180 Bwk1 ENSRNOG00000032178 Cenpa ENSRNOG00000020433 Actn4 ENSRNOG00000019935 Armc5 ENSRNOG00000000587 Bxdc1 ENSRNOG00000021246 Cenpb ENSRNOG00000007504 Actr10 ENSRNOG00000037709 Armcx1 ENSRNOG00000002316 C1ql4 ENSRNOG00000006792 Cep57 ENSRNOG00000019725 Actr1a ENSRNOG00000037735 Armcx1 ENSRNOG00000007300 C1qtnf6 ENSRNOG00000015711 Cetn3 ENSRNOG00000016789 Actr1b ENSRNOG00000031174 Arnt ENSRNOG00000018899 C5 ENSRNOG00000000419 Cfb ENSRNOG00000003206 Actr3 ENSRNOG00000002904 Arr3 ENSRNOG00000017297 Cab39 ENSRNOG00000019326 Cfdp1 ENSRNOG00000010265 Ada ENSRNOG00000030404 Arrb1 ENSRNOG00000011603 Cab39l ENSRNOG00000020660 Cfl1 ENSRNOG00000002753 Adam11 ENSRNOG00000028350 Arse ENSRNOG00000011607 Cab39l ENSRNOG00000000718 Cggbp1 ENSRNOG00000028036 Adamts7 ENSRNOG00000007476 Asam ENSRNOG00000015835 Cacna2d2 ENSRNOG00000013211 Chchd3 ENSRNOG00000012098 Adcyap1r1 ENSRNOG00000017967 Asb13 ENSRNOG00000012362 Cacng3 ENSRNOG00000022466 Chchd4 ENSRNOG00000007990 Adipor2 ENSRNOG00000009197 Asb4 ENSRNOG00000003262 Cacng4 ENSRNOG00000002267 Chic2 ENSRNOG00000010849 Adprhl2 ENSRNOG00000024786 Asb6 ENSRNOG00000030840 Cadm2 ENSRNOG00000011404 Chkb ENSRNOG00000018985 Adrbk1 ENSRNOG00000012157 Ascc3l1 ENSRNOG00000015447 Calcoco1 ENSRNOG00000034071 Chmp4bl1 ENSRNOG00000018655 Adsl ENSRNOG00000004294 Ascl1 ENSRNOG00000028376 Calcoco2 ENSRNOG00000004014 Chmp6 ENSRNOG00000004481 Adss ENSRNOG00000003747 Asna1 ENSRNOG00000010233 Cald1 ENSRNOG00000009411 Chn2 ENSRNOG00000029133 Aes ENSRNOG00000020202 Asrgl1 ENSRNOG00000003029 Calr ENSRNOG00000026643 Chordc1 ENSRNOG00000026994 Afg3l1 ENSRNOG00000024414 Ate1 ENSRNOG00000013260 Calr3 ENSRNOG00000018286 Chrna1 ENSRNOG00000000108 Aga ENSRNOG00000024444 Ate1 ENSRNOG00000018337 Caly ENSRNOG00000017424 Chrna2 ENSRNOG00000012764 Agap3 ENSRNOG00000017801 Atf4 ENSRNOG00000008741 Camsap1l1 ENSRNOG00000019527 Chrnd ENSRNOG00000015619 Agfg1 ENSRNOG00000024632 Atf6 ENSRNOG00000017766 Car12 ENSRNOG00000007989 Chst1 ENSRNOG00000017731 Agpat4 ENSRNOG00000000431 Atf6b ENSRNOG00000002610 Carhsp1 ENSRNOG00000008885 Chst11 ENSRNOG00000018077 Agpat6 ENSRNOG00000011873 Atg14 ENSRNOG00000015021 Carkd ENSRNOG00000016267 Chst15 ENSRNOG00000002159 Agpat9 ENSRNOG00000021028 Atg2a ENSRNOG00000020651 Cars ENSRNOG00000022485 Chuk ENSRNOG00000001547 Agps ENSRNOG00000002094 Atg3 ENSRNOG00000017712 Cartpt ENSRNOG00000012638 Ciao1 ENSRNOG00000005023 Agr2 ENSRNOG00000018403 Atg4b ENSRNOG00000027630 Casc1 ENSRNOG00000016234 Ciapin1 ENSRNOG00000018445 Agt ENSRNOG00000017154 Atp11a ENSRNOG00000016357 Casc4 ENSRNOG00000009942 Cib2 ENSRNOG00000023856 Agxt ENSRNOG00000001285 Atp2a2 ENSRNOG00000010475 Casp3 ENSRNOG00000003189 Cited1 ENSRNOG00000005318 Ahsa2 ENSRNOG00000018543 Atp4b ENSRNOG00000012331 Casp8 ENSRNOG00000012193 Cited2 ENSRNOG00000010753 Aig1 ENSRNOG00000002840 Atp5b ENSRNOG00000012944 Casp9 ENSRNOG00000010872 Ckb ENSRNOG00000001213 Aire ENSRNOG00000007235 Atp5g1 ENSRNOG00000002265 Casr ENSRNOG00000020360 Clcc1 ENSRNOG00000002899 Akap10 ENSRNOG00000000064 Atp5i ENSRNOG00000008364 Cat ENSRNOG00000016917 CLCN1_RAT ENSRNOG00000019549 Akap12 ENSRNOG00000020596 Atp5sl ENSRNOG00000006694 Cav1 ENSRNOG00000010263 Cldn11 ENSRNOG00000006355 Akap8l ENSRNOG00000006542 Atp6v0c ENSRNOG00000014647 Cbfb ENSRNOG00000039862 Cldn12 ENSRNOG00000016727 Akr1a1 ENSRNOG00000017235 Atp6v0d1 ENSRNOG00000001982 Cblb ENSRNOG00000000728 Clic2 ENSRNOG00000009875 Akr1b7 ENSRNOG00000008218 Atp6v0e2 ENSRNOG00000013654 Cbln2 ENSRNOG00000025772 Clic5 ENSRNOG00000017780 Akr7a2 ENSRNOG00000001992 Atp6v1a ENSRNOG00000030904 CBPB1_RAT ENSRNOG00000020821 Clip3 ENSRNOG00000020289 Akt1s1 ENSRNOG00000011891 Atp6v1b2 ENSRNOG00000032165 Cbr1 ENSRNOG00000025768 Clk1 ENSRNOG00000001989 Alcam ENSRNOG00000004846 Atp6v1c1 ENSRNOG00000024411 Cbr4 ENSRNOG00000030126 Clk3 ENSRNOG00000015267 Aldh18a1 ENSRNOG00000007392 Atp6v1f ENSRNOG00000011814 Cbx3 ENSRNOG00000012788 Clns1a ENSRNOG00000002342 Aldh3a2 ENSRNOG00000000840 Atp6v1g2 ENSRNOG00000013892 Cby1 ENSRNOG00000018255 Clptm1 ENSRNOG00000023538 Aldh5a1 ENSRNOG00000003719 Atpaf2 ENSRNOG00000006747 Cc2d1a ENSRNOG00000030225 Clpx ENSRNOG00000023647 Aldoa ENSRNOG00000010027 Atr ENSRNOG00000008634 Cc2d1b ENSRNOG00000016398 Clstn1 ENSRNOG00000014511 Alg10 ENSRNOG00000020670 Atrip ENSRNOG00000003901 Ccdc104 ENSRNOG00000014635 Clta ENSRNOG00000011528 Alg14 ENSRNOG00000014637 Atxn10 ENSRNOG00000012922 Ccdc147 ENSRNOG00000013178 Cmip ENSRNOG00000028341 Alkbh5 ENSRNOG00000005470 Atxn3 ENSRNOG00000023216 Ccdc17 ENSRNOG00000010951 Cmtm6 ENSRNOG00000000008 Alx4 ENSRNOG00000007842 Aup1 ENSRNOG00000010846 Ccdc22 ENSRNOG00000010239 Cnbp ENSRNOG00000017422 Ambra1 ENSRNOG00000004479 Aurka ENSRNOG00000001930 Ccdc50 ENSRNOG00000030119 Cnga2 ENSRNOG00000000585 Amd1 ENSRNOG00000019156 Aurkaip1 ENSRNOG00000005102 Ccdc53 ENSRNOG00000011559 Cnn3 ENSRNOG00000006460 Amdhd2 ENSRNOG00000034287 Auts2l ENSRNOG00000031653 Ccdc58 ENSRNOG00000006207 Cno ENSRNOG00000008487 Amotl2 ENSRNOG00000006891 Avpr1b ENSRNOG00000013767 Ccdc61 ENSRNOG00000005326 Cnrip1

145

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000008270 Cntrob ENSRNOG00000017883 D3ZDC5_RAT ENSRNOG00000014619 D4A321_RAT ENSRNOG00000020772 Dhx8 ENSRNOG00000019918 Coasy ENSRNOG00000021503 D3ZDN1_RAT ENSRNOG00000028274 D4A352_RAT ENSRNOG00000002735 Dhx9 ENSRNOG00000017745 Cog4 ENSRNOG00000027250 D3ZE52_RAT ENSRNOG00000019254 D4A382_RAT ENSRNOG00000009125 Dis3 ENSRNOG00000015686 Col4a3bp ENSRNOG00000000262 D3ZEI3_RAT ENSRNOG00000017213 D4A3G2_RAT ENSRNOG00000026787 Disp2 ENSRNOG00000019615 COLQ_RAT ENSRNOG00000009219 D3ZEI4_RAT ENSRNOG00000028523 D4A3L5_RAT ENSRNOG00000006364 Dld ENSRNOG00000009281 Commd1 ENSRNOG00000010144 D3ZEP6_RAT ENSRNOG00000008338 D4A3M7_RAT ENSRNOG00000014011 Dll4 ENSRNOG00000004484 Commd5 ENSRNOG00000038448 D3ZF22_RAT ENSRNOG00000017151 D4A3V4_RAT ENSRNOG00000033085 DLP1_RAT ENSRNOG00000010538 Commd7 ENSRNOG00000024157 D3ZFA1_RAT ENSRNOG00000006982 D4A3W0_RAT ENSRNOG00000010905 Dlx5 ENSRNOG00000002320 Commd8 ENSRNOG00000023073 D3ZFA7_RAT ENSRNOG00000008445 D4A3W8_RAT ENSRNOG00000010835 Dmbx1 ENSRNOG00000013968 Comtd1 ENSRNOG00000025142 D3ZFL6_RAT ENSRNOG00000032312 D4A402_RAT ENSRNOG00000037923 Dmrtc1c ENSRNOG00000012000 Copb1 ENSRNOG00000012457 D3ZFQ8_RAT ENSRNOG00000037871 D4A441_RAT ENSRNOG00000005908 Dmtf1 ENSRNOG00000003315 Cops3 ENSRNOG00000012148 D3ZFR5_RAT ENSRNOG00000031485 D4A476_RAT ENSRNOG00000012106 Dnaja4 ENSRNOG00000023650 Cops4 ENSRNOG00000012651 D3ZFS5_RAT ENSRNOG00000023213 D4A494_RAT ENSRNOG00000001803 Dnajb11 ENSRNOG00000006499 Cops5 ENSRNOG00000007820 D3ZFX2_RAT ENSRNOG00000038100 D4A4A4_RAT ENSRNOG00000000130 Dnajb5 ENSRNOG00000001346 Cops6 ENSRNOG00000029863 D3ZFX8_RAT ENSRNOG00000025558 D4A4D8_RAT ENSRNOG00000008802 Dnajc11 ENSRNOG00000019635 Cops8 ENSRNOG00000009303 D3ZG58_RAT ENSRNOG00000037947 D4A4J6_RAT ENSRNOG00000006844 Dnajc14 ENSRNOG00000001171 Coq5 ENSRNOG00000014368 D3ZG60_RAT ENSRNOG00000034066 D4A4S3_RAT ENSRNOG00000009063 Dnajc15 ENSRNOG00000021828 Coro1b ENSRNOG00000039057 D3ZGU0_RAT ENSRNOG00000006600 D4A564_RAT ENSRNOG00000012368 Dnajc17 ENSRNOG00000014496 Coro6 ENSRNOG00000004604 D3ZH43_RAT ENSRNOG00000003028 D4A599_RAT ENSRNOG00000019995 Dnajc18 ENSRNOG00000002458 Cox11 ENSRNOG00000007281 D3ZHA0_RAT ENSRNOG00000030929 D4A5P1_RAT ENSRNOG00000004842 Dnajc24 ENSRNOG00000038951 Cox17 ENSRNOG00000040094 D3ZHC7_RAT ENSRNOG00000022876 D4A5Y4_RAT ENSRNOG00000031721 Dnajc30 ENSRNOG00000003052 Cox18 ENSRNOG00000039320 D3ZHZ1_RAT ENSRNOG00000014857 D4A6E8_RAT ENSRNOG00000033835 Dnm1 ENSRNOG00000001290 Cox19 ENSRNOG00000003935 D3ZI63_RAT ENSRNOG00000006028 D4A709_RAT ENSRNOG00000010625 Dnmt3b ENSRNOG00000017817 Cox4i1 ENSRNOG00000025680 D3ZID4_RAT ENSRNOG00000017443 D4A7B6_RAT ENSRNOG00000004259 Dohh ENSRNOG00000001170 Cox6a1 ENSRNOG00000036726 D3ZIY9_RAT ENSRNOG00000008560 D4A7I7_RAT ENSRNOG00000038190 Dok6 ENSRNOG00000010807 Cox6c ENSRNOG00000038318 D3ZJ40_RAT ENSRNOG00000039674 D4A7L5_RAT ENSRNOG00000016989 Dolk ENSRNOG00000030237 Cox7c ENSRNOG00000023718 D3ZJI1_RAT ENSRNOG00000008405 D4A7N9_RAT ENSRNOG00000026431 Dopey2 ENSRNOG00000021177 Cox8a ENSRNOG00000018877 D3ZJW5_RAT ENSRNOG00000007024 D4A7X3_RAT ENSRNOG00000023612 Doxl1 ENSRNOG00000011913 Cp ENSRNOG00000022436 D3ZKA4_RAT ENSRNOG00000037811 D4A887_RAT ENSRNOG00000009799 Dpagt1 ENSRNOG00000010467 Cpa5 ENSRNOG00000016206 D3ZKE1_RAT ENSRNOG00000039874 D4A8E2_RAT ENSRNOG00000010993 Dpm1 ENSRNOG00000015118 Cpped1 ENSRNOG00000033366 D3ZKT3_RAT ENSRNOG00000000247 D4A9F2_RAT ENSRNOG00000018992 Dpysl3 ENSRNOG00000019712 Cpsf3l ENSRNOG00000016534 D3ZKX0_RAT ENSRNOG00000037016 D4A9L8_RAT ENSRNOG00000008996 Dpysl5 ENSRNOG00000005927 Cpsf6 ENSRNOG00000016505 D3ZKX8_RAT ENSRNOG00000037023 D4A9L8_RAT ENSRNOG00000000070 Dr1 ENSRNOG00000006534 Crbn ENSRNOG00000007532 D3ZKZ0_RAT ENSRNOG00000037071 D4A9L8_RAT ENSRNOG00000020527 Drap1 ENSRNOG00000012826 Creb3l2 ENSRNOG00000040303 D3ZL39_RAT ENSRNOG00000023869 D4A9V1_RAT ENSRNOG00000018590 Drg1 ENSRNOG00000014900 CREM_RAT ENSRNOG00000007811 D3ZLE4_RAT ENSRNOG00000028166 D4AA35_RAT ENSRNOG00000001681 Dscr3 ENSRNOG00000011145 Crhr2 ENSRNOG00000014664 D3ZLK3_RAT ENSRNOG00000029245 D4AA82_RAT ENSRNOG00000021298 Dstyk ENSRNOG00000015215 Cript ENSRNOG00000016834 D3ZLR5_RAT ENSRNOG00000027061 D4AAD0_RAT ENSRNOG00000008746 Dtd1 ENSRNOG00000006779 Crot ENSRNOG00000037608 D3ZMB3_RAT ENSRNOG00000015824 D4AB04_RAT ENSRNOG00000001432 Dtx2 ENSRNOG00000022421 Crtc1 ENSRNOG00000002336 D3ZMK4_RAT ENSRNOG00000039346 D4AB39_RAT ENSRNOG00000003977 Dusp1 ENSRNOG00000016138 Crtc2 ENSRNOG00000031326 D3ZMY0_RAT ENSRNOG00000010746 D4ABC0_RAT ENSRNOG00000017915 Dvl2 ENSRNOG00000007478 Cry2 ENSRNOG00000023290 D3ZN25_RAT ENSRNOG00000015847 D4ABE8_RAT ENSRNOG00000001708 Dvl3 ENSRNOG00000011573 Csad ENSRNOG00000011491 D3ZN27_RAT ENSRNOG00000024987 D4ABJ0_RAT ENSRNOG00000010434 Dync1li1 ENSRNOG00000007665 Cse1l ENSRNOG00000002270 D3ZN89_RAT ENSRNOG00000039322 D4ABL5_RAT ENSRNOG00000025791 Dync1li2 ENSRNOG00000019374 Csk ENSRNOG00000008392 D3ZNN0_RAT ENSRNOG00000022629 D4ABN1_RAT ENSRNOG00000001166 Dynll1 ENSRNOG00000017106 Csnk1a1 ENSRNOG00000002041 D3ZP61_RAT ENSRNOG00000008807 D4ABP8_RAT ENSRNOG00000018207 Dynlt1 ENSRNOG00000018529 Csnk1g2 ENSRNOG00000040240 D3ZPF8_RAT ENSRNOG00000033921 D4ABR3_RAT ENSRNOG00000006894 Dyt1 ENSRNOG00000011933 Csnk2a2 ENSRNOG00000008970 D3ZPN0_RAT ENSRNOG00000019352 D4ABX9_RAT ENSRNOG00000009224 ENSRNOG00000004775 Cstf1 ENSRNOG00000028785 D3ZPS9_RAT ENSRNOG00000009566 D4AC30_RAT ENSRNOG00000004398 E9PSK6_RAT ENSRNOG00000032945 Ctdspl ENSRNOG00000040214 D3ZQ01_RAT ENSRNOG00000010418 D4AC98_RAT ENSRNOG00000006440 E9PSL5_RAT ENSRNOG00000010658 Cth ENSRNOG00000040209 D3ZQ05_RAT ENSRNOG00000010483 D4ACD7_RAT ENSRNOG00000002452 E9PSP7_RAT ENSRNOG00000010336 Ctla4 ENSRNOG00000018132 D3ZQ35_RAT ENSRNOG00000016141 D4ACL0_RAT ENSRNOG00000030729 E9PSV0_RAT ENSRNOG00000016313 Ctnnbip1 ENSRNOG00000006051 D3ZQ54_RAT ENSRNOG00000011621 D4ACR0_RAT ENSRNOG00000030783 E9PSV0_RAT ENSRNOG00000004257 Ctps2 ENSRNOG00000037207 D3ZQC6_RAT ENSRNOG00000009662 D4ACX0_RAT ENSRNOG00000034001 E9PT18_RAT ENSRNOG00000017195 Ctr9 ENSRNOG00000028669 D3ZQF6_RAT ENSRNOG00000033733 D4ADM4_RAT ENSRNOG00000009252 E9PT51_RAT ENSRNOG00000015857 Ctsa ENSRNOG00000005884 D3ZQL1_RAT ENSRNOG00000039606 D4ADW9_RAT ENSRNOG00000018860 E9PTB3_RAT ENSRNOG00000020206 Ctsd ENSRNOG00000015226 D3ZR49_RAT ENSRNOG00000039607 D4ADW9_RAT ENSRNOG00000004104 E9PTG0_RAT ENSRNOG00000019708 Ctsf ENSRNOG00000031689 D3ZRI0_RAT ENSRNOG00000039665 D4ADW9_RAT ENSRNOG00000010673 E9PTG2_RAT ENSRNOG00000020647 Ctsg ENSRNOG00000007244 D3ZRX0_RAT ENSRNOG00000010298 D4AE14_RAT ENSRNOG00000030247 E9PTK9_RAT ENSRNOG00000023661 Cugbp2 ENSRNOG00000028817 D3ZRY5_RAT ENSRNOG00000010125 D4AE49_RAT ENSRNOG00000004106 E9PTM2_RAT ENSRNOG00000015292 Cul2 ENSRNOG00000003505 D3ZSJ9_RAT ENSRNOG00000013434 D4AE98_RAT ENSRNOG00000006576 E9PTV4_RAT ENSRNOG00000019649 Cul4a ENSRNOG00000032243 D3ZSP9_RAT ENSRNOG00000039426 D4AEB1_RAT ENSRNOG00000024025 Edc4 ENSRNOG00000000481 Cuta ENSRNOG00000029623 D3ZT50_RAT ENSRNOG00000039411 D4AEJ2_RAT ENSRNOG00000014361 Edn1 ENSRNOG00000002174 Cwh43 ENSRNOG00000038119 D3ZT53_RAT ENSRNOG00000039400 D4AEM2_RAT ENSRNOG00000009390 Edn2 ENSRNOG00000002792 Cxcl2 ENSRNOG00000025129 D3ZT77_RAT ENSRNOG00000020373 Dap3 ENSRNOG00000009439 Eef1a1 ENSRNOG00000003866 Cxcr4 ENSRNOG00000030116 D3ZT82_RAT ENSRNOG00000003743 Dars ENSRNOG00000012477 Eef1a2 ENSRNOG00000019622 Cxcr7 ENSRNOG00000014872 D3ZTC8_RAT ENSRNOG00000002813 Dars2 ENSRNOG00000024186 Eef1b2 ENSRNOG00000011142 Cyb5b ENSRNOG00000036635 D3ZTM8_RAT ENSRNOG00000007771 Dbil5 ENSRNOG00000016390 Eef1e1 ENSRNOG00000024553 Cyb5d2 ENSRNOG00000009778 D3ZTP3_RAT ENSRNOG00000026974 Dbndd1 ENSRNOG00000006931 Eepd1 ENSRNOG00000009620 Cybrd1 ENSRNOG00000000922 D3ZTR4_RAT ENSRNOG00000012378 Dbnl ENSRNOG00000009782 Efcab7 ENSRNOG00000010452 Cycs ENSRNOG00000039120 D3ZU68_RAT ENSRNOG00000015029 Dbt ENSRNOG00000013783 Efhd2 ENSRNOG00000011541 Cygb ENSRNOG00000039130 D3ZU68_RAT ENSRNOG00000018825 Dcaf11 ENSRNOG00000032723 Eftud1 ENSRNOG00000018236 Cym ENSRNOG00000029678 D3ZUE7_RAT ENSRNOG00000016550 Dclk2 ENSRNOG00000020947 Egln2 ENSRNOG00000020035 Cyp17a1 ENSRNOG00000004032 D3ZUJ3_RAT ENSRNOG00000033026 Dclk3 ENSRNOG00000005053 Egln3 ENSRNOG00000019500 Cyp1a1 ENSRNOG00000027067 D3ZUN6_RAT ENSRNOG00000016015 Dcp1a ENSRNOG00000019422 Egr1 ENSRNOG00000011053 Cyp2u1 ENSRNOG00000039200 D3ZV53_RAT ENSRNOG00000025481 Dctn2 ENSRNOG00000000640 Egr2 ENSRNOG00000029478 Cyp4f39 ENSRNOG00000022561 D3ZV58_RAT ENSRNOG00000014208 Dctn3 ENSRNOG00000011346 Ehd2 ENSRNOG00000001303 Cytsa ENSRNOG00000009339 D3ZV60_RAT ENSRNOG00000014431 Dctn3l1 ENSRNOG00000011370 Ehd2 ENSRNOG00000006557 D3Z8C3_RAT ENSRNOG00000027762 D3ZWM6_RAT ENSRNOG00000018048 Dctn5 ENSRNOG00000007242 Ehmt1 ENSRNOG00000002291 D3Z8C5_RAT ENSRNOG00000018621 D3ZWP2_RAT ENSRNOG00000014037 Dcun1d3 ENSRNOG00000019310 Eid2 ENSRNOG00000025885 D3Z8J4_RAT ENSRNOG00000008455 D3ZWX1_RAT ENSRNOG00000032104 Dcx ENSRNOG00000018848 Eif1b ENSRNOG00000026880 D3Z8K5_RAT ENSRNOG00000014890 D3ZXF8_RAT ENSRNOG00000026542 Dd25 ENSRNOG00000005804 Eif3d ENSRNOG00000002883 D3Z8M7_RAT ENSRNOG00000036686 D3ZXH8_RAT ENSRNOG00000039417 Dda1 ENSRNOG00000027690 Eif3e ENSRNOG00000011908 D3Z8N9_RAT ENSRNOG00000010106 D3ZXJ6_RAT ENSRNOG00000020715 Ddb1 ENSRNOG00000011020 Eif3s6ip ENSRNOG00000012264 D3Z8W2_RAT ENSRNOG00000024245 D3ZXU0_RAT ENSRNOG00000009481 Ddhd1 ENSRNOG00000030628 Eif4a1 ENSRNOG00000017047 D3Z914_RAT ENSRNOG00000001194 D3ZY39_RAT ENSRNOG00000000577 Ddit4 ENSRNOG00000010103 Eif4b ENSRNOG00000014105 D3Z942_RAT ENSRNOG00000029190 D3ZYH3_RAT ENSRNOG00000001239 Ddt ENSRNOG00000000555 Eif4ebp2 ENSRNOG00000015294 D3Z9U1_RAT ENSRNOG00000033342 D3ZYL6_RAT ENSRNOG00000006652 Ddx1 ENSRNOG00000010218 Eif5 ENSRNOG00000021954 D3Z9Z3_RAT ENSRNOG00000015426 D3ZYS1_RAT ENSRNOG00000012500 Ddx10 ENSRNOG00000023601 Elavl4 ENSRNOG00000004033 D3ZAG0_RAT ENSRNOG00000031974 D3ZZG0_RAT ENSRNOG00000008081 Ddx27 ENSRNOG00000019824 Ell ENSRNOG00000012323 D3ZAX5_RAT ENSRNOG00000012552 D3ZZQ6_RAT ENSRNOG00000030680 Ddx5 ENSRNOG00000027089 Ell2 ENSRNOG00000018066 D3ZB65_RAT ENSRNOG00000021157 D3ZZR3_RAT ENSRNOG00000000396 Ddx50 ENSRNOG00000018747 Elmo2 ENSRNOG00000009829 D3ZBB1_RAT ENSRNOG00000012655 D4A065_RAT ENSRNOG00000017960 Deaf1 ENSRNOG00000014284 Elof1 ENSRNOG00000038372 D3ZBB9_RAT ENSRNOG00000003464 D4A0C3_RAT ENSRNOG00000036901 Defb26 ENSRNOG00000010468 Elovl6 ENSRNOG00000026481 D3ZBJ9_RAT ENSRNOG00000003538 D4A0C5_RAT ENSRNOG00000038760 Defb41 ENSRNOG00000008246 Emilin1 ENSRNOG00000000316 D3ZBN6_RAT ENSRNOG00000014794 D4A0Q5_RAT ENSRNOG00000038149 Defb9 ENSRNOG00000006846 En2 ENSRNOG00000009193 D3ZBP7_RAT ENSRNOG00000029955 D4A151_RAT ENSRNOG00000011716 Degs2 ENSRNOG00000016541 Enc1 ENSRNOG00000013218 D3ZC08_RAT ENSRNOG00000039819 D4A170_RAT ENSRNOG00000025030 Dffb ENSRNOG00000013141 Eno2 ENSRNOG00000031668 D3ZC45_RAT ENSRNOG00000039839 D4A170_RAT ENSRNOG00000000283 Dgcr14 ENSRNOG00000002262 Enoph1 ENSRNOG00000024867 D3ZC60_RAT ENSRNOG00000038473 D4A1Z9_RAT ENSRNOG00000026705 Dgki ENSRNOG00000016883 Entpd7 ENSRNOG00000030639 D3ZCY2_RAT ENSRNOG00000037131 D4A295_RAT ENSRNOG00000011617 Dguok ENSRNOG00000009239 Entpd8 ENSRNOG00000017657 D3ZCY7_RAT ENSRNOG00000014245 D4A2I2_RAT ENSRNOG00000020883 Dhdh ENSRNOG00000013213 Epha4 ENSRNOG00000000275 D3ZD52_RAT ENSRNOG00000003758 D4A2Z9_RAT ENSRNOG00000003844 Dhx15 ENSRNOG00000015753 Epn1 ENSRNOG00000003729 D3ZDC1_RAT ENSRNOG00000033722 D4A306_RAT ENSRNOG00000022171 Dhx37 ENSRNOG00000014248 ERBB4_RAT

146

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000013180 Ercc3 ENSRNOG00000023968 F1M0R6_RAT ENSRNOG00000000066 Fbxo28 ENSRNOG00000018237 Gstp1 ENSRNOG00000011448 Eri1 ENSRNOG00000024503 F1M0U5_RAT ENSRNOG00000014852 Fbxo30 ENSRNOG00000028188 GSTT2_RAT ENSRNOG00000012911 Erlin1 ENSRNOG00000039202 F1M126_RAT ENSRNOG00000001126 Fbxw8 ENSRNOG00000001242 Gstt3 ENSRNOG00000014031 Esco1 ENSRNOG00000038161 F1M174_RAT ENSRNOG00000039415 Fchsd1 ENSRNOG00000029316 Gtf2f2 ENSRNOG00000024717 Espnl ENSRNOG00000014806 F1M1E4_RAT ENSRNOG00000019319 Fchsd2 ENSRNOG00000029326 Gtf2f2 ENSRNOG00000005343 Esr2 ENSRNOG00000038124 F1M1E9_RAT ENSRNOG00000003135 Fcrlb ENSRNOG00000012360 Gtf2h1 ENSRNOG00000009437 Ewsr1 ENSRNOG00000038111 F1M1F7_RAT ENSRNOG00000021314 Fdft1 ENSRNOG00000018230 Gtf2h2 ENSRNOG00000003876 Exo1 ENSRNOG00000038817 F1M1P4_RAT ENSRNOG00000010743 Fdxacb1 ENSRNOG00000018007 Gtf2h5 ENSRNOG00000009617 Exoc7 ENSRNOG00000012461 F1M1S6_RAT ENSRNOG00000007077 Fem1b ENSRNOG00000001479 Gtf2i ENSRNOG00000019766 Exoc8 ENSRNOG00000011939 F1M1T6_RAT ENSRNOG00000006075 Fez1 ENSRNOG00000016218 Gtf3c1 ENSRNOG00000012348 Exosc4 ENSRNOG00000036771 F1M1U6_RAT ENSRNOG00000016225 Fgd3 ENSRNOG00000002091 Gtf3c3 ENSRNOG00000004413 Exosc7 ENSRNOG00000015626 F1M1V4_RAT ENSRNOG00000019855 Fgf23 ENSRNOG00000012784 Gtf3c4 ENSRNOG00000008944 Ext2 ENSRNOG00000039181 F1M1W5_RAT ENSRNOG00000011471 Fgf9 ENSRNOG00000005408 Gtlf3b ENSRNOG00000018524 Ezr ENSRNOG00000034029 F1M1X4_RAT ENSRNOG00000016050 Fgfr1 ENSRNOG00000016217 Gtpbp4 ENSRNOG00000014353 F1LN31_RAT ENSRNOG00000028866 F1M2F6_RAT ENSRNOG00000025074 Fgg ENSRNOG00000008418 Gtpbp5 ENSRNOG00000009419 F1LND3_RAT ENSRNOG00000028871 F1M2F6_RAT ENSRNOG00000000875 Fhl1 ENSRNOG00000016148 Gtse1 ENSRNOG00000011799 F1LNU5_RAT ENSRNOG00000022780 F1M2G5_RAT ENSRNOG00000002275 Fip1l1 ENSRNOG00000037934 GUAA_RAT ENSRNOG00000027871 F1LPE3_RAT ENSRNOG00000038962 F1M360_RAT ENSRNOG00000028129 Fktn ENSRNOG00000015623 Guca1b ENSRNOG00000014076 F1LPR3_RAT ENSRNOG00000038993 F1M360_RAT ENSRNOG00000008904 Fli1 ENSRNOG00000027722 H1fx ENSRNOG00000005998 F1LSF2_RAT ENSRNOG00000039056 F1M360_RAT ENSRNOG00000022428 Flrt1 ENSRNOG00000010800 Hadhb ENSRNOG00000038552 F1LSY9_RAT ENSRNOG00000038961 F1M364_RAT ENSRNOG00000014462 Fnta ENSRNOG00000021029 Hamp ENSRNOG00000038584 F1LSY9_RAT ENSRNOG00000023765 F1M3A4_RAT ENSRNOG00000008015 Fos ENSRNOG00000014819 Hap1 ENSRNOG00000038605 F1LSY9_RAT ENSRNOG00000018845 F1M3G1_RAT ENSRNOG00000023497 Foxe1 ENSRNOG00000020493 Hapln4 ENSRNOG00000027776 F1LT81_RAT ENSRNOG00000023561 F1M3G8_RAT ENSRNOG00000009000 Foxj2 ENSRNOG00000023534 Haus8 ENSRNOG00000009032 F1LT96_RAT ENSRNOG00000037185 F1M3S4_RAT ENSRNOG00000010870 Foxn1 ENSRNOG00000031443 Havcr2 ENSRNOG00000009152 F1LTA2_RAT ENSRNOG00000033459 F1M3X4_RAT ENSRNOG00000007942 Fscn3 ENSRNOG00000020044 Hcca2 ENSRNOG00000022196 F1LTC9_RAT ENSRNOG00000030145 F1M3Z2_RAT ENSRNOG00000011631 Fst ENSRNOG00000020444 Hcn3 ENSRNOG00000038768 F1LTD9_RAT ENSRNOG00000005788 F1M4B7_RAT ENSRNOG00000006565 Fstl4 ENSRNOG00000019618 HDAC3_RAT ENSRNOG00000000599 F1LTF8_RAT ENSRNOG00000039784 F1M4F0_RAT ENSRNOG00000022619 Fth1 ENSRNOG00000021686 Heatr1 ENSRNOG00000023070 F1LTG9_RAT ENSRNOG00000032210 F1M4J8_RAT ENSRNOG00000024066 Fundc2 ENSRNOG00000012466 Heca ENSRNOG00000000662 F1LTK4_RAT ENSRNOG00000028955 F1M4R9_RAT ENSRNOG00000023360 Fus ENSRNOG00000006905 Hectd1 ENSRNOG00000022966 F1LTM2_RAT ENSRNOG00000030872 F1M4T0_RAT ENSRNOG00000009274 Fut11 ENSRNOG00000003213 Helz ENSRNOG00000034266 F1LTP5_RAT ENSRNOG00000040148 F1M4U8_RAT ENSRNOG00000011876 Fxr2 ENSRNOG00000015458 Hemk1 ENSRNOG00000032218 F1LTR6_RAT ENSRNOG00000012089 F1M4W7_RAT ENSRNOG00000021079 Fxyd1 ENSRNOG00000021097 HEPS_RAT ENSRNOG00000025898 F1LTS3_RAT ENSRNOG00000037136 F1M4X1_RAT ENSRNOG00000001452 Fzd9 ENSRNOG00000013718 Herc2 ENSRNOG00000027696 F1LTW7_RAT ENSRNOG00000021437 F1M566_RAT ENSRNOG00000006019 G0s2 ENSRNOG00000000381 Herc4 ENSRNOG00000025881 F1LU19_RAT ENSRNOG00000010354 F1M5H4_RAT ENSRNOG00000013186 G3bp1 ENSRNOG00000014133 Hesx1 ENSRNOG00000028920 F1LU73_RAT ENSRNOG00000020807 F1M5K2_RAT ENSRNOG00000001549 Gabpa ENSRNOG00000003203 Hexim1 ENSRNOG00000040171 F1LU89_RAT ENSRNOG00000037240 F1M5S6_RAT ENSRNOG00000010659 Gabpb1 ENSRNOG00000021287 Hexim2 ENSRNOG00000040206 F1LU89_RAT ENSRNOG00000024325 F1M6D8_RAT ENSRNOG00000002349 Gabra2 ENSRNOG00000016967 Hfe ENSRNOG00000038681 F1LUA3_RAT ENSRNOG00000037317 F1M6N2_RAT ENSRNOG00000002327 Gabrb1 ENSRNOG00000001863 Hic2 ENSRNOG00000038707 F1LUA3_RAT ENSRNOG00000019579 F1M6R5_RAT ENSRNOG00000003011 Gadd45gip1 ENSRNOG00000017372 Higd2a ENSRNOG00000038714 F1LUA3_RAT ENSRNOG00000028961 F1M750_RAT ENSRNOG00000015156 Gal ENSRNOG00000020835 Hipk4 ENSRNOG00000038724 F1LUA3_RAT ENSRNOG00000028971 F1M750_RAT ENSRNOG00000009712 Gale ENSRNOG00000018073 Hist1h1b ENSRNOG00000038747 F1LUA3_RAT ENSRNOG00000038814 F1M756_RAT ENSRNOG00000012671 Gan ENSRNOG00000027842 Hist1h2bd ENSRNOG00000000519 F1LUL1_RAT ENSRNOG00000037095 F1M7U3_RAT ENSRNOG00000019724 Ganab ENSRNOG00000021198 Hist1h2bh ENSRNOG00000029729 F1LUQ2_RAT ENSRNOG00000005323 F1M804_RAT ENSRNOG00000016364 Gba2 ENSRNOG00000029609 Hist1h2bl ENSRNOG00000033041 F1LUQ7_RAT ENSRNOG00000028470 F1M812_RAT ENSRNOG00000028768 Gbp4 ENSRNOG00000031790 Hist3h2bb ENSRNOG00000033044 F1LUQ7_RAT ENSRNOG00000009081 F1M8A2_RAT ENSRNOG00000011535 Gcsh ENSRNOG00000006116 Hk2 ENSRNOG00000000254 F1LUT1_RAT ENSRNOG00000021989 F1M8D1_RAT ENSRNOG00000015331 Gdf3 ENSRNOG00000026235 Hk3 ENSRNOG00000032784 F1LUV2_RAT ENSRNOG00000013421 F1M8Y6_RAT ENSRNOG00000037245 Gdi1 ENSRNOG00000016905 Hmg20a ENSRNOG00000028940 F1LUY3_RAT ENSRNOG00000015658 F1M8Z8_RAT ENSRNOG00000037265 Gdi1 ENSRNOG00000016122 Hmgcr ENSRNOG00000018429 F1LV33_RAT ENSRNOG00000036899 F1M904_RAT ENSRNOG00000037269 Gdi1 ENSRNOG00000028286 Hmgn1 ENSRNOG00000034178 F1LV59_RAT ENSRNOG00000037010 F1M970_RAT ENSRNOG00000018091 Gdi2 ENSRNOG00000033313 Hmgn2 ENSRNOG00000030704 F1LV74_RAT ENSRNOG00000039719 F1M992_RAT ENSRNOG00000002800 Gdpd2 ENSRNOG00000003773 Hmox2 ENSRNOG00000029020 F1LV75_RAT ENSRNOG00000013502 F1M9F5_RAT ENSRNOG00000008897 Gga1 ENSRNOG00000020637 Hmx3 ENSRNOG00000025470 F1LVB4_RAT ENSRNOG00000030019 F1M9H2_RAT ENSRNOG00000007351 Ggh ENSRNOG00000003661 Hn1 ENSRNOG00000038621 F1LVD6_RAT ENSRNOG00000024492 F1M9P0_RAT ENSRNOG00000016767 Ggps1 ENSRNOG00000026849 Hnrnph3 ENSRNOG00000038665 F1LVD6_RAT ENSRNOG00000039561 F1M9Q9_RAT ENSRNOG00000007782 Ghrh ENSRNOG00000020235 Hnrnpl ENSRNOG00000026228 F1LVG0_RAT ENSRNOG00000039542 F1M9S6_RAT ENSRNOG00000033338 Gimap6 ENSRNOG00000011910 Hnrnpr ENSRNOG00000005867 F1LVK5_RAT ENSRNOG00000038883 F1MAI9_RAT ENSRNOG00000003864 Gipc1 ENSRNOG00000020683 Hnrnpul1 ENSRNOG00000022823 F1LVR9_RAT ENSRNOG00000033667 F1MAN0_RAT ENSRNOG00000019369 Giyd2 ENSRNOG00000003399 Hnrph1 ENSRNOG00000003129 F1LVS9_RAT ENSRNOG00000002914 F1MAP8_RAT ENSRNOG00000009256 Gkn2 ENSRNOG00000007928 Hoxa13 ENSRNOG00000017951 F1LVV3_RAT ENSRNOG00000000814 Fabp7 ENSRNOG00000025120 Gli1 ENSRNOG00000007491 Hoxb13 ENSRNOG00000022467 F1LVY8_RAT ENSRNOG00000017607 Faf2 ENSRNOG00000007261 Gli2 ENSRNOG00000007823 Hoxb6 ENSRNOG00000020523 F1LW41_RAT ENSRNOG00000014727 Fahd1 ENSRNOG00000000541 Glo1 ENSRNOG00000028619 Hoxc8 ENSRNOG00000036780 F1LW47_RAT ENSRNOG00000013974 Fahd2a ENSRNOG00000004206 Glrx5 ENSRNOG00000005492 Hpcal1 ENSRNOG00000018698 F1LW69_RAT ENSRNOG00000004441 Faim3 ENSRNOG00000013023 Gltscr2 ENSRNOG00000005437 Hrsp12 ENSRNOG00000023563 F1LW79_RAT ENSRNOG00000027368 Fam100a ENSRNOG00000017838 Gmcl1 ENSRNOG00000014672 Hs3st6 ENSRNOG00000025961 F1LW87_RAT ENSRNOG00000010029 Fam100b ENSRNOG00000019482 Gnao1 ENSRNOG00000002212 Hsd17b13 ENSRNOG00000026407 F1LW90_RAT ENSRNOG00000002851 Fam104a ENSRNOG00000002390 Gnb2l1 ENSRNOG00000020949 Hsd17b14 ENSRNOG00000038247 F1LWA6_RAT ENSRNOG00000018212 Fam108a1 ENSRNOG00000025040 Gng10 ENSRNOG00000015840 Hsd17b4 ENSRNOG00000038315 F1LWA6_RAT ENSRNOG00000001834 Fam128b ENSRNOG00000015936 Gng5 ENSRNOG00000021732 Hsf1 ENSRNOG00000032689 F1LWP3_RAT ENSRNOG00000009163 Fam133b ENSRNOG00000009430 Gnl2 ENSRNOG00000001193 Hsf2bp ENSRNOG00000029584 F1LWP7_RAT ENSRNOG00000020384 Fam13b1 ENSRNOG00000037437 Golga3 ENSRNOG00000026963 Hsp90b1 ENSRNOG00000011231 F1LX29_RAT ENSRNOG00000019162 Fam158a ENSRNOG00000017956 Golga7 ENSRNOG00000010819 Hspa4l ENSRNOG00000007788 F1LX89_RAT ENSRNOG00000019684 Fam173a ENSRNOG00000024213 GOLI4_RAT ENSRNOG00000018294 Hspa5 ENSRNOG00000037324 F1LXD1_RAT ENSRNOG00000022347 Fam173b ENSRNOG00000020297 Gon4l ENSRNOG00000019525 Hspa9 ENSRNOG00000040323 F1LXG4_RAT ENSRNOG00000002165 Fam175a ENSRNOG00000016356 Got1 ENSRNOG00000029079 Hspb7 ENSRNOG00000019966 F1LXK0_RAT ENSRNOG00000014482 Fam178a ENSRNOG00000018969 Gpatch4 ENSRNOG00000014525 Hspd1 ENSRNOG00000034064 F1LXK3_RAT ENSRNOG00000004415 Fam179b ENSRNOG00000017702 Gpld1 ENSRNOG00000000902 Hsph1 ENSRNOG00000040294 F1LXM7_RAT ENSRNOG00000003704 Fam184b ENSRNOG00000007083 Gpn2 ENSRNOG00000027761 Htatsf1 ENSRNOG00000013016 F1LXN9_RAT ENSRNOG00000025545 Fam18a ENSRNOG00000027658 Gpr101 ENSRNOG00000022448 Htra2 ENSRNOG00000029308 F1LXR9_RAT ENSRNOG00000039807 Fam193b ENSRNOG00000023863 Gpr139 ENSRNOG00000011073 Htt ENSRNOG00000040018 F1LXY7_RAT ENSRNOG00000039528 Fam32a ENSRNOG00000014793 Gpr149 ENSRNOG00000005141 Hus1 ENSRNOG00000034090 F1LY44_RAT ENSRNOG00000004553 Fam36a ENSRNOG00000008603 Gpr25 ENSRNOG00000021750 Id1 ENSRNOG00000035079 F1LYA9_RAT ENSRNOG00000013913 Fam38a ENSRNOG00000039255 Gpr31 ENSRNOG00000026124 Id3 ENSRNOG00000030060 F1LYB4_RAT ENSRNOG00000018293 Fam40a ENSRNOG00000022176 Gpr75 ENSRNOG00000015020 Idh1 ENSRNOG00000028833 F1LYH0_RAT ENSRNOG00000010230 Fam45a ENSRNOG00000036834 Gpr84 ENSRNOG00000013949 Idh2 ENSRNOG00000037540 F1LYI7_RAT ENSRNOG00000017548 Fam53a ENSRNOG00000024636 Gpr85 ENSRNOG00000007316 Idh3B ENSRNOG00000037549 F1LYQ4_RAT ENSRNOG00000004532 Fam69b ENSRNOG00000026953 Gpr88 ENSRNOG00000016690 Idi1 ENSRNOG00000037534 F1LYR4_RAT ENSRNOG00000017513 Fam73b ENSRNOG00000008412 Gprc5a ENSRNOG00000002837 Ier2 ENSRNOG00000000708 F1LYU5_RAT ENSRNOG00000008271 Fam91a1 ENSRNOG00000018666 Gpsm1 ENSRNOG00000000827 Ier3 ENSRNOG00000039978 F1LZ68_RAT ENSRNOG00000017119 Fam96a ENSRNOG00000013604 Gpx4 ENSRNOG00000036604 Ifit2 ENSRNOG00000037936 F1LZD1_RAT ENSRNOG00000011865 Fam96b ENSRNOG00000021106 Gramd1a ENSRNOG00000016150 Ifrd2 ENSRNOG00000011515 F1LZI2_RAT ENSRNOG00000027249 Fancl ENSRNOG00000007682 Gria3 ENSRNOG00000008075 Ift74 ENSRNOG00000040222 F1LZK7_RAT ENSRNOG00000014119 Farsb ENSRNOG00000006957 Gria4 ENSRNOG00000016957 Igfbp2 ENSRNOG00000004912 F1LZX9_RAT ENSRNOG00000027422 Fastkd3 ENSRNOG00000006174 Grid2 ENSRNOG00000013456 Ighmbp2 ENSRNOG00000030250 F1LZZ2_RAT ENSRNOG00000020368 Fbxl12 ENSRNOG00000008992 Grik3 ENSRNOG00000030509 Igsf21 ENSRNOG00000025015 F1M007_RAT ENSRNOG00000000021 Fbxl14 ENSRNOG00000020310 Grik5 ENSRNOG00000007604 Igsf8 ENSRNOG00000030564 F1M022_RAT ENSRNOG00000019509 Fbxl15 ENSRNOG00000016999 Grp ENSRNOG00000016725 Ikbkap ENSRNOG00000031825 F1M083_RAT ENSRNOG00000005261 Fbxl5 ENSRNOG00000006593 Grpel1 ENSRNOG00000009204 Il17re ENSRNOG00000039096 F1M0C9_RAT ENSRNOG00000025497 Fbxl6 ENSRNOG00000029726 Gstm1 ENSRNOG00000017376 Il21 ENSRNOG00000030479 F1M0R5_RAT ENSRNOG00000012634 Fbxo10 ENSRNOG00000019221 Gstm4 ENSRNOG00000012259 Il22ra2

147

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000014269 Il8rb ENSRNOG00000016161 Lman2 ENSRNOG00000037857 LOC686393 ENSRNOG00000017137 Mettl10 ENSRNOG00000018993 Ilk ENSRNOG00000000230 Lmf1 ENSRNOG00000013802 LOC686689 ENSRNOG00000024809 Mettl11a ENSRNOG00000020115 Ilkap ENSRNOG00000009205 Lmo4 ENSRNOG00000034107 LOC686765 ENSRNOG00000025415 Mettl11b ENSRNOG00000005104 Imp5 ENSRNOG00000001593 Lnp ENSRNOG00000024964 LOC687994 ENSRNOG00000015984 Mfap1a ENSRNOG00000031965 Impdh2 ENSRNOG00000015597 LOC100125364 ENSRNOG00000037419 LOC688702 ENSRNOG00000015428 Mff ENSRNOG00000013480 Ing2 ENSRNOG00000030355 LOC100125385 ENSRNOG00000040092 LOC688786 ENSRNOG00000017510 Mfge8 ENSRNOG00000014320 Inhba ENSRNOG00000009958 LOC100151767 ENSRNOG00000039588 LOC688802 ENSRNOG00000014008 Mfsd2 ENSRNOG00000002439 Inhbb ENSRNOG00000037310 LOC100270669 ENSRNOG00000020118 LOC688903 ENSRNOG00000006378 Mga ENSRNOG00000024272 Ino80d ENSRNOG00000039399 LOC100359766 ENSRNOG00000037485 LOC688924 ENSRNOG00000004234 Mgat2 ENSRNOG00000019960 Ino80e ENSRNOG00000033058 LOC100359825 ENSRNOG00000020427 LOC688966 ENSRNOG00000003614 Mgat5 ENSRNOG00000007902 Inpp5b ENSRNOG00000031995 LOC100359861 ENSRNOG00000036641 LOC689065 ENSRNOG00000024954 Mgat5b ENSRNOG00000007903 Inpp5b ENSRNOG00000003192 LOC100360253 ENSRNOG00000036656 LOC689065 ENSRNOG00000031448 MGC114440 ENSRNOG00000019730 Inppl1 ENSRNOG00000019122 LOC100360302 ENSRNOG00000038807 LOC689425 ENSRNOG00000019519 MGC125086 ENSRNOG00000003576 Ints2 ENSRNOG00000030992 LOC100360329 ENSRNOG00000009138 LOC689589 ENSRNOG00000026672 MGC94199 ENSRNOG00000015153 Ints3 ENSRNOG00000027230 LOC100360334 ENSRNOG00000022030 LOC689656 ENSRNOG00000015983 MGC94335 ENSRNOG00000026005 Ints5 ENSRNOG00000002980 LOC100360406 ENSRNOG00000023725 LOC689756 ENSRNOG00000017071 MGC94542 ENSRNOG00000010556 Intu ENSRNOG00000019884 LOC100360446 ENSRNOG00000028416 LOC689959 ENSRNOG00000024780 MGC95210 ENSRNOG00000019932 Ip6k1 ENSRNOG00000003354 LOC100360473 ENSRNOG00000008485 LOC689963 ENSRNOG00000017822 Mgea5 ENSRNOG00000020361 Ip6k2 ENSRNOG00000033149 LOC100360477 ENSRNOG00000037323 LOC690041 ENSRNOG00000033189 Micb ENSRNOG00000019758 IPO13_RAT ENSRNOG00000032830 LOC100360507 ENSRNOG00000031946 LOC690138 ENSRNOG00000003176 Mixl1 ENSRNOG00000010427 Ipo7 ENSRNOG00000003650 LOC100360582 ENSRNOG00000025713 LOC690140 ENSRNOG00000025701 Mki67ip ENSRNOG00000016183 Ipp ENSRNOG00000033913 LOC100360642 ENSRNOG00000039489 LOC690368 ENSRNOG00000011524 Mkln1 ENSRNOG00000005965 Irak4 ENSRNOG00000037930 LOC100360645 ENSRNOG00000039507 LOC690368 ENSRNOG00000018938 Mkx ENSRNOG00000008144 Irf1 ENSRNOG00000001556 LOC100360986 ENSRNOG00000015914 LOC690372 ENSRNOG00000021725 Mlec ENSRNOG00000017414 Irf7 ENSRNOG00000013510 LOC100361056 ENSRNOG00000013437 LOC691083 ENSRNOG00000016589 Mlf2 ENSRNOG00000012742 Irx2 ENSRNOG00000029588 LOC100361231 ENSRNOG00000015271 LOC691135 ENSRNOG00000033809 Mlh1 ENSRNOG00000011180 Irx5 ENSRNOG00000011932 LOC100361269 ENSRNOG00000008911 LOC691317 ENSRNOG00000019983 Mlx ENSRNOG00000000701 Iscu ENSRNOG00000018354 LOC100361421 ENSRNOG00000036925 LOC691362 ENSRNOG00000014522 Mlycd ENSRNOG00000011402 Isg20l2 ENSRNOG00000007792 LOC100361513 ENSRNOG00000008053 LOC691889 ENSRNOG00000012622 Mmp15 ENSRNOG00000006199 Ispd ENSRNOG00000031802 LOC100361856 ENSRNOG00000026982 LOC691921 ENSRNOG00000017756 Mmp21 ENSRNOG00000010021 Isy1 ENSRNOG00000025515 LOC100361909 ENSRNOG00000037178 LOC691952 ENSRNOG00000027489 Mn1 ENSRNOG00000016271 Itm2b ENSRNOG00000005535 LOC100361923 ENSRNOG00000012164 LPPR2_RAT ENSRNOG00000002894 Mnt ENSRNOG00000002969 Itpkb ENSRNOG00000007758 LOC100362350 ENSRNOG00000019869 Lrfn1 ENSRNOG00000010996 Mobkl1a ENSRNOG00000002001 Itsn1 ENSRNOG00000024266 LOC100362495 ENSRNOG00000022726 Lrit2 ENSRNOG00000014692 Mogat1 ENSRNOG00000002618 Ivns1abp ENSRNOG00000000373 LOC100362857 ENSRNOG00000009313 Lrpap1 ENSRNOG00000011552 Mon1b ENSRNOG00000014630 Iws1 ENSRNOG00000016973 LOC100362950 ENSRNOG00000016128 Lrrc14 ENSRNOG00000004185 Mon2 ENSRNOG00000009149 Jam3 ENSRNOG00000004196 LOC100363173 ENSRNOG00000020080 Lrrc18 ENSRNOG00000019624 Morc2 ENSRNOG00000019729 Jmjd8 ENSRNOG00000037466 LOC100363238 ENSRNOG00000013642 Lrrc41 ENSRNOG00000014642 Morn1 ENSRNOG00000016379 Jtb ENSRNOG00000037468 LOC100363238 ENSRNOG00000009983 Lrrc42 ENSRNOG00000026111 Morn5 ENSRNOG00000026293 Jun ENSRNOG00000017695 LOC100363379 ENSRNOG00000019418 Lrrc4b ENSRNOG00000004863 Mpped2 ENSRNOG00000019568 Jund ENSRNOG00000024112 LOC100363819 ENSRNOG00000009143 Lrrc57 ENSRNOG00000021524 Mrap ENSRNOG00000019456 Kars ENSRNOG00000038122 LOC100363906 ENSRNOG00000025296 Lrrc8a ENSRNOG00000013426 Mrgprf ENSRNOG00000016058 Kazald1 ENSRNOG00000026569 LOC100364115 ENSRNOG00000021047 Lrrfip2 ENSRNOG00000026211 Mri1 ENSRNOG00000011550 Kcnab2 ENSRNOG00000039247 LOC100364244 ENSRNOG00000006093 LRRTM1 ENSRNOG00000036695 Mrpl12 ENSRNOG00000011380 Kcnc1 ENSRNOG00000000576 LOC100364587 ENSRNOG00000026466 Lrrtm3 ENSRNOG00000008566 Mrpl15 ENSRNOG00000004077 Kcnc2 ENSRNOG00000013191 LOC100364603 ENSRNOG00000021133 Lsm14a ENSRNOG00000014582 Mrpl18 ENSRNOG00000039544 Kcnd1 ENSRNOG00000003220 LOC100364712 ENSRNOG00000000838 Lta ENSRNOG00000018057 Mrpl2 ENSRNOG00000007705 Kcnj10 ENSRNOG00000012312 LOC100364770 ENSRNOG00000004494 Lta4h ENSRNOG00000003724 Mrpl27 ENSRNOG00000004713 Kcnj16 ENSRNOG00000018787 LOC100364844 ENSRNOG00000019264 Ltbr ENSRNOG00000009078 Mrpl37 ENSRNOG00000013463 Kcnj8 ENSRNOG00000033298 LOC100364868 ENSRNOG00000015217 Ltv1 ENSRNOG00000029875 Mrpl41 ENSRNOG00000019937 Kcnk1 ENSRNOG00000017753 LOC100364929 ENSRNOG00000020488 Luc7l ENSRNOG00000015231 Mrpl44 ENSRNOG00000015669 Kctd11 ENSRNOG00000019612 LOC100365011 ENSRNOG00000006001 Luc7l2 ENSRNOG00000018547 Mrpl46 ENSRNOG00000023764 Kctd2 ENSRNOG00000008382 LOC100365033 ENSRNOG00000005374 Lyar ENSRNOG00000011639 Mrpl47 ENSRNOG00000021082 Kdelr1 ENSRNOG00000033556 LOC100365546 ENSRNOG00000018534 Lyg1 ENSRNOG00000019165 Mrpl51 ENSRNOG00000019145 Kdm2a ENSRNOG00000032524 LOC100365568 ENSRNOG00000016200 Lzic ENSRNOG00000008477 Mrpl53 ENSRNOG00000020878 Keap1 ENSRNOG00000000217 LOC100365881 ENSRNOG00000009134 Mad2l2 ENSRNOG00000020869 mrpl9 ENSRNOG00000009918 KHDR1_RAT ENSRNOG00000010587 LOC100365941 ENSRNOG00000001277 Mafk ENSRNOG00000019949 Mrps12 ENSRNOG00000016715 Kif11 ENSRNOG00000022795 LOC292543 ENSRNOG00000012559 Man1b1 ENSRNOG00000006898 Mrps16 ENSRNOG00000001455 Kif13a ENSRNOG00000030584 LOC292801 ENSRNOG00000013476 Manba ENSRNOG00000019511 Mrps18a ENSRNOG00000030317 Kif1b ENSRNOG00000031539 LOC304725 ENSRNOG00000031453 Manbal ENSRNOG00000000804 Mrps18b ENSRNOG00000014000 Kif2a ENSRNOG00000025157 LOC310177 ENSRNOG00000017428 Map1b ENSRNOG00000010363 Mrps23 ENSRNOG00000010361 Kif3b ENSRNOG00000015222 LOC310721 ENSRNOG00000010552 Map2k1ip1 ENSRNOG00000032630 Mrps28 ENSRNOG00000011394 Kif3c ENSRNOG00000013203 LOC312273 ENSRNOG00000006612 Map2k3 ENSRNOG00000011839 Mrps31 ENSRNOG00000017466 Kif5b ENSRNOG00000015575 LOC312502 ENSRNOG00000005724 Map3k7 ENSRNOG00000020932 Ms4a10 ENSRNOG00000004680 Kif5c ENSRNOG00000008857 LOC314655 ENSRNOG00000016054 Map3k7ip2 ENSRNOG00000023021 Msl2 ENSRNOG00000014087 Kifc3 ENSRNOG00000007206 LOC361016 ENSRNOG00000010237 Map7d1 ENSRNOG00000039786 Msmb ENSRNOG00000026690 Kin ENSRNOG00000017758 LOC361985 ENSRNOG00000005176 Map7d2 ENSRNOG00000012440 Msra ENSRNOG00000002227 Kit ENSRNOG00000024346 LOC363060 ENSRNOG00000009381 Mapk6 ENSRNOG00000020357 Msto1 ENSRNOG00000005386 Kitlg ENSRNOG00000033227 LOC367050 ENSRNOG00000032828 Mapk8ip2 ENSRNOG00000006870 Mtdh ENSRNOG00000011572 Klc1 ENSRNOG00000033228 LOC367050 ENSRNOG00000001345 Mapkapk5 ENSRNOG00000004492 Mterfd1 ENSRNOG00000018168 Klc4 ENSRNOG00000022555 LOC497899 ENSRNOG00000011798 Mapre1 ENSRNOG00000025724 Mtf1 ENSRNOG00000015822 Klf13 ENSRNOG00000002116 LOC498555 ENSRNOG00000016472 March4 ENSRNOG00000000075 Mtf2 ENSRNOG00000014205 Klf2 ENSRNOG00000024221 LOC499465 ENSRNOG00000000579 Marcks ENSRNOG00000005602 Mthfd1 ENSRNOG00000016885 Klf6 ENSRNOG00000010896 LOC499749 ENSRNOG00000015648 Mars2 ENSRNOG00000002697 Mtmr1 ENSRNOG00000012961 Klf7 ENSRNOG00000022860 LOC499779 ENSRNOG00000014971 Mas1 ENSRNOG00000023985 Mtmr15 ENSRNOG00000010267 Klhdc10 ENSRNOG00000016220 LOC499782 ENSRNOG00000001827 Masp1 ENSRNOG00000007120 Mtmr3 ENSRNOG00000016921 Klhl11 ENSRNOG00000027955 LOC499900 ENSRNOG00000022753 Mast3 ENSRNOG00000011857 Mtpn ENSRNOG00000006515 Klhl15 ENSRNOG00000007224 LOC500034 ENSRNOG00000008049 Max ENSRNOG00000009001 Mtss1 ENSRNOG00000020302 Klhl17 ENSRNOG00000026958 LOC500077 ENSRNOG00000024104 Mbd1 ENSRNOG00000017500 Mtss1l ENSRNOG00000010959 Klhl25 ENSRNOG00000030522 LOC500331 ENSRNOG00000006209 Mbd6 ENSRNOG00000016010 Mul1 ENSRNOG00000020105 Klhl30 ENSRNOG00000021765 LOC500893 ENSRNOG00000001357 Mblac1 ENSRNOG00000033208 Mup4 ENSRNOG00000012061 KPCB_RAT ENSRNOG00000018559 LOC501180 ENSRNOG00000015173 Mbtps1 ENSRNOG00000033567 Musk ENSRNOG00000011628 Krt27 ENSRNOG00000036666 LOC619574 ENSRNOG00000010539 Mcat ENSRNOG00000013376 Mvd ENSRNOG00000008057 Krt7 ENSRNOG00000001724 LOC678704 ENSRNOG00000018895 MCHR1_RAT ENSRNOG00000020182 Mvp ENSRNOG00000014370 Krt9 ENSRNOG00000015229 LOC678741 ENSRNOG00000001272 Mcm3ap ENSRNOG00000001959 Mx1 ENSRNOG00000036838 Krtap31‐1 ENSRNOG00000021944 LOC679690 ENSRNOG00000000975 Mcoln1 ENSRNOG00000034078 Mxi1 ENSRNOG00000019629 LAMP1_RAT ENSRNOG00000010184 LOC679934 ENSRNOG00000000618 Mdga2 ENSRNOG00000016588 Myadm ENSRNOG00000006810 Lancl2 ENSRNOG00000013069 LOC680531 ENSRNOG00000012891 Mdh1b ENSRNOG00000015236 Mybbp1a ENSRNOG00000004760 Lars2 ENSRNOG00000039749 LOC680885 ENSRNOG00000001440 Mdh2 ENSRNOG00000004500 Myc ENSRNOG00000013082 LCAP_RAT ENSRNOG00000002036 LOC681004 ENSRNOG00000009696 Mdm4 ENSRNOG00000010479 Mycbp2 ENSRNOG00000013000 Ldhb ENSRNOG00000021373 LOC681219 ENSRNOG00000019840 Mdp1 ENSRNOG00000037402 Myeov2 ENSRNOG00000033503 Ldlrad2 ENSRNOG00000005934 LOC681849 ENSRNOG00000012645 Mecom ENSRNOG00000020955 Myl3 ENSRNOG00000038794 Leng4 ENSRNOG00000002463 LOC682752 ENSRNOG00000005606 Med1 ENSRNOG00000004072 Myo1c ENSRNOG00000006059 Lep ENSRNOG00000031332 LOC682793 ENSRNOG00000003792 Med14 ENSRNOG00000014815 Myoz2 ENSRNOG00000001250 Lfng ENSRNOG00000031512 LOC684745 ENSRNOG00000013422 Med23 ENSRNOG00000004925 MYPT1_RAT ENSRNOG00000007089 Lgmn ENSRNOG00000025365 LOC684861 ENSRNOG00000020363 Med25 ENSRNOG00000022664 Myst2 ENSRNOG00000005715 Lgr4 ENSRNOG00000024505 LOC684993 ENSRNOG00000012270 Med26 ENSRNOG00000017346 Myt1 ENSRNOG00000034076 Lhfpl3 ENSRNOG00000014480 LOC685079 ENSRNOG00000013933 Med27 ENSRNOG00000001108 N4bp2l2 ENSRNOG00000007727 Lhfpl4 ENSRNOG00000014410 LOC685572 ENSRNOG00000013674 Megf10 ENSRNOG00000001350 Naa25 ENSRNOG00000010357 Lhx9 ENSRNOG00000039841 LOC685669 ENSRNOG00000020498 Megf8 ENSRNOG00000033396 Naaladl2 ENSRNOG00000002203 Lin54 ENSRNOG00000030545 LOC685793 ENSRNOG00000005932 Megf9 ENSRNOG00000020736 Nadsyn1 ENSRNOG00000005131 Lin7c ENSRNOG00000005533 LOC686013 ENSRNOG00000004606 Meis1 ENSRNOG00000008307 Nanp ENSRNOG00000021213 Lix1l ENSRNOG00000019266 LOC686289 ENSRNOG00000004730 Meis2 ENSRNOG00000021553 Nap5 ENSRNOG00000003948 Llgl1 ENSRNOG00000033330 LOC686314 ENSRNOG00000013598 Melk ENSRNOG00000001494 Napa

148

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000004753 Napb ENSRNOG00000029053 Olr1237 ENSRNOG00000012012 Pinx1 ENSRNOG00000020979 Psmb4 ENSRNOG00000019854 Napsa ENSRNOG00000029633 Olr1340 ENSRNOG00000021068 Pip5k1a ENSRNOG00000012026 Psmc2 ENSRNOG00000036664 Narf ENSRNOG00000030290 Olr1409 ENSRNOG00000000665 Pitpnb ENSRNOG00000011414 Psmc3 ENSRNOG00000019522 Narfl ENSRNOG00000038413 Olr1433 ENSRNOG00000015683 Pitpnc1 ENSRNOG00000018994 Psmc4 ENSRNOG00000014192 Nat12 ENSRNOG00000038446 Olr1433 ENSRNOG00000016511 Pitrm1 ENSRNOG00000017730 Psmd1 ENSRNOG00000007280 Nat15 ENSRNOG00000032324 Olr1461 ENSRNOG00000010681 Pitx2 ENSRNOG00000003117 Psmd12 ENSRNOG00000023605 Ncbp1 ENSRNOG00000032366 Olr1461 ENSRNOG00000011329 Pkm2 ENSRNOG00000014109 Psmd13 ENSRNOG00000018273 Ncl ENSRNOG00000022244 Olr1462 ENSRNOG00000025892 Pkn3 ENSRNOG00000018809 Psmd5 ENSRNOG00000018288 Ncoa6 ENSRNOG00000033537 Olr1640 ENSRNOG00000001825 Pkp2 ENSRNOG00000006751 Psmd6 ENSRNOG00000001004 Ncor2 ENSRNOG00000039434 Olr1645 ENSRNOG00000019859 Pla2g15 ENSRNOG00000036806 Psme3 ENSRNOG00000004139 Ndel1 ENSRNOG00000031007 Olr347 ENSRNOG00000016945 Pla2g2a ENSRNOG00000006609 Psme4 ENSRNOG00000024022 Ndfip2 ENSRNOG00000032117 Olr384 ENSRNOG00000007447 Pla2g4b ENSRNOG00000000925 Psph ENSRNOG00000016489 Ndnl2 ENSRNOG00000007954 Olr417 ENSRNOG00000010086 Plagl2 ENSRNOG00000016413 Pstpip1 ENSRNOG00000010616 Ndor1 ENSRNOG00000032273 Olr436 ENSRNOG00000014276 Plce1 ENSRNOG00000013231 Ptafr ENSRNOG00000014224 Ndufa3 ENSRNOG00000009624 Olr462 ENSRNOG00000016340 Plcg1 ENSRNOG00000010827 Ptbp2 ENSRNOG00000008569 Ndufa6 ENSRNOG00000033774 Olr594 ENSRNOG00000037160 Pldn ENSRNOG00000009484 Ptcd3 ENSRNOG00000006939 Ndufa7 ENSRNOG00000032256 Olr832 ENSRNOG00000005214 Plek ENSRNOG00000019354 Ptch1 ENSRNOG00000018129 Ndufab1 ENSRNOG00000032760 Olr865 ENSRNOG00000019247 Plekhj1 ENSRNOG00000020723 Pten ENSRNOG00000014568 Ndufb10 ENSRNOG00000030573 Olr932 ENSRNOG00000028521 Plekhm1 ENSRNOG00000031535 Ptgdr ENSRNOG00000009364 Ndufb9 ENSRNOG00000031369 Olr954 ENSRNOG00000011951 Plk2 ENSRNOG00000031307 Ptgdrl ENSRNOG00000009236 Necap1 ENSRNOG00000014107 Omg ENSRNOG00000027914 Plscr3 ENSRNOG00000002642 Ptges3 ENSRNOG00000008427 Necap2 ENSRNOG00000025890 Opa3 ENSRNOG00000005358 Pmm1 ENSRNOG00000018584 Ptma ENSRNOG00000006683 Nedd4 ENSRNOG00000022386 Opalin ENSRNOG00000020318 Pnkp ENSRNOG00000020358 Ptov1 ENSRNOG00000021410 Negr1 ENSRNOG00000023809 Opcml ENSRNOG00000016863 Pnmal2 ENSRNOG00000013415 Ptpn18 ENSRNOG00000007001 Nek9 ENSRNOG00000003059 Optc ENSRNOG00000007793 Pnrc1 ENSRNOG00000017453 Ptpn2 ENSRNOG00000018681 NEST_RAT ENSRNOG00000005021 Orc4l ENSRNOG00000016255 Podxl2 ENSRNOG00000020862 Ptpn23 ENSRNOG00000017994 Nfatc2ip ENSRNOG00000009333 Osgep ENSRNOG00000019150 Polb ENSRNOG00000017600 Ptpn9 ENSRNOG00000018618 Nfatc3 ENSRNOG00000004001 Osgepl1 ENSRNOG00000018765 Pold4 ENSRNOG00000004483 Ptprr ENSRNOG00000009795 Nfib ENSRNOG00000009117 Otub2 ENSRNOG00000015392 Pole3 ENSRNOG00000005277 Ptprv ENSRNOG00000002983 Nfix ENSRNOG00000016950 Otud1 ENSRNOG00000013728 Polg2 ENSRNOG00000006030 Ptprz1 ENSRNOG00000019907 Nfkbie ENSRNOG00000006636 Otud6b ENSRNOG00000019195 Polh ENSRNOG00000009960 Puf60 ENSRNOG00000014703 Nfkbil2 ENSRNOG00000013136 Oxsr1 ENSRNOG00000002471 Polq ENSRNOG00000019062 Pura ENSRNOG00000018069 Ngdn ENSRNOG00000004904 Pa2g4 ENSRNOG00000012664 Polr1e ENSRNOG00000037500 Pus1 ENSRNOG00000028822 Ngfrap1 ENSRNOG00000022267 Pabpc1l2b ENSRNOG00000015720 Polr2c ENSRNOG00000037512 Pus1 ENSRNOG00000028830 Ngfrap1 ENSRNOG00000014527 Pabpc2 ENSRNOG00000016231 Polr2d ENSRNOG00000010572 Pus7 ENSRNOG00000013553 NGRN_RAT ENSRNOG00000017367 Pabpc3 ENSRNOG00000010008 Polr3a ENSRNOG00000037446 Pxmp2 ENSRNOG00000018162 Nhej1 ENSRNOG00000014092 Paip2b ENSRNOG00000010028 Polr3d ENSRNOG00000016975 Pxmp4 ENSRNOG00000016948 Nhlrc2 ENSRNOG00000005509 Pak7 ENSRNOG00000007548 Polr3f ENSRNOG00000007583 Pygb ENSRNOG00000039113 Nipsnap3a ENSRNOG00000025126 Palb2 ENSRNOG00000016260 Polr3g ENSRNOG00000006005 Q566D1_RAT ENSRNOG00000018823 Nisch ENSRNOG00000016508 Palmd ENSRNOG00000017843 Polr3k ENSRNOG00000023456 Q5XIR0_RAT ENSRNOG00000031834 Nkain4 ENSRNOG00000018944 Pank1 ENSRNOG00000024879 Polrmt ENSRNOG00000020011 Q66HF5_RAT ENSRNOG00000018095 Nkiras2 ENSRNOG00000007419 Pank3 ENSRNOG00000033343 POLS2_RAT ENSRNOG00000039396 Q6MG96_RAT ENSRNOG00000008644 Nkx2‐1 ENSRNOG00000002035 Paqr3 ENSRNOG00000001442 Por ENSRNOG00000030870 Q6QI20_RAT ENSRNOG00000015082 Nlrp10 ENSRNOG00000003721 Paqr4 ENSRNOG00000011500 Pou2af1 ENSRNOG00000030298 Q6QI85_RAT ENSRNOG00000014812 Nlrp12 ENSRNOG00000032374 Paqr9 ENSRNOG00000038909 Pou3f3 ENSRNOG00000023687 Q78P65_RAT ENSRNOG00000008771 Nlrx1 ENSRNOG00000032437 Pard3 ENSRNOG00000038936 Pou3f3 ENSRNOG00000003253 Qdpr ENSRNOG00000002693 Nme1 ENSRNOG00000017675 Pard6g ENSRNOG00000002784 Pou3f4 ENSRNOG00000015413 Qpctl ENSRNOG00000020721 Nme6 ENSRNOG00000023271 PARL_RAT ENSRNOG00000012167 Pou4f2 ENSRNOG00000016980 Qprt ENSRNOG00000002989 Nmt1 ENSRNOG00000008892 Parp2 ENSRNOG00000018842 Pou4f3 ENSRNOG00000011302 Rab11a ENSRNOG00000021890 Nob1 ENSRNOG00000007327 Pars2 ENSRNOG00000001269 PP1G_RAT ENSRNOG00000018972 Rab18 ENSRNOG00000013965 Noc3l ENSRNOG00000008826 Pax9 ENSRNOG00000000558 Ppa1 ENSRNOG00000003923 Rab21 ENSRNOG00000014818 Nol8 ENSRNOG00000000566 Pcbd1 ENSRNOG00000010068 Ppapdc3 ENSRNOG00000005762 Rab22a ENSRNOG00000018704 Nolc1 ENSRNOG00000017708 Pcbp1 ENSRNOG00000016781 Ppib ENSRNOG00000005963 Rab2a ENSRNOG00000017284 Nop16 ENSRNOG00000001245 Pcbp3 ENSRNOG00000027408 Ppid ENSRNOG00000006698 Rab33a ENSRNOG00000018453 Nop2 ENSRNOG00000012367 Pcdh7 ENSRNOG00000007673 Ppig ENSRNOG00000022014 Rab35 ENSRNOG00000007128 Nop56 ENSRNOG00000030352 Pcdhga9 ENSRNOG00000015552 Ppil4 ENSRNOG00000036661 Rab40b ENSRNOG00000001130 Nos1 ENSRNOG00000010155 Pcm1 ENSRNOG00000004216 Ppil5 ENSRNOG00000017467 Rab4a ENSRNOG00000020543 Nosip ENSRNOG00000006280 Pcsk9 ENSRNOG00000024730 Ppm1e ENSRNOG00000018176 Rab6a ENSRNOG00000008697 Nov ENSRNOG00000016704 Pcyox1 ENSRNOG00000004314 PPM1H_RAT ENSRNOG00000009198 Rab6b ENSRNOG00000031440 Nova1 ENSRNOG00000008484 Pdcl ENSRNOG00000003567 Ppox ENSRNOG00000018009 Rab8b ENSRNOG00000007640 Npbwr1 ENSRNOG00000002169 Pdcl2 ENSRNOG00000018708 Ppp1ca ENSRNOG00000030443 Rab9a ENSRNOG00000013895 Npdc1 ENSRNOG00000021655 Pddc1 ENSRNOG00000000780 Ppp1r11 ENSRNOG00000002437 Rab9b ENSRNOG00000004616 Npm1 ENSRNOG00000013264 Pde12 ENSRNOG00000027959 Ppp1r12c ENSRNOG00000018591 Rabepk ENSRNOG00000016156 Nptxr ENSRNOG00000006154 Pde1a ENSRNOG00000016368 Ppp1r14c ENSRNOG00000013947 Rabl2b ENSRNOG00000009768 Npy ENSRNOG00000025042 Pde3a ENSRNOG00000028493 Ppp1r15b ENSRNOG00000031346 Rabl5 ENSRNOG00000012772 Nqo1 ENSRNOG00000019518 Pde4c ENSRNOG00000008869 Ppp1r9a ENSRNOG00000013461 Ralbp1 ENSRNOG00000010308 Nr2f2 ENSRNOG00000013048 Pde7a ENSRNOG00000015182 Ppp2cb ENSRNOG00000018000 Ranbp10 ENSRNOG00000005600 Nr4a2 ENSRNOG00000010280 Pde8b ENSRNOG00000000068 Ppp2r5a ENSRNOG00000014420 Rap2b ENSRNOG00000005964 Nr4a3 ENSRNOG00000001312 Pdgfa ENSRNOG00000004973 Ppp2r5c ENSRNOG00000021581 Rapgef2 ENSRNOG00000024722 NRX3A_RAT ENSRNOG00000017197 Pdgfb ENSRNOG00000012616 Ppt1 ENSRNOG00000029185 Rasa1 ENSRNOG00000017254 Nsun2 ENSRNOG00000007895 Pdhb ENSRNOG00000021440 Pptc7 ENSRNOG00000004917 Rasal2 ENSRNOG00000023720 Ntm ENSRNOG00000005632 Pdzrn3 ENSRNOG00000017204 PQLC1_RAT ENSRNOG00000033744 Rasgrp4 ENSRNOG00000018674 Ntrk3 ENSRNOG00000006854 Pea15a ENSRNOG00000005126 Pqlc3 ENSRNOG00000016611 RASH_RAT ENSRNOG00000024929 Nudcd1 ENSRNOG00000013972 Pef1 ENSRNOG00000010630 Prcp ENSRNOG00000007972 RBBP9_RAT ENSRNOG00000003225 Nudcd2 ENSRNOG00000019268 Pelp1 ENSRNOG00000003763 Prdx4 ENSRNOG00000019723 Rbm12 ENSRNOG00000029786 Nudt11 ENSRNOG00000005193 Pfas ENSRNOG00000007141 Preb ENSRNOG00000016925 Rbm24 ENSRNOG00000003224 Nudt16l1 ENSRNOG00000012985 Pfdn5 ENSRNOG00000012364 Prickle2 ENSRNOG00000005468 Rbm28 ENSRNOG00000012777 Nudt19 ENSRNOG00000000473 Pfdn6 ENSRNOG00000001142 Prkab1 ENSRNOG00000019848 Rbm39 ENSRNOG00000013110 Nudt2 ENSRNOG00000004162 Pfkfb2 ENSRNOG00000016434 Prkd2 ENSRNOG00000019669 Rbm4 ENSRNOG00000009094 Nudt4 ENSRNOG00000018911 Pfkfb3 ENSRNOG00000016616 Prl7a3 ENSRNOG00000010595 Rbm45 ENSRNOG00000017741 Nudt5 ENSRNOG00000003975 Pfn1 ENSRNOG00000012046 Prmt5 ENSRNOG00000018153 Rbm5 ENSRNOG00000020867 Numbl ENSRNOG00000037443 Pgam5 ENSRNOG00000017187 Prmt6 ENSRNOG00000021215 Rbm8a ENSRNOG00000017919 Nup133 ENSRNOG00000018798 PGCB_RAT ENSRNOG00000020900 Prodh2 ENSRNOG00000012138 Rbmxrtl ENSRNOG00000013411 Nup155 ENSRNOG00000009889 Pgm1 ENSRNOG00000018396 Prpf18 ENSRNOG00000026295 Rbpjl ENSRNOG00000025185 Nup188 ENSRNOG00000019639 Pgpep1 ENSRNOG00000009451 Prpf38a ENSRNOG00000007797 Rbpsuh ENSRNOG00000010852 Nup205 ENSRNOG00000014051 Pgrmc2 ENSRNOG00000004864 Prpf40a ENSRNOG00000019214 Rbx1 ENSRNOG00000003673 Nup85 ENSRNOG00000014459 Phax ENSRNOG00000003495 Prpf8 ENSRNOG00000015780 Rcn2 ENSRNOG00000012644 Nupl1 ENSRNOG00000012999 Phb2 ENSRNOG00000019744 Prr16 ENSRNOG00000029651 Rdh2 ENSRNOG00000018945 Nutf2 ENSRNOG00000005775 Phf14 ENSRNOG00000025806 Prr3 ENSRNOG00000030888 Rela ENSRNOG00000019069 Nxf1 ENSRNOG00000013067 Phf21b ENSRNOG00000006299 Prrx2 ENSRNOG00000008239 Repin1 ENSRNOG00000004705 O88596_RAT ENSRNOG00000024170 Phf5a ENSRNOG00000025184 Prss35 ENSRNOG00000017940 Rere ENSRNOG00000031726 Oas1e ENSRNOG00000004019 Phlda1 ENSRNOG00000030073 Prss44 ENSRNOG00000017916 Rexo1 ENSRNOG00000001370 Oas1h ENSRNOG00000007979 Phospho2 ENSRNOG00000030114 Prss44 ENSRNOG00000001816 Rfc4 ENSRNOG00000033220 Oas1k ENSRNOG00000016723 Phpt1 ENSRNOG00000000571 Psap ENSRNOG00000006614 Rfx7 ENSRNOG00000019459 Oaz1 ENSRNOG00000017299 Phrf1 ENSRNOG00000013971 Psat1 ENSRNOG00000028382 Rfxap ENSRNOG00000023480 Obfc2b ENSRNOG00000000274 Phyhipl ENSRNOG00000019290 Pskh1 ENSRNOG00000039567 Rg9mtd1 ENSRNOG00000023372 Odam ENSRNOG00000013669 PI3R4_RAT ENSRNOG00000011745 Psma1 ENSRNOG00000011025 Rg9mtd2 ENSRNOG00000001081 Ogfod2 ENSRNOG00000021024 Pi4kb ENSRNOG00000007851 Psma3 ENSRNOG00000001211 RGD1303003 ENSRNOG00000028658 Olig2 ENSRNOG00000034272 Pias1 ENSRNOG00000013493 Psma4 ENSRNOG00000019281 RGD1303130 ENSRNOG00000031289 Olr1095 ENSRNOG00000020230 Pias4 ENSRNOG00000007114 Psma6 ENSRNOG00000002944 RGD1304587 ENSRNOG00000039921 Olr1130 ENSRNOG00000003070 PIGL_RAT ENSRNOG00000001488 Psmb1 ENSRNOG00000006986 RGD1304624 ENSRNOG00000033982 Olr1162 ENSRNOG00000007735 Pigm ENSRNOG00000019494 Psmb10 ENSRNOG00000006888 RGD1304792 ENSRNOG00000011563 Olr1199 ENSRNOG00000006858 Pigy ENSRNOG00000011463 Psmb2 ENSRNOG00000037552 RGD1304953 ENSRNOG00000032146 Olr1225 ENSRNOG00000019228 Pik3r2 ENSRNOG00000012938 Psmb3 ENSRNOG00000037599 RGD1304953

149

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000010090 RGD1305225 ENSRNOG00000002972 RGD1565283 ENSRNOG00000027513 RTEL1_RAT ENSRNOG00000000170 Slc30a4 ENSRNOG00000009830 RGD1305500 ENSRNOG00000019397 RGD1565363 ENSRNOG00000005183 Rtf1 ENSRNOG00000015393 Slc32a1 ENSRNOG00000002691 RGD1305609 ENSRNOG00000023436 RGD1565455 ENSRNOG00000009022 Rtkn ENSRNOG00000010023 Slc33a1 ENSRNOG00000010653 RGD1306119 ENSRNOG00000021237 RGD1565616 ENSRNOG00000004794 RTN1_RAT ENSRNOG00000015627 Slc35a3 ENSRNOG00000010466 RGD1306404 ENSRNOG00000039522 RGD1565779 ENSRNOG00000021202 Rtn3 ENSRNOG00000004510 Slc35b1 ENSRNOG00000015095 RGD1306437 ENSRNOG00000029415 RGD1565784 ENSRNOG00000037167 Rtp3 ENSRNOG00000019900 Slc35b2 ENSRNOG00000022745 RGD1306502 ENSRNOG00000039157 RGD1565815 ENSRNOG00000037169 Rtp3 ENSRNOG00000016174 Slc35b3 ENSRNOG00000005572 RGD1306782 ENSRNOG00000004120 RGD1565844 ENSRNOG00000038200 Rttn ENSRNOG00000022967 Slc35d1 ENSRNOG00000017728 RGD1306820 ENSRNOG00000027014 RGD1565972 ENSRNOG00000038219 Rttn ENSRNOG00000023015 Slc35d1 ENSRNOG00000005769 RGD1306862 ENSRNOG00000023459 RGD1566001 ENSRNOG00000008593 Ryk ENSRNOG00000004168 Slc35e4 ENSRNOG00000025141 RGD1307071 ENSRNOG00000036971 RGD1566320 ENSRNOG00000023226 S100a10 ENSRNOG00000012356 Slc36a1 ENSRNOG00000003664 RGD1307161 ENSRNOG00000028651 RGD1566386 ENSRNOG00000004222 S100g ENSRNOG00000021310 Slc36a3 ENSRNOG00000015508 RGD1307235 ENSRNOG00000014668 RGD621098 ENSRNOG00000013683 S1pr1 ENSRNOG00000005291 Slc38a1 ENSRNOG00000032192 RGD1307325 ENSRNOG00000019276 RGD735029 ENSRNOG00000013907 Sall1 ENSRNOG00000016344 Slc39a1 ENSRNOG00000010194 RGD1307392 ENSRNOG00000018325 RGD735175 ENSRNOG00000027995 Samd7 ENSRNOG00000028703 Slc39a6 ENSRNOG00000013919 RGD1307682 ENSRNOG00000007949 Rgn ENSRNOG00000010732 Sap18 ENSRNOG00000012508 Slc39a8 ENSRNOG00000010126 RGD1307704 ENSRNOG00000016309 Rgp1 ENSRNOG00000002575 Sap30l ENSRNOG00000005052 Slc39a9 ENSRNOG00000002580 RGD1307890 ENSRNOG00000012396 Rgr ENSRNOG00000018090 Saps1 ENSRNOG00000018487 Slc3a2 ENSRNOG00000012679 RGD1308299 ENSRNOG00000003895 Rgs1 ENSRNOG00000004820 Sar1b ENSRNOG00000005687 Slc6a14 ENSRNOG00000008898 RGD1308380 ENSRNOG00000027024 Rgs16 ENSRNOG00000000702 Sart3 ENSRNOG00000009019 Slc6a6 ENSRNOG00000017792 RGD1308544 ENSRNOG00000003687 Rgs2 ENSRNOG00000001064 Sbno1 ENSRNOG00000019484 Slc6a9 ENSRNOG00000009055 RGD1308612 ENSRNOG00000021403 Rhob ENSRNOG00000008305 Sc5dl ENSRNOG00000018824 Slc7a5 ENSRNOG00000006795 RGD1308874 ENSRNOG00000015415 Rhoq ENSRNOG00000019987 Scand1 ENSRNOG00000019943 Slc7a6 ENSRNOG00000040040 RGD1309085 ENSRNOG00000007597 Rhpn1 ENSRNOG00000006864 Scaper ENSRNOG00000020049 Slc7a6os ENSRNOG00000028236 RGD1309104 ENSRNOG00000014049 Riok1 ENSRNOG00000002225 Scarb2 ENSRNOG00000029871 Slc8a3 ENSRNOG00000025501 RGD1309483 ENSRNOG00000012692 Riok2 ENSRNOG00000031203 Scfd1 ENSRNOG00000015567 Slc9a2 ENSRNOG00000002545 RGD1309748 ENSRNOG00000032635 RL8_RAT ENSRNOG00000020196 Scgb1a1 ENSRNOG00000009005 Slco2a1 ENSRNOG00000018295 RGD1309922 ENSRNOG00000015920 Rln1 ENSRNOG00000026912 Scgb3a2 ENSRNOG00000011562 Slitrk2 ENSRNOG00000014186 RGD1310257 ENSRNOG00000019497 RM17_RAT ENSRNOG00000020083 Scly ENSRNOG00000009649 Slitrk5 ENSRNOG00000027451 RGD1310270 ENSRNOG00000019501 Rmnd1 ENSRNOG00000009636 Scrn1 ENSRNOG00000030539 Slpi ENSRNOG00000039183 RGD1310335 ENSRNOG00000028422 Rmnd5a ENSRNOG00000023668 Scyl1 ENSRNOG00000008620 Smad3 ENSRNOG00000016447 RGD1310553 ENSRNOG00000013373 Rmt1 ENSRNOG00000025318 Scyl3 ENSRNOG00000022870 Smad5 ENSRNOG00000003328 RGD1310686 ENSRNOG00000032133 Rnase2 ENSRNOG00000022229 Sdad1 ENSRNOG00000011421 Smap2 ENSRNOG00000008781 RGD1310769 ENSRNOG00000008584 Rnaseh1 ENSRNOG00000004936 Sdc2 ENSRNOG00000006391 Smarcad1 ENSRNOG00000006370 RGD1310852 ENSRNOG00000027017 Rnasel ENSRNOG00000011927 SDC3_RAT ENSRNOG00000034268 Smarcd1 ENSRNOG00000004723 RGD1310899 ENSRNOG00000004624 Rnd3 ENSRNOG00000014297 Sdc4 ENSRNOG00000014173 Smc3 ENSRNOG00000005083 RGD1311072 ENSRNOG00000001172 Rnf10 ENSRNOG00000013252 Sdccag10 ENSRNOG00000003712 Smek2 ENSRNOG00000002553 RGD1311122 ENSRNOG00000007272 Rnf103 ENSRNOG00000018876 Sdccag3 ENSRNOG00000019590 Smg5 ENSRNOG00000009312 RGD1311188 ENSRNOG00000015645 Rnf138 ENSRNOG00000020544 Sdr39u1 ENSRNOG00000007495 Smpx ENSRNOG00000001225 RGD1311257 ENSRNOG00000017900 rnf141 ENSRNOG00000002722 Sec14l1 ENSRNOG00000015157 Smtnl2 ENSRNOG00000021525 RGD1311357 ENSRNOG00000007370 Rnf144a ENSRNOG00000004555 Sec14l4 ENSRNOG00000007671 Smu1 ENSRNOG00000005840 RGD1311501 ENSRNOG00000011588 Rnf146 ENSRNOG00000004657 Sec23a ENSRNOG00000001867 Snap29 ENSRNOG00000020199 RGD1311517 ENSRNOG00000001602 Rnf160 ENSRNOG00000020411 Sec23ip ENSRNOG00000022472 Snap47 ENSRNOG00000005447 RGD1311564 ENSRNOG00000019325 Rnf185 ENSRNOG00000023373 Sec24b ENSRNOG00000013356 Snapap ENSRNOG00000020436 RGD1311703 ENSRNOG00000006087 Rnf20 ENSRNOG00000013743 Sec61a1 ENSRNOG00000010825 Snapc3 ENSRNOG00000020076 RGD1311783 ENSRNOG00000013323 Rnf217 ENSRNOG00000020992 Selenbp1 ENSRNOG00000008656 Snca ENSRNOG00000001753 RGD1311861 ENSRNOG00000018840 Rnf40 ENSRNOG00000014624 Selk ENSRNOG00000006500 Snf8 ENSRNOG00000007756 RGD1359310 ENSRNOG00000035642 rno‐let‐7i ENSRNOG00000037924 Selm ENSRNOG00000041111 snoR38 ENSRNOG00000005929 RGD1359378 ENSRNOG00000035550 rno‐mir‐125b‐1 ENSRNOG00000024794 Senp5 ENSRNOG00000034963 SNORA14 ENSRNOG00000002999 RGD1359380 ENSRNOG00000035491 rno‐mir‐137 ENSRNOG00000017952 Sept2 ENSRNOG00000034689 SNORA16 ENSRNOG00000017309 RGD1359529 ENSRNOG00000035494 rno‐mir‐181a‐1 ENSRNOG00000006545 Sept7 ENSRNOG00000034703 SNORA16 ENSRNOG00000012469 RGD1359616 ENSRNOG00000035575 rno‐mir‐30b ENSRNOG00000003137 Sept12 ENSRNOG00000040539 SNORA17 ENSRNOG00000011170 RGD1359634 ENSRNOG00000035506 rno‐mir‐326 ENSRNOG00000013548 Sepw1 ENSRNOG00000040574 SNORA17 ENSRNOG00000019415 RGD1559505 ENSRNOG00000036268 rno‐mir‐362 ENSRNOG00000017945 Serf1 ENSRNOG00000040583 SNORA17 ENSRNOG00000028926 RGD1559662 ENSRNOG00000040395 rno‐mir‐484 ENSRNOG00000009552 Serinc3 ENSRNOG00000040697 SNORA17 ENSRNOG00000028928 RGD1559662 ENSRNOG00000035551 rno‐mir‐9‐1 ENSRNOG00000001414 Serpine1 ENSRNOG00000040760 SNORA17 ENSRNOG00000030048 RGD1560207 ENSRNOG00000035628 rno‐mir‐9‐2 ENSRNOG00000037690 Sertad3 ENSRNOG00000040810 SNORA17 ENSRNOG00000009183 RGD1560286 ENSRNOG00000017310 Rnpc3 ENSRNOG00000016208 Setbp1 ENSRNOG00000040874 SNORA17 ENSRNOG00000005366 RGD1560464 ENSRNOG00000003125 Rogdi ENSRNOG00000006587 Setd3 ENSRNOG00000040894 SNORA17 ENSRNOG00000033673 RGD1560513 ENSRNOG00000023484 Rpap2 ENSRNOG00000021143 Setdb1 ENSRNOG00000040906 SNORA17 ENSRNOG00000018289 RGD1560648 ENSRNOG00000027149 Rpl10 ENSRNOG00000006806 Setmar ENSRNOG00000041127 SNORA17 ENSRNOG00000027649 RGD1560902 ENSRNOG00000008140 Rpl15 ENSRNOG00000007629 Sf3a3 ENSRNOG00000041137 SNORA17 ENSRNOG00000023220 RGD1560909 ENSRNOG00000021035 Rpl18 ENSRNOG00000013516 Sf3b1 ENSRNOG00000035693 SNORA18 ENSRNOG00000023221 RGD1560909 ENSRNOG00000018795 Rpl18a ENSRNOG00000014908 Sf3b5 ENSRNOG00000036028 SNORA2 ENSRNOG00000006283 RGD1560936 ENSRNOG00000004741 Rpl19 ENSRNOG00000005513 Sfrs5 ENSRNOG00000035784 SNORA21 ENSRNOG00000032132 RGD1560997 ENSRNOG00000000957 Rpl21 ENSRNOG00000027360 Sfrs7 ENSRNOG00000034328 SNORA24 ENSRNOG00000037667 RGD1561147 ENSRNOG00000004214 Rpl26 ENSRNOG00000015442 Sfxn3 ENSRNOG00000040763 SNORA26 ENSRNOG00000037670 RGD1561147 ENSRNOG00000014214 Rpl27a ENSRNOG00000014628 Sgk196 ENSRNOG00000035202 SNORA30 ENSRNOG00000027803 RGD1561311 ENSRNOG00000017127 Rpl28 ENSRNOG00000019891 Sgta ENSRNOG00000041148 SNORA31 ENSRNOG00000001062 RGD1561318 ENSRNOG00000011138 Rpl29 ENSRNOG00000011937 Sgtb ENSRNOG00000040729 SNORA40 ENSRNOG00000031381 RGD1561333 ENSRNOG00000013508 Rpl31 ENSRNOG00000001425 Sh2b2 ENSRNOG00000040739 SNORA40 ENSRNOG00000038239 RGD1561507 ENSRNOG00000026921 Rpl32‐ps1 ENSRNOG00000022552 Sh2d3c ENSRNOG00000034602 SNORA41 ENSRNOG00000025033 RGD1561635 ENSRNOG00000014272 Rpl35 ENSRNOG00000019316 Sh3bp4 ENSRNOG00000035058 SNORA51 ENSRNOG00000018801 RGD1561676 ENSRNOG00000023529 Rpl5 ENSRNOG00000002737 Sh3bp5l ENSRNOG00000041836 SNORA61 ENSRNOG00000029850 RGD1561826 ENSRNOG00000006992 Rpl7 ENSRNOG00000017295 Sh3glb2 ENSRNOG00000035051 SNORA71 ENSRNOG00000021249 RGD1561852 ENSRNOG00000004497 Rpl8 ENSRNOG00000012812 Sharpin ENSRNOG00000041548 SNORA76 ENSRNOG00000030835 RGD1561875 ENSRNOG00000000954 Rpo1‐3 ENSRNOG00000016877 Shisa7 ENSRNOG00000041593 SNORA79 ENSRNOG00000033106 RGD1561956 ENSRNOG00000000786 Rpp21 ENSRNOG00000012478 Shox2 ENSRNOG00000034818 SNORD115 ENSRNOG00000037401 RGD1562139 ENSRNOG00000016226 Rpp40 ENSRNOG00000037339 Siglec10 ENSRNOG00000035197 SNORD115 ENSRNOG00000027833 RGD1562174 ENSRNOG00000016411 Rps12 ENSRNOG00000014604 Sigmar1 ENSRNOG00000035259 SNORD115 ENSRNOG00000006827 RGD1562342 ENSRNOG00000018774 Rps14 ENSRNOG00000001189 Sik1 ENSRNOG00000035295 SNORD115 ENSRNOG00000011739 RGD1562639 ENSRNOG00000024603 Rps15 ENSRNOG00000017502 Sike ENSRNOG00000035669 SNORD115 ENSRNOG00000038243 RGD1563048 ENSRNOG00000018320 Rps15a ENSRNOG00000019791 Sipa1l2 ENSRNOG00000035683 SNORD115 ENSRNOG00000038474 RGD1563169 ENSRNOG00000028505 Rps18 ENSRNOG00000020102 Sirt2 ENSRNOG00000035728 SNORD115 ENSRNOG00000012085 RGD1563216 ENSRNOG00000014179 Rps2 ENSRNOG00000013828 Sirt3 ENSRNOG00000035835 SNORD115 ENSRNOG00000031532 RGD1563220 ENSRNOG00000006325 Rps21 ENSRNOG00000018006 Skor2 ENSRNOG00000035944 SNORD115 ENSRNOG00000008934 RGD1563224 ENSRNOG00000016580 Rps23 ENSRNOG00000002271 Slain2 ENSRNOG00000035966 SNORD115 ENSRNOG00000008827 RGD1563296 ENSRNOG00000005517 Rps26 ENSRNOG00000005196 Slc12a6 ENSRNOG00000036107 SNORD115 ENSRNOG00000023656 RGD1563859 ENSRNOG00000016961 Rps27 ENSRNOG00000000962 Slc15a4 ENSRNOG00000040590 SNORD115 ENSRNOG00000010845 RGD1563962 ENSRNOG00000007663 Rps6 ENSRNOG00000018785 Slc16a13 ENSRNOG00000040804 SNORD115 ENSRNOG00000003068 RGD1564036 ENSRNOG00000006632 Rps6ka3 ENSRNOG00000009330 Slc17a5 ENSRNOG00000040596 SNORD12 ENSRNOG00000037220 RGD1564051 ENSRNOG00000004362 Rps6ka5 ENSRNOG00000010275 Slc17a9 ENSRNOG00000035002 SNORD14 ENSRNOG00000037234 RGD1564051 ENSRNOG00000021689 Rps6kb2 ENSRNOG00000002839 Slc19a2 ENSRNOG00000034872 SNORD15 ENSRNOG00000033611 RGD1564058 ENSRNOG00000010528 Rpusd2 ENSRNOG00000005248 Slc1a4 ENSRNOG00000041162 SNORD22 ENSRNOG00000030956 RGD1564261 ENSRNOG00000007051 Rraga ENSRNOG00000018567 Slc20a1 ENSRNOG00000041164 SNORD22 ENSRNOG00000038819 RGD1564308 ENSRNOG00000007331 Rragd ENSRNOG00000016414 Slc22a17 ENSRNOG00000034855 SNORD33 ENSRNOG00000038824 RGD1564308 ENSRNOG00000010159 Rrh ENSRNOG00000008432 Slc22a5 ENSRNOG00000035374 SNORD46 ENSRNOG00000012453 RGD1564560 ENSRNOG00000001203 Rrp1 ENSRNOG00000020288 Slc25a20 ENSRNOG00000034674 SNORD48 ENSRNOG00000021687 RGD1564664 ENSRNOG00000022896 Rrp7a ENSRNOG00000010592 Slc25a27 ENSRNOG00000035937 SNORD49 ENSRNOG00000025523 RGD1564887 ENSRNOG00000012927 Rrp9 ENSRNOG00000016751 Slc25a28 ENSRNOG00000034892 SNORD50 ENSRNOG00000030962 RGD1564937 ENSRNOG00000007630 Rsl24d1 ENSRNOG00000004351 Slc25a29 ENSRNOG00000034932 SNORD87 ENSRNOG00000004004 RGD1565082 ENSRNOG00000000723 RT1‐CE5 ENSRNOG00000025269 Slc25a44 ENSRNOG00000041514 SNORD88 ENSRNOG00000018102 RGD1565095 ENSRNOG00000000795 RT1‐N3 ENSRNOG00000020450 Slc26a6 ENSRNOG00000041481 SNORD93 ENSRNOG00000038325 RGD1565252 ENSRNOG00000014575 Rtcd1 ENSRNOG00000005302 Slc2a9 ENSRNOG00000041509 SNORD93

150

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000035992 snoU2‐30 ENSRNOG00000017349 Tbc1d10b ENSRNOG00000001049 Tpt1 ENSRNOG00000015734 Ube3a ENSRNOG00000041985 snoU2‐30 ENSRNOG00000015970 Tbc1d13 ENSRNOG00000029095 Trabd ENSRNOG00000010702 Ube3c ENSRNOG00000036171 snoU6‐53 ENSRNOG00000005766 Tbc1d20 ENSRNOG00000013169 Traf4 ENSRNOG00000000921 Ubl3 ENSRNOG00000001501 Snrpa ENSRNOG00000008737 Tbc1d21 ENSRNOG00000009835 Tram1l1 ENSRNOG00000004477 Ublcp1 ENSRNOG00000006961 Snrpb ENSRNOG00000029667 Tbce ENSRNOG00000021091 Trank1 ENSRNOG00000005564 Ubn2 ENSRNOG00000005426 Snupn ENSRNOG00000009370 Tbkbp1 ENSRNOG00000005418 Trap1 ENSRNOG00000019933 Ubqln4 ENSRNOG00000026884 Snx17 ENSRNOG00000009427 Tbx21 ENSRNOG00000014581 Trappc2l ENSRNOG00000019666 Ubxn1 ENSRNOG00000014719 Snx19 ENSRNOG00000004814 Tceb2 ENSRNOG00000010550 Trappc3 ENSRNOG00000003625 Ubxn4 ENSRNOG00000017832 Snx2 ENSRNOG00000010902 Tceb3 ENSRNOG00000002915 TRI11_RAT ENSRNOG00000003636 Ubxn4 ENSRNOG00000017382 Snx33 ENSRNOG00000018849 Tcerg1 ENSRNOG00000004110 Trib2 ENSRNOG00000015109 Ubxn8 ENSRNOG00000002946 Socs3 ENSRNOG00000016700 Tcf21 ENSRNOG00000007319 Trib3 ENSRNOG00000003545 Uchl5 ENSRNOG00000028504 Socs5 ENSRNOG00000012405 Tcf4 ENSRNOG00000008922 Trim14 ENSRNOG00000038176 Ufm1 ENSRNOG00000011204 Socs7 ENSRNOG00000032395 Tcfcp2 ENSRNOG00000021771 Trim29 ENSRNOG00000002643 Ugdh ENSRNOG00000002115 Sod1 ENSRNOG00000004280 Tcn2 ENSRNOG00000010303 Trim32 ENSRNOG00000014901 Uggt1 ENSRNOG00000033983 Sohlh1 ENSRNOG00000014160 Tcp1 ENSRNOG00000006248 Trim37 ENSRNOG00000001797 Umps ENSRNOG00000011313 Sorcs1 ENSRNOG00000007587 Tcp11l2 ENSRNOG00000000785 Trim39 ENSRNOG00000012357 Unc45a ENSRNOG00000012199 ENSRNOG00000014952 Tctn3 ENSRNOG00000008215 Trim47 ENSRNOG00000023593 Upf2 ENSRNOG00000018588 Sox4 ENSRNOG00000025693 Tdrd6 ENSRNOG00000019968 Trim8 ENSRNOG00000016952 Uqcr11 ENSRNOG00000014084 Sp1 ENSRNOG00000000506 Tead3 ENSRNOG00000014274 Trit1 ENSRNOG00000032134 Uqcrc1 ENSRNOG00000022769 Sp100 ENSRNOG00000001010 Tecpr1 ENSRNOG00000008978 Trmt12 ENSRNOG00000036742 Uqcrc2 ENSRNOG00000010078 Spag1 ENSRNOG00000001019 Tect2 ENSRNOG00000001885 Trmt2a ENSRNOG00000012550 Uqcrh ENSRNOG00000027419 Spag16 ENSRNOG00000016774 Telo2 ENSRNOG00000021270 Trmt6 ENSRNOG00000007233 Uqcrq ENSRNOG00000004246 Spag7 ENSRNOG00000039398 Tesb ENSRNOG00000027233 Trpc5 ENSRNOG00000007890 Usp1 ENSRNOG00000009207 Spata2 ENSRNOG00000013659 Tex2 ENSRNOG00000006324 Trpc6 ENSRNOG00000016509 Usp10 ENSRNOG00000003273 Spata20 ENSRNOG00000038496 Tex21 ENSRNOG00000020714 Trpm4 ENSRNOG00000023202 Usp15 ENSRNOG00000019976 Spata24 ENSRNOG00000030625 Tf ENSRNOG00000001195 Trpv4 ENSRNOG00000032492 Usp22 ENSRNOG00000019963 Spats1 ENSRNOG00000002695 Tfb2m ENSRNOG00000017321 Trub1 ENSRNOG00000005802 Usp24 ENSRNOG00000015019 Spg21 ENSRNOG00000000663 Tfip11 ENSRNOG00000011470 Tsc1 ENSRNOG00000000909 Uspl1 ENSRNOG00000031710 Spink2 ENSRNOG00000016182 Tgfa ENSRNOG00000027784 Tsku ENSRNOG00000005823 Utp20 ENSRNOG00000031711 Spink2 ENSRNOG00000012956 Tgm2 ENSRNOG00000018122 TSN17_RAT ENSRNOG00000004387 Utp23 ENSRNOG00000016920 Spire2 ENSRNOG00000007158 TGT_RAT ENSRNOG00000016474 Tspan3 ENSRNOG00000006989 Vamp2 ENSRNOG00000029903 Spock3 ENSRNOG00000019109 Thap11 ENSRNOG00000029810 Tspan4 ENSRNOG00000016477 Vangl1 ENSRNOG00000034303 Spon1 ENSRNOG00000037967 Thap7 ENSRNOG00000010549 Tspo ENSRNOG00000010457 Vash1 ENSRNOG00000004686 Spop ENSRNOG00000038019 Thap7 ENSRNOG00000000549 Tspyl1 ENSRNOG00000006980 Vcpip1 ENSRNOG00000033794 Sppl2b ENSRNOG00000009066 Thra ENSRNOG00000000547 Tspyl4 ENSRNOG00000013505 Vdac2 ENSRNOG00000005209 Spred1 ENSRNOG00000008291 Thumpd2 ENSRNOG00000009285 Tssc1 ENSRNOG00000019277 Vdac3 ENSRNOG00000012862 Spsb4 ENSRNOG00000006941 Thumpd3 ENSRNOG00000000186 Tst ENSRNOG00000021156 Vegfb ENSRNOG00000015396 Sptan1 ENSRNOG00000016813 Tia1 ENSRNOG00000018884 Ttc13 ENSRNOG00000012427 Veph1 ENSRNOG00000010882 Sptlc1 ENSRNOG00000008686 Tigd5 ENSRNOG00000011261 Ttc14 ENSRNOG00000000405 Vgll2 ENSRNOG00000012210 Sptlc2 ENSRNOG00000007883 Timm10 ENSRNOG00000009039 Ttc15 ENSRNOG00000007822 Vgll4 ENSRNOG00000009550 Sqle ENSRNOG00000019682 Timm13 ENSRNOG00000010495 Ttc17 ENSRNOG00000010258 Vhl ENSRNOG00000002216 Srd5a3 ENSRNOG00000007988 Timm22 ENSRNOG00000002977 Ttc19 ENSRNOG00000018087 Vim ENSRNOG00000018232 Srf ENSRNOG00000001058 Timm44 ENSRNOG00000017473 Ttc25 ENSRNOG00000019399 Vkorc1 ENSRNOG00000020204 Srp19 ENSRNOG00000013835 Timm8a2 ENSRNOG00000013217 Ttc33 ENSRNOG00000033483 Vom1r44 ENSRNOG00000009351 Srp68 ENSRNOG00000003048 Tiprl ENSRNOG00000022623 Ttll12 ENSRNOG00000030117 Vom1r92 ENSRNOG00000003211 Srp9 ENSRNOG00000016064 Tkt ENSRNOG00000004939 Ttll6 ENSRNOG00000032691 Vom1r96 ENSRNOG00000010543 Srpr ENSRNOG00000012579 Tlcd1 ENSRNOG00000009076 Ttpal ENSRNOG00000032583 Vom2r1 ENSRNOG00000003715 Srpx2 ENSRNOG00000013013 TLE3_RAT ENSRNOG00000024244 Ttyh3 ENSRNOG00000029660 Vom2r50 ENSRNOG00000007998 Ssb ENSRNOG00000010522 Tlr4 ENSRNOG00000004559 Tuba1a ENSRNOG00000016443 Vps13d ENSRNOG00000012100 Ssbp1 ENSRNOG00000016120 Tlx1 ENSRNOG00000017445 Tubb2b ENSRNOG00000013689 Vps18 ENSRNOG00000007920 Ssbp3 ENSRNOG00000016559 Tm2d2 ENSRNOG00000000821 Tubb5 ENSRNOG00000007356 Vps24 ENSRNOG00000008825 Ssrp1 ENSRNOG00000012751 Tm9sf2 ENSRNOG00000020213 Tubg1 ENSRNOG00000014633 Vps28 ENSRNOG00000012736 Sssca1 ENSRNOG00000004312 Tmbim4 ENSRNOG00000006494 Tubg2 ENSRNOG00000012001 Vps37a ENSRNOG00000019070 St13 ENSRNOG00000000033 Tmcc2 ENSRNOG00000022507 Twf1 ENSRNOG00000001086 Vps37b ENSRNOG00000017932 St3gal2 ENSRNOG00000003928 Tmco1 ENSRNOG00000025925 Txk ENSRNOG00000000470 Vps52 ENSRNOG00000014018 St8sia1 ENSRNOG00000017718 Tmco6 ENSRNOG00000027604 TXND3_RAT ENSRNOG00000006895 Vps53 ENSRNOG00000026026 Stam ENSRNOG00000007901 Tmed10 ENSRNOG00000000133 Txndc15 ENSRNOG00000011540 Vta1 ENSRNOG00000012227 Stambp ENSRNOG00000005016 Tmed4 ENSRNOG00000018593 Txndc9 ENSRNOG00000025110 Vwa3a ENSRNOG00000002014 Stap1 ENSRNOG00000000073 TMED5_RAT ENSRNOG00000024352 Tyw1 ENSRNOG00000008720 Wbp1 ENSRNOG00000001090 Stard13 ENSRNOG00000021882 Tmed9 ENSRNOG00000034619 U1 ENSRNOG00000015080 Wdfy1 ENSRNOG00000025023 Stat6 ENSRNOG00000008034 Tmeff1 ENSRNOG00000034665 U1 ENSRNOG00000010042 Wdfy2 ENSRNOG00000033557 Stfa2l1 ENSRNOG00000025669 Tmem104 ENSRNOG00000034765 U1 ENSRNOG00000028498 Wdr1 ENSRNOG00000002956 Stim2 ENSRNOG00000006206 Tmem106b ENSRNOG00000034785 U1 ENSRNOG00000039587 Wdr13 ENSRNOG00000004217 Stk10 ENSRNOG00000021899 Tmem115 ENSRNOG00000034806 U1 ENSRNOG00000012379 WDR18_RAT ENSRNOG00000014287 Stk11 ENSRNOG00000000700 Tmem119 ENSRNOG00000034911 U1 ENSRNOG00000007333 Wdr20a ENSRNOG00000018181 Stk25 ENSRNOG00000001441 Tmem120a ENSRNOG00000035063 U1 ENSRNOG00000019713 Wdr24 ENSRNOG00000006002 Stk35 ENSRNOG00000022727 Tmem127 ENSRNOG00000035170 U1 ENSRNOG00000019670 Wdr3 ENSRNOG00000016810 Stmn1 ENSRNOG00000017329 Tmem129 ENSRNOG00000035664 U1 ENSRNOG00000011382 Wdr33 ENSRNOG00000008530 Stoml1 ENSRNOG00000021338 Tmem132a ENSRNOG00000036506 U1 ENSRNOG00000001181 Wdr4 ENSRNOG00000009535 Stoml2 ENSRNOG00000022153 Tmem134 ENSRNOG00000040710 U11 ENSRNOG00000026316 Wdr43 ENSRNOG00000009590 Stox2 ENSRNOG00000013238 Tmem14a ENSRNOG00000035384 U12 ENSRNOG00000036662 Wdr45l ENSRNOG00000008637 Strada ENSRNOG00000019958 Tmem151b ENSRNOG00000035983 U2 ENSRNOG00000020185 Wdr6 ENSRNOG00000004806 Strn ENSRNOG00000028945 Tmem182 ENSRNOG00000008607 U2surp ENSRNOG00000014893 Wdr63 ENSRNOG00000005585 Strn3 ENSRNOG00000003594 Tmem183a ENSRNOG00000035067 U3 ENSRNOG00000022491 Wdr76 ENSRNOG00000031896 Stt3a ENSRNOG00000022802 Tmem184b ENSRNOG00000035069 U3 ENSRNOG00000003243 Wdr81 ENSRNOG00000019302 Stx4 ENSRNOG00000012860 Tmem184c ENSRNOG00000036336 U4 ENSRNOG00000008377 Wdtc1 ENSRNOG00000018847 Stx5 ENSRNOG00000003915 Tmem206 ENSRNOG00000041081 U4 ENSRNOG00000026369 Wfdc15a ENSRNOG00000023366 Styxl1 ENSRNOG00000008564 Tmem222 ENSRNOG00000034376 U5 ENSRNOG00000028113 Whamm ENSRNOG00000011558 Sub1 ENSRNOG00000010895 Tmem30a ENSRNOG00000035786 U5 ENSRNOG00000015474 Whsc2 ENSRNOG00000005587 Suclg1 ENSRNOG00000003075 Tmem39a ENSRNOG00000034326 U6 ENSRNOG00000030821 Wibg ENSRNOG00000001139 Suds3 ENSRNOG00000010752 Tmem41b ENSRNOG00000034343 U6 ENSRNOG00000003845 Wnt3 ENSRNOG00000009037 Sulf1 ENSRNOG00000003967 Tmem49 ENSRNOG00000034659 U6 ENSRNOG00000015618 Wnt5a ENSRNOG00000011953 Supt16h ENSRNOG00000004421 Tmem5 ENSRNOG00000034867 U6 ENSRNOG00000010520 Wrap53 ENSRNOG00000007845 Supt4h1 ENSRNOG00000012329 Tmem66 ENSRNOG00000035006 U6 ENSRNOG00000001629 Wrb ENSRNOG00000005167 Surf2 ENSRNOG00000018061 Tmem80 ENSRNOG00000035236 U6 ENSRNOG00000012929 Wsb1 ENSRNOG00000016525 Susd3 ENSRNOG00000038607 Tmem86b ENSRNOG00000035816 U6 ENSRNOG00000007869 Wscd1 ENSRNOG00000008714 Svs1 ENSRNOG00000036869 Tmem88b ENSRNOG00000035853 U6 ENSRNOG00000008065 Wwc1 ENSRNOG00000017742 Syf2 ENSRNOG00000003865 Tmigd1 ENSRNOG00000036031 U6 ENSRNOG00000000988 Xab2 ENSRNOG00000020287 Syt15 ENSRNOG00000009069 Tnfaip1 ENSRNOG00000036033 U6 ENSRNOG00000028636 Xkr5 ENSRNOG00000017136 SYT17_RAT ENSRNOG00000031312 Tnfrsf1a ENSRNOG00000036143 U6 ENSRNOG00000012084 Xpnpep1 ENSRNOG00000019318 Syt3 ENSRNOG00000014464 Tnfsf13b ENSRNOG00000036161 U6 ENSRNOG00000010137 Xpo4 ENSRNOG00000020245 Syt8 ENSRNOG00000012422 Tnik ENSRNOG00000040969 U6 ENSRNOG00000019915 Xrcc1 ENSRNOG00000003585 Sytl5 ENSRNOG00000024177 Tob2 ENSRNOG00000041161 U6 ENSRNOG00000016105 Xrcc5 ENSRNOG00000020950 Syvn1 ENSRNOG00000019861 Tollip ENSRNOG00000041872 U6 ENSRNOG00000006392 Xrcc6 ENSRNOG00000004229 Tac2 ENSRNOG00000014058 Tomm22 ENSRNOG00000041888 U6 ENSRNOG00000006405 Xrcc6 ENSRNOG00000016423 Tacc1 ENSRNOG00000001845 Top3b ENSRNOG00000041948 U6 ENSRNOG00000034309 Y_RNA ENSRNOG00000008597 TADA3_RAT ENSRNOG00000006435 Tor1b ENSRNOG00000019974 Uba52 ENSRNOG00000035828 Y_RNA ENSRNOG00000024601 Taf11 ENSRNOG00000010777 Tox ENSRNOG00000012710 Ubac2 ENSRNOG00000007213 Yars ENSRNOG00000004506 Taf1b ENSRNOG00000028649 Tox3 ENSRNOG00000028756 Ubc ENSRNOG00000010512 Yipf1 ENSRNOG00000015249 Taf8 ENSRNOG00000010756 Tp53 ENSRNOG00000032690 Ube2e2 ENSRNOG00000005610 Yipf4 ENSRNOG00000025394 TANC1_RAT ENSRNOG00000008738 Tp53i11 ENSRNOG00000004544 Ube2e3 ENSRNOG00000006642 Yipf6 ENSRNOG00000003174 Tapt1 ENSRNOG00000001924 Tp63 ENSRNOG00000001862 Ube2l3 ENSRNOG00000014785 Ykt6 ENSRNOG00000019023 Tars ENSRNOG00000013387 Tpcn2 ENSRNOG00000010727 Ube2r2 ENSRNOG00000001996 Ythdc1 ENSRNOG00000028135 Tas2r39 ENSRNOG00000018701 Tpsg1 ENSRNOG00000016930 Ube2s ENSRNOG00000010945 Ywhab

151

Ensembl Gene ID Associated Gene Name Ensembl Gene ID Associated Gene Name ENSRNOG00000026119 Ywhah ENSRNOG00000026760 "Gene name not found" ENSRNOG00000008104 Ywhaq ENSRNOG00000027273 "Gene name not found" ENSRNOG00000000308 Zbtb24 ENSRNOG00000028446 "Gene name not found" ENSRNOG00000009346 Zbtb26 ENSRNOG00000029958 "Gene name not found" ENSRNOG00000004306 Zbtb39 ENSRNOG00000030959 "Gene name not found" ENSRNOG00000014689 Zbtb4 ENSRNOG00000030967 "Gene name not found" ENSRNOG00000027459 Zbtb45 ENSRNOG00000031242 "Gene name not found" ENSRNOG00000009595 Zbtb48 ENSRNOG00000031601 "Gene name not found" ENSRNOG00000007684 Zc3h3 ENSRNOG00000031632 "Gene name not found" ENSRNOG00000017647 Zc3h8 ENSRNOG00000032652 "Gene name not found" ENSRNOG00000013944 Zc3hav1l ENSRNOG00000033951 "Gene name not found" ENSRNOG00000005625 Zc4h2 ENSRNOG00000034058 "Gene name not found" ENSRNOG00000018226 Zcchc14 ENSRNOG00000036318 "Gene name not found" ENSRNOG00000012266 Zcchc17 ENSRNOG00000036404 "Gene name not found" ENSRNOG00000002882 Zcchc2 ENSRNOG00000036420 "Gene name not found" ENSRNOG00000007520 Zcchc3 ENSRNOG00000036522 "Gene name not found" ENSRNOG00000029049 Zdhhc14 ENSRNOG00000037520 "Gene name not found" ENSRNOG00000002751 Zdhhc15 ENSRNOG00000037674 "Gene name not found" ENSRNOG00000011285 Zdhhc22 ENSRNOG00000039074 "Gene name not found" ENSRNOG00000021891 Zdhhc8 ENSRNOG00000039256 "Gene name not found" ENSRNOG00000025832 Zer1 ENSRNOG00000039274 "Gene name not found" ENSRNOG00000032917 Zfand2a ENSRNOG00000039275 "Gene name not found" ENSRNOG00000029022 Zfp112 ENSRNOG00000039520 "Gene name not found" ENSRNOG00000010087 Zfp143 ENSRNOG00000039782 "Gene name not found" ENSRNOG00000029336 Zfp180 ENSRNOG00000040387 "Gene name not found" ENSRNOG00000000236 Zfp207 ENSRNOG00000040425 "Gene name not found" ENSRNOG00000011544 Zfp219 ENSRNOG00000041202 "Gene name not found" ENSRNOG00000032625 Zfp235 ENSRNOG00000041204 "Gene name not found" ENSRNOG00000018481 Zfp259 ENSRNOG00000041215 "Gene name not found" ENSRNOG00000018709 Zfp278 ENSRNOG00000041221 "Gene name not found" ENSRNOG00000005009 Zfp3 ENSRNOG00000041238 "Gene name not found" ENSRNOG00000003461 Zfp330 ENSRNOG00000041261 "Gene name not found" ENSRNOG00000017290 Zfp335 ENSRNOG00000041266 "Gene name not found" ENSRNOG00000029205 Zfp354c ENSRNOG00000041291 "Gene name not found" ENSRNOG00000019673 Zfp36 ENSRNOG00000041301 "Gene name not found" ENSRNOG00000005067 Zfp36l2 ENSRNOG00000041351 "Gene name not found" ENSRNOG00000017066 Zfp384 ENSRNOG00000041356 "Gene name not found" ENSRNOG00000019065 Zfp385b ENSRNOG00000041372 "Gene name not found" ENSRNOG00000008378 Zfp414 ENSRNOG00000041388 "Gene name not found" ENSRNOG00000019802 Zfp428 ENSRNOG00000041405 "Gene name not found" ENSRNOG00000012718 Zfp451 ENSRNOG00000041409 "Gene name not found" ENSRNOG00000031576 Zfp455 ENSRNOG00000041411 "Gene name not found" ENSRNOG00000017986 Zfp458 ENSRNOG00000041434 "Gene name not found" ENSRNOG00000034184 Zfp583 ENSRNOG00000041445 "Gene name not found" ENSRNOG00000012762 Zfp64 ENSRNOG00000041449 "Gene name not found" ENSRNOG00000022742 Zfp772 ENSRNOG00000041455 "Gene name not found" ENSRNOG00000006925 Zfp786 ENSRNOG00000041475 "Gene name not found" ENSRNOG00000012524 Zfp91 ENSRNOG00000041506 "Gene name not found" ENSRNOG00000008614 Zfyve1 ENSRNOG00000041510 "Gene name not found" ENSRNOG00000014644 Zic1 ENSRNOG00000041523 "Gene name not found" ENSRNOG00000014397 Zic2 ENSRNOG00000041531 "Gene name not found" ENSRNOG00000014871 Zic4 ENSRNOG00000041552 "Gene name not found" ENSRNOG00000001335 Zkscan1 ENSRNOG00000041589 "Gene name not found" ENSRNOG00000016016 Zmat2 ENSRNOG00000041591 "Gene name not found" ENSRNOG00000012054 Zmpste24 ENSRNOG00000041612 "Gene name not found" ENSRNOG00000013855 Zmym1 ENSRNOG00000041806 "Gene name not found" ENSRNOG00000019154 Zmynd8 ENSRNOG00000041824 "Gene name not found" ENSRNOG00000021872 Znf213 ENSRNOG00000041971 "Gene name not found" ENSRNOG00000016971 Znf23 ENSRNOG00000000983 Znf394 ENSRNOG00000014237 Znf503 ENSRNOG00000013565 Znf507 ENSRNOG00000016565 Znf524 ENSRNOG00000016608 Znf579 ENSRNOG00000016392 Znf593 ENSRNOG00000012434 Znf598 ENSRNOG00000023197 Znf608 ENSRNOG00000032447 Znf641 ENSRNOG00000007398 Znf691 ENSRNOG00000014199 Znf703 ENSRNOG00000014278 Znf710 ENSRNOG00000008362 Znf775 ENSRNOG00000007578 Znf830 ENSRNOG00000021794 Znhit2 ENSRNOG00000030049 Znhit6 ENSRNOG00000000779 Znrd1 ENSRNOG00000039234 Zscan21 ENSRNOG00000015525 Zswim3 ENSRNOG00000007661 Zw10 ENSRNOG00000000819 "Gene name not found" ENSRNOG00000002576 "Gene name not found" ENSRNOG00000002789 "Gene name not found" ENSRNOG00000003182 "Gene name not found" ENSRNOG00000003641 "Gene name not found" ENSRNOG00000004188 "Gene name not found" ENSRNOG00000004329 "Gene name not found" ENSRNOG00000004765 "Gene name not found" ENSRNOG00000005116 "Gene name not found" ENSRNOG00000006900 "Gene name not found" ENSRNOG00000008914 "Gene name not found" ENSRNOG00000014534 "Gene name not found" ENSRNOG00000015378 "Gene name not found" ENSRNOG00000016498 "Gene name not found" ENSRNOG00000020046 "Gene name not found" ENSRNOG00000021957 "Gene name not found" ENSRNOG00000022457 "Gene name not found" ENSRNOG00000022560 "Gene name not found" ENSRNOG00000022566 "Gene name not found" ENSRNOG00000022592 "Gene name not found" ENSRNOG00000023121 "Gene name not found" ENSRNOG00000024256 "Gene name not found" ENSRNOG00000025375 "Gene name not found" ENSRNOG00000025598 "Gene name not found" ENSRNOG00000026401 "Gene name not found"

152

Table1: Epigenetically regulated genes after VPA treatment. A list of all genes enriched in acetyl marks within 3 kb from each TSS after three days of differentiation and treatment with VPA. All peaks were generated due to enrichment read densities compared to the input control. Specific peak locations and distance from each TSS are included.

153

Table2: Epigenetically regulated microRNAs after VPA treatment. A list of all microRNAs enriched in acetyl marks within 3 kb from each TSS. Included are microRNAs (mir-125b, mir-30b, mir-9) that have been previously identified with brain development and specifically neurogenesis (Krichevsky et al., 2003; Gao, 2010; Song et al., 2011). All peaks were generated due to enrichment read densities compared to the input control. Specific peak locations and distance from each TSS are included.

154

Table2: GO analysis of epigenetically regulated categories in response to ADD over expression. A list of all cellular component GO terms enriched in acetyl marks one day after ADD transfections. Epitope-tagged transcription factors to genomic promoters by chromatin immunoprecipitation sequencing identified potential target sites of the combination treatment. Using the ChIPpeakAnno algorithm (Zhu et al., 2010), all peaks were annotated to their nearest gene and then assigned to a gene ontology biological process. Each GO term was given a Benjamini & Hochberg (BH) adjusted p-value based on the number of enriched counts within the dataset. The GO categories are ranked according to the lowest adjusted p-values. These results suggest that the exogenously expressed transcription factor combination is binding near neuronal component regulatory regions. All peaks were generated due to enrichment read densities compared to an input control. GO terms were ranked according to the Benjamini-

Hochberg (BH) adjusted p-value.

155

VI. METHODS

Rat cell culture and differentiation

Generation of precursor clones (L2.2 and L2.3) from embryonic rat cortical cultures and their culturing conditions as described previously (Li et al., 2004). Fischer clones were generated similarly from a GFP positive Fischer rat (Li et al., 2011). Fischer clones were isolated from either dorsal or ventral regions of the E14.5 forebrain. Briefly, immortalized clones were cultured overnight on laminin coated glass coverslips (20

µg/ml) in bFGF2 containing serum-free medium, the medium was then removed and replaced with culture medium lacking bFGF2. After maintenance for the number of days indicated, cultures were then cross-linked with 4% paraformaldehyde and stained with cell type specific markers.

Western blot

Cell cultures were harvested and lysed using RIPA buffer (50 mM Tris, HCl (pH

7.6), 150 mM NaCl, 1% NP-40, 0.1% SDS). 10-20 μg of protein were ran on Invitrogen

NuPAGE gels and transferred using the IBlot system (Invitrogen, Carlsbad, CA) following manufactures protocols. Blots were then assayed using desired antibodies.

Gene expression profiling

All five clones were subjected to a three point time course differentiation series where total RNA was extracted at each time point for microarray analysis. Clones were thawed and grown as previously described as neurospheres in bFGF containing culture media.

156

Depending on growth rate, neurospheres were dissociated using trypsin/EDTA, washed

and aliquotted into batches of approximately 4x106 cells each. All five clones with

biological replicates (n=3) were plated onto laminin (20 μg/ml) coated 10 cm tissue culture dishes and grown as monolayers. Cells were allowed to attach overnight and 3 time points were chosen to represent undifferentiated (Day 0) and differentiated phenotypes (Days 3,7). Differentiation was induced by bFGF withdrawal with the addition of 0.5% FBS as a survival factor. At each time point, cells were lysed with 1ml

Trizol™ (Invitrogen of Carlsbad, CA) and stored at -80° C. Total RNA was extracted using Invitrogen’s protocol and concentrated using a NanoDrop spectrophotometer.

RNA quality was measured on the 2100 Bioanalyzer (Agilent, Santa Clara, CA) and confirmed that all samples (n=45) had an RNA integrity number above 7.5.

Approximately 11 μg from each sample was aliquotted and given to our collaborator,

Bristol-Myers Squibb (BMS - New York, NY) for custom microarray analysis.

Chromatin immunoprecipitation

Until recently, all ChIP protocols required millions of cells for each immunoprecipitation. This requirement severely limited rare cell populations and differentiated neuronal cell lines that were post-mitotic. To overcome this barrier we performed ChIP using Invitrogen’s MAGnify ™ kit which utilizes magnetic bead pull downs and requires far less cells per immunoprecipitation (IP) than previously reported protocols. There are many benefits to using a low cell ChIP kit such as the ability to IP multiple histone marks or transcription factors on the same biological sample and the ability to investigate chromatin profiles of differentiated cultures, which have been notoriously difficult to culture in mass quantities.

157

All 3 clones were cultured as neurospheres and chromatin immunoprecipitation

(ChIP) was performed with three histone marks correlating with gene permission

(H3K4me3, H3K9/14 ac) or repression (H3K27me3). We prepared neurospheres of our three clones (GE6, GE2, CTX8) by culturing in suspension in the presence of bFGF.

After sufficient growth they were dissociated with trypsin/EDTA, counted and distributed into 3x106 aliquots. Differentiated conditions were instead distributed into 1x106 aliquots.

Each aliquot was fixed with 1% fresh formaldehyde, quenched with 2.5M , flash frozen and stored at -80° C.

To keep the samples consistent, each culture was counted, cross-linked and washed. Cell pellets flash frozen and stored in -80° C. ChIP sequencing requires chromatin fragments no longer than 250 base pairs, so it was necessary to shear the

DNA consistently. Cell lysates were sonicated using Diagenode’s Bioruptor in 30 second pulses on/off. Samples were validated using a 2% agarose gel and pulse times were optimized to approximately 15 minutes, consistently producing randomly sheared fragments averaging 250 base pairs. Post sonication, cell lysates were quantified using a Nanodrop spectrophotometer. To account for any cell counting errors that may have occurred during the ChIP preparation, we normalized all concentrations to the clone’s largest quantity. Once the chromatin was sheared to the appropriate lengths, the immunoprecipitations were performed. 4µg of each antibody was conjugated to protein

A/G magnetic beads an added to the sheared and diluted chromatin. An IgG antibody and non immunoprecipitated chromatin sample were used as both negative and positive input controls respectively. Approximately 50 thousand cells were used per immunoprecipitations and a non immunoprecipitated sample from each treatment was saved as an input control. Enriched chromatin from each sample was reverse-cross- linked and the remaining DNA fragments were purified and were stored at -20°C.

158

ChIP enrichment and analysis

ChIP enrichment analysis was performed using qPCR with designed primers at specific neuronal or glial promoter regions and Power SYBR™green master mix.

Promoter sequences to key transcription factor were selected using the USCS genome browser. 250 base pairs upstream and downstream of the transcription start site were used to design primers using Primer Express 2.0 or Primer 3. Amplicons only in the range of 50 to 150 base pairs were selected due to the sonicated chromatin average of approximately 250 base pairs. Selected primers were initially validated to produce good overall amplification using 10 fold diluted chromatin from previous ChIP assays or rat genomic DNA. Amplicons were also verified to not contain any additional peaks on a dissociation curve. qPCRs of ChIP samples were run on the ABI 7900HT in 384 well plate format. To obtain relative enrichment of chromatin compared to its positive input control, we used the standard curve method using an input dilution series as our standards. This produced a quantity of each sample compared to its own input.

ChIP sequencing and analysis

ChIP sequencing libraries were constructed from anti-histone immunoprecipitated chromatin using the SOLiD™ DNA fragment library kit (Invitrogen) following the standard ChIP-Seq protocol. Libraries containing samples by clone type, treatment and temporal period were bar-coded, applied to beads with emulsion PCR, enriched, and sequenced using the SOLiD™ System v3.5 at the Waksman Genomics

Laboratory of Rutgers University. A sample prepared without immunoprecipitation from the control was used as the input control. Sequenced reads and quality strings were

159

aligned to the rat genome (rn4) using Bowtie 0.12.5 (Langmead et al., 2009) to identify the single, best-quality match location. Results were converted to BAM format using

Samtools (Li et al., 2009) and could. To properly visualize the small sequence alignments and separate the signal to noise ratios from the input control, ChIP peak files were generated based on the read alignment densities. The peaks were selected, comparing all time points and conditions against the input control, using Find Peaks 4.0

(Fejes et al., 2008). Tracks were created and visualized using the UCSC Genome

Browser where subsets of peaks were chosen for qPCR validation. Each peak file was designated its own track within a user defined browser session. These were then easily viewed by stacking each track by time period and histone enrichment mark. Permissive acetyl peaks were pseudo colored green and repressive H3K27me3 marks were pseudo colored red. Peak filtering was completed using a combination of the ChIPpeakAnno algorithm (Zhu et al., 2010), a plugin from the R Statistical Package.

microRNA mimics

Clones were amplified as neurospheres, dissociated and transfected with 250 ng of each

Premir™ in a 96 well format using Lonza’s nucleofector. A scrambled RNA and no DNA template were used as negative controls. Transfected cells were allowed to attach and recover overnight and were differentiated by bFGF withdrawal approximately 16-24 hours post electroporation. Percentage of Tuj1 positive cells were quantified using flow cytometry. Cell events were properly gated based on cell size and granularity and approximately 10,000 events were captured and quantified per sample.

160

ADD transfections

Transfections into rat NSC clones were done via electroporation in the amaxa

96-well shuttle system (Lonza) using the Rat Neuron Nucleofector Kit (VHPG-1003).

Observed transfection efficiencies using the 96-well shuttle system were consistently

>60% for all NSCs. Transfections were done following amaxa standard protocols.

0.5x106 to 1x106 rat precursor clones were nucleofected per well using plasmid DNA. To

begin the reprogramming process, CTX8 precursors were propagated as neurospheres

for three to four days depending on their growth rate and were supplemented with bFGF

daily in order to keep cells dividing and stable. Neurosphere colonies were dissociated,

quantified and transfected in suspension with Lonza’s 96 well nucleofector. The Ascl1,

Dlx1, Dlx5 (ADD) plasmid constructs were built by cloning the human coding regions of

each into a chicken actin promoting plasmid vector (CAG-Ascl1, CAG-Dlx1, CAG-Dlx5 )–

(Origene). Approximately 1µg of each plasmid or puc19, used as a filler DNA control,

was transfected into approximately 1x106 dissociated CTX8 cells. To monitor the

transfection efficiencies, 1µg of Ds-Red was co-transfected into both conditions and was

visible within 12-16 hours using epi-fluorescence microscopy. Immediately following the

transfections, cells were re-suspended into 96 well plates containing fresh culture media.

This increased the viability percentage and allowed the cells to recover. Next, the

electroporated cells were plated onto laminin coated glass coverslips for immunostaining

or tissue culture plates for co-culture and electrophysiology experiments. Differentiation

was induced approximately 12 to 16 hours post transfection by bFGF withdrawal.

161

Electrophysiology

Recordings were done with an Axopatch 200 amplifier. Pipette resistance was typically in the 4-7 MΩ range. Inward current was checked for in voltage clamp with a holding potential of -70 mV and testing potentials ranging from -60 mV to +30 mV in steps of 10 mV. Sweeps were filtered at 2 kHz and lasted 400 msecs (100 μsec sampling period) with 2 seconds between each sweep. Action potentials were tested for in current clamp, filtered at 2 kHz. The sweep length was 100 milliseconds and there were 2 seconds between each sweep. Negative current was injected to maintain the Vm at -80 mV and increasing test current was injected to elicit an action potential. The presence of synaptic currents was also investigated in voltage clamp.. Sweeps of 1.8 seconds (450 μsec sampling period) were filtered at 5 kHz and taken every 2 seconds for a total of 5 minutes.

162

VII. REFERENCES

Alberts, B. (2002). Molecular biology of the cell (4th ed.). New York: Garland Science.

Allen, N. D. (2008). Temporal and epigenetic regulation of neurodevelopmental plasticity. Philos Trans R Soc Lond B Biol Sci, 363(1489), 23-38.

Amariglio, N., Hirshberg, A., Scheithauer, B. W., Cohen, Y., Loewenthal, R., Trakhtenbrot, L., et al. (2009). Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS medicine, 6(2), e1000029.

Ambros, V. (2001). microRNAs: tiny regulators with great potential. Cell, 107(7), 823- 826.

Anderson, S., Mione, M., Yun, K., & Rubenstein, J. L. (1999). Differential origins of neocortical projection and local circuit neurons: role of Dlx genes in neocortical interneuronogenesis. Cerebral cortex, 9(6), 646-654.

Barnabe-Heider, F., Wasylnka, J. A., Fernandes, K. J., Porsche, C., Sendtner, M., Kaplan, D. R., et al. (2005). Evidence that embryonic neurons regulate the onset of cortical gliogenesis via cardiotrophin-1. Neuron, 48(2), 253-265.

Bar-Nur, O., Russ, H. A., Efrat, S., & Benvenisty, N. (2011). Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet Beta cells. Cell stem cell, 9(1), 17-23.

Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E., Wang, Z., et al. (2007). High-resolution profiling of histone methylations in the . Cell, 129(4), 823- 837.

Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2), 281-297.

Battiste, J., Helms, A. W., Kim, E. J., Savage, T. K., Lagace, D. C., Mandyam, C. D., et al. (2007). Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord. Development, 134(2), 285-293.

Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P., & Izaurralde, E. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes & development, 20(14), 1885-1898.

Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., et al. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell, 125(2), 315-326.

Bertrand, N., Castro, D. S., & Guillemot, F. (2002). and the specification of neural cell types. Nat Rev Neurosci, 3(7), 517-530.

Birchmeier, C. (2009). ErbB receptors and the development of the nervous system.

Experimental cell research, 315(4), 611-618.

Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes Dev, 16(1), 6- 21.

163

Bird, A. (2007). Perceptions of epigenetics. Nature, 447(7143), 396-398.

Briscoe, J., Chen, Y., Jessell, T. M., & Struhl, G. (2001). A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Molecular cell, 7(6), 1279-1291.

Buck, M. J., & Lieb, J. D. (2004). ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. [Review]. Genomics, 83(3), 349-360.

Butt, S. J., Cobos, I., Golden, J., Kessaris, N., Pachnis, V., & Anderson, S. (2007). Transcriptional regulation of cortical interneuron development. The Journal of neuroscience : the official journal of the Society for Neuroscience, 27(44), 11847-11850.

Butt, S. J., Fuccillo, M., Nery, S., Noctor, S., Kriegstein, A., Corbin, J. G., et al. (2005). The temporal and spatial origins of cortical interneurons predict their physiological subtype. Neuron, 48(4), 591-604.

Cahoy, J. D., Emery, B., Kaushal, A., Foo, L. C., Zamanian, J. L., Christopherson, K. S., et al. (2008). A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci, 28(1), 264- 278.

Carrington, J. C., & Ambros, V. (2003). Role of microRNAs in plant and animal development. Science, 301(5631), 336-338.

Casarosa, S., Fode, C., & Guillemot, F. (1999). Mash1 regulates neurogenesis in the ventral telencephalon. Development, 126(3), 525-534.

Cavalli, G., & Paro, R. (1999). Epigenetic inheritance of active chromatin after removal of the main transactivator. Science, 286(5441), 955-958.

Cedar, H., & Bergman, Y. (2009). Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet, 10(5), 295-304.

Chenn, A. (2009). A top-NOTCH way to make astrocytes. Dev Cell, 16(2), 158-159.

Craig, A. M., Banker, G., Chang, W., McGrath, M. E., & Serpinskaya, A. S. (1996). Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience, 16(10), 3166-3177.

Cui, K., Zang, C., Roh, T. Y., Schones, D. E., Childs, R. W., Peng, W., et al. (2009). Chromatin signatures in multipotent human hematopoietic stem cells indicate the fate of bivalent genes during differentiation. Cell Stem Cell, 4(1), 80-93.

Dahlstrand, J., Lardelli, M., & Lendahl, U. (1995). Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system. Brain research. Developmental brain research, 84(1), 109-129.

Das, G. D. (1977). Gliogenesis during embryonic development in the rat. Experientia, 33(12), 1648-1649.

164

Davis, A. A., & Temple, S. (1994). A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature, 372(6503), 263-266.

De Filippis, L., Lamorte, G., Snyder, E. Y., Malgaroli, A., & Vescovi, A. L. (2007). A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes. Stem cells, 25(9), 2312-2321.

Delaunay, D., Heydon, K., Cumano, A., Schwab, M. H., Thomas, J. L., Suter, U., et al. (2008). Early neuronal and glial fate restriction of embryonic neural stem cells. The Journal of neuroscience : the official journal of the Society for Neuroscience, 28(10), 2551-2562.

Deneen, B., Ho, R., Lukaszewicz, A., Hochstim, C. J., Gronostajski, R. M., & Anderson, D. J. (2006). The transcription factor NFIA controls the onset of gliogenesis in the developing spinal cord. Neuron, 52(6), 953-968.

Dhara, S. K., & Stice, S. L. (2008). Neural differentiation of human embryonic stem cells. Journal of cellular biochemistry, 105(3), 633-640.

Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M., & Alvarez-Buylla, A. (1999). Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell, 97(6), 703-716.

Dougherty, E. R., Brun, M., Trent, J. M., & Bittner, M. L. (2009). Conditioning-based modeling of contextual genomic regulation. IEEE/ACM transactions on computational biology and bioinformatics / IEEE, ACM, 6(2), 310-320.

Dykxhoorn, D. M., Novina, C. D., & Sharp, P. A. (2003). Killing the messenger: short RNAs that silence gene expression. Nature reviews. Molecular cell biology, 4(6), 457- 467.

Eaton, M. J., & Wolfe, S. Q. (2009). Clinical feasibility for cell therapy using human neuronal cell line to treat neuropathic behavioral hypersensitivity following spinal cord injury in rats. Journal of rehabilitation research and development, 46(1), 145-165.

Egger, G., Liang, G., Aparicio, A., & Jones, P. A. (2004). Epigenetics in human disease and prospects for epigenetic therapy. Nature, 429(6990), 457-463.

Eisenstat, D. D., Liu, J. K., Mione, M., Zhong, W., Yu, G., Anderson, S. A., et al. (1999). DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation. The Journal of comparative neurology, 414(2), 217-237.

Elkabetz, Y., & Studer, L. (2008). Human ESC-derived neural rosettes and neural stem cell progression. Cold Spring Harbor symposia on quantitative biology, 73, 377-387.

Enokido, Y., Suzuki, E., Iwasawa, K., Namekata, K., Okazawa, H., & Kimura, H. (2005). Cystathionine beta-synthase, a key enzyme for homocysteine metabolism, is preferentially expressed in the radial glia/astrocyte lineage of developing mouse CNS. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 19(13), 1854-1856.

165

Fazzari, P., Paternain, A. V., Valiente, M., Pla, R., Lujan, R., Lloyd, K., et al. (2010). Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature, 464(7293), 1376-1380.

Fedele, V., Roybon, L., Nordstrom, U., Li, J. Y., & Brundin, P. (2011). Neurogenesis in the R6/2 mouse model of Huntington's disease is impaired at the level of NeuroD1. Neuroscience, 173, 76-81.

Feinberg, A. P. (2008). Epigenetics at the epicenter of modern medicine. JAMA, 299(11), 1345-1350.

Fritsch, B., Qashu, F., Figueiredo, T. H., Aroniadou-Anderjaska, V., Rogawski, M. A., & Braga, M. F. (2009). Pathological alterations in GABAergic interneurons and reduced tonic inhibition in the basolateral amygdala during epileptogenesis. Neuroscience, 163(1), 415-429.

Gabay, L., Lowell, S., Rubin, L. L., & Anderson, D. J. (2003). Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron, 40(3), 485-499.

Gaiano, N., & Fishell, G. (2002). The role of notch in promoting glial and neural stem cell fates. Annu Rev Neurosci, 25, 471-490.

Gan, Q., Yoshida, T., McDonald, O. G., & Owens, G. K. (2007). Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells. Stem Cells, 25(1), 2-9.

Giraldez, A. J., Mishima, Y., Rihel, J., Grocock, R. J., Van Dongen, S., Inoue, K., et al. (2006). MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science, 312(5770), 75-79.

Glaser, K. B. (2007). HDAC inhibitors: clinical update and mechanism-based potential. Biochemical pharmacology, 74(5), 659-671.

Goff, L. A., Boucher, S., Ricupero, C. L., Fenstermacher, S., Swerdel, M., Chase, L. G., et al. (2008). Differentiating human multipotent mesenchymal stromal cells regulate microRNAs: prediction of microRNA regulation by PDGF during osteogenesis. Experimental hematology, 36(10), 1354-1369.

Goff, L. A., Davila, J., Jornsten, R., Keles, S., & Hart, R. P. (2007). Bioinformatic analysis of neural stem cell differentiation. Journal of biomolecular techniques : JBT, 18(4), 205- 212.

Goldberg, A. D., Allis, C. D., & Bernstein, E. (2007). Epigenetics: a landscape takes shape. Cell, 128(4), 635-638.

Gregg, C., & Weiss, S. (2005). CNTF/LIF/gp130 receptor complex signaling maintains a VZ precursor differentiation gradient in the developing ventral forebrain. Development, 132(3), 565-578.

Gronostajski, R. M. (2000). Roles of the NFI/CTF gene family in transcription and development. Gene, 249(1-2), 31-45.

166

Guillemot, F. (2005). Cellular and molecular control of neurogenesis in the mammalian telencephalon. Curr Opin Cell Biol, 17(6), 639-647.

Guillemot, F. (2007). Cell fate specification in the mammalian telencephalon. [Review]. Progress in neurobiology, 83(1), 37-52.

Guo, L., & Lu, Z. (2010). The fate of miRNA* strand through evolutionary analysis: implication for degradation as merely carrier strand or potential regulatory molecule? PloS one, 5(6), e11387.

Hall, I. M., Shankaranarayana, G. D., Noma, K., Ayoub, N., Cohen, A., & Grewal, S. I. (2002). Establishment and maintenance of a heterochromatin domain. Science, 297(5590), 2232-2237.

Hamby, M. E., Coskun, V., & Sun, Y. E. (2008). Transcriptional regulation of neuronal differentiation: the epigenetic layer of complexity. Biochim Biophys Acta, 1779(8), 432- 437.

Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H., & Kim, V. N. (2004). The Drosha- DGCR8 complex in primary microRNA processing. Genes & development, 18(24), 3016- 3027.

Hebbes, T. R., Thorne, A. W., & Crane-Robinson, C. (1988). A direct link between core histone acetylation and transcriptionally active chromatin. The EMBO journal, 7(5), 1395- 1402.

Hockfield, S., & McKay, R. D. (1985). Identification of major cell classes in the developing mammalian nervous system. The Journal of neuroscience : the official journal of the Society for Neuroscience, 5(12), 3310-3328.

Hong, L., Schroth, G. P., Matthews, H. R., Yau, P., & Bradbury, E. M. (1993). Studies of the DNA binding properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA. The Journal of biological chemistry, 268(1), 305-314.

Hong, S. H., Rampalli, S., Lee, J. B., McNicol, J., Collins, T., Draper, J. S., et al. (2011). Cell fate potential of human pluripotent stem cells is encoded by histone modifications. Cell stem cell, 9(1), 24-36.

Horton, S., Meredith, A., Richardson, J. A., & Johnson, J. E. (1999). Correct coordination of neuronal differentiation events in ventral forebrain requires the bHLH factor MASH1. Molecular and cellular neurosciences, 14(4-5), 355-369.

Hsieh, J., Nakashima, K., Kuwabara, T., Mejia, E., & Gage, F. H. (2004). Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proceedings of the National Academy of Sciences of the United States of America, 101(47), 16659-16664.

Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A. E., et al. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature biotechnology, 26(7), 795-797.

167

Jakovcevski, I., Mayer, N., & Zecevic, N. (2011). Multiple origins of human neocortical interneurons are supported by distinct expression of transcription factors. Cerebral cortex, 21(8), 1771-1782.

Jandial, R., & Levy, M. L. (2011). Cellular alchemy: induced pluripotent stem cells retain epigenetic memory. World neurosurgery, 75(1), 5-6.

Jenuwein, T., & Allis, C. D. (2001). Translating the histone code. Science, 293(5532), 1074-1080.

Johnson, D. S., Mortazavi, A., Myers, R. M., & Wold, B. (2007). Genome-wide mapping of in vivo protein-DNA interactions. Science, 316(5830), 1497-1502.

Johnstone, R. W. (2002). Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nature reviews. Drug discovery, 1(4), 287-299.

Kapsimali, M., Kloosterman, W. P., de Bruijn, E., Rosa, F., Plasterk, R. H., & Wilson, S. W. (2007). MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system. Genome biology, 8(8), R173.

Kawase-Koga, Y., Otaegi, G., & Sun, T. (2009). Different timings of Dicer deletion affect neurogenesis and gliogenesis in the developing mouse central nervous system. Developmental dynamics : an official publication of the American Association of Anatomists, 238(11), 2800-2812.

Kazantsev, A. G., & Thompson, L. M. (2008). Therapeutic application of histone deacetylase inhibitors for central nervous system disorders. Nature reviews. Drug discovery, 7(10), 854-868.

Keirstead, H. S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K., et al. (2005). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience, 25(19), 4694-4705.

Kim, H. J., Leeds, P., & Chuang, D. M. (2009). The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain. Journal of neurochemistry, 110(4), 1226- 1240.

Kouzarides, T. (2007). Chromatin modifications and their function. Cell, 128(4), 693-705.

Kramer, O. H., Gottlicher, M., & Heinzel, T. (2001). Histone deacetylase as a therapeutic target. Trends in endocrinology and metabolism: TEM, 12(7), 294-300.

Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K., & Kosik, K. S. (2003). A microRNA array reveals extensive regulation of microRNAs during brain development. RNA, 9(10), 1274-1281.

Kriegstein, A. R., & Noctor, S. C. (2004). Patterns of neuronal migration in the embryonic cortex. Trends in neurosciences, 27(7), 392-399.

Kunkel, D. D., Hendrickson, A. E., Wu, J. Y., & Schwartzkroin, P. A. (1986). Glutamic acid decarboxylase (GAD) immunocytochemistry of developing rabbit hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience, 6(2), 541- 552.

168

Langhammer, C. G., Previtera, M. L., Sweet, E. S., Sran, S. S., Chen, M., & Firestein, B. L. (2010). Automated Sholl analysis of digitized neuronal morphology at multiple scales: Whole cell Sholl analysis versus Sholl analysis of arbor subregions. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 77(12), 1160-1168.

Le, M. T., Xie, H., Zhou, B., Chia, P. H., Rizk, P., Um, M., et al. (2009). MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets. Molecular and cellular biology, 29(19), 5290-5305.

Le, T. N., Du, G., Fonseca, M., Zhou, Q. P., Wigle, J. T., & Eisenstat, D. D. (2007). Dlx homeobox genes promote cortical interneuron migration from the basal forebrain by direct repression of the semaphorin receptor neuropilin-2. The Journal of biological chemistry, 282(26), 19071-19081.

Lee, J. C., Mayer-Proschel, M., & Rao, M. S. (2000). Gliogenesis in the central nervous system. Glia, 30(2), 105-121.

Levesque, J. C., & Parent, A. (2005). GABAergic interneurons in human subthalamic nucleus. Movement disorders : official journal of the Movement Disorder Society, 20(5), 574-584.

Lewis, B. P., Burge, C. B., & Bartel, D. P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 120(1), 15-20.

Li, H., Babiarz, J., Woodbury, J., Kane-Goldsmith, N., & Grumet, M. (2004). Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors. Developmental biology, 271(2), 225-238.

Li, H., & Grumet, M. (2007). BMP and LIF signaling coordinately regulate lineage restriction of radial glia in the developing forebrain. Glia, 55(1), 24-35.

Li, H., Hader, A. T., Han, Y. R., Wong, J. A., Babiarz, J., Ricupero, C. L., et al. (2011). Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture. Developmental neurobiology.

Li, H., Han, Y. R., Bi, C., Davila, J., Goff, L. A., Thompson, K., et al. (2008). Functional differentiation of a clone resembling embryonic cortical interneuron progenitors. Developmental neurobiology, 68(14), 1549-1564.

Lieb, J. D., Liu, X., Botstein, D., & Brown, P. O. (2001). Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nature genetics, 28(4), 327-334.

Lim, D. A., Huang, Y. C., Swigut, T., Mirick, A. L., Garcia-Verdugo, J. M., Wysocka, J., et al. (2009). Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells. Nature, 458(7237), 529-533.

Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J., et al. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science, 305(5689), 1437-1441.

169

Liu, Y., & Rao, M. S. (2004). Glial progenitors in the CNS and possible lineage relationships among them. Biol Cell, 96(4), 279-290.

Lowenstein, D. H., Thomas, M. J., Smith, D. H., & McIntosh, T. K. (1992). Selective vulnerability of dentate hilar neurons following traumatic brain injury: a potential mechanistic link between head trauma and disorders of the hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience, 12(12), 4846-4853.

Marano, J. E., Sun, D., Zama, A. M., Young, W., & Uzumcu, M. (2008). Orthotopic transplantation of neonatal GFP rat ovary as experimental model to study ovarian development and toxicology. Reproductive toxicology, 26(3-4), 191-196.

Marin, O., & Rubenstein, J. L. (2003). Cell migration in the forebrain. Annual review of neuroscience, 26, 441-483.

Marmorstein, R., & Trievel, R. C. (2009). Histone modifying enzymes: structures, mechanisms, and specificities. Biochimica et biophysica acta, 1789(1), 58-68.

Matsuda, T., & Cepko, C. L. (2004). Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proceedings of the National Academy of Sciences of the United States of America, 101(1), 16-22.

Mehler, M. F. (2008). Epigenetics and the nervous system. Ann Neurol, 64(6), 602-617.

Meister, G., Landthaler, M., Dorsett, Y., & Tuschl, T. (2004). Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA, 10(3), 544-550.

Mellios, N., Galdzicka, M., Ginns, E., Baker, S. P., Rogaev, E., Xu, J., et al. (2010). Gender-Specific Reduction of Estrogen-Sensitive Small RNA, miR-30b, in Subjects With Schizophrenia. Schizophrenia bulletin.

Meshorer, E., & Misteli, T. (2006). Chromatin in pluripotent embryonic stem cells and differentiation. Nature reviews. Molecular cell biology, 7(7), 540-546.

Meyer, N., & Penn, L. Z. (2008). Reflecting on 25 years with MYC. Nature reviews. Cancer, 8(12), 976-990.

Miller, F. D., & Gauthier, A. S. (2007). Timing is everything: making neurons versus glia in the developing cortex. Neuron, 54(3), 357-369.

Miska, E. A., Alvarez-Saavedra, E., Townsend, M., Yoshii, A., Sestan, N., Rakic, P., et al. (2004). Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol, 5(9), R68.

Mohn, F., Weber, M., Rebhan, M., Roloff, T. C., Richter, J., Stadler, M. B., et al. (2008). Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol Cell, 30(6), 755-766.

Morshead, C. M., Reynolds, B. A., Craig, C. G., McBurney, M. W., Staines, W. A., Morassutti, D., et al. (1994). Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron, 13(5), 1071-1082.

170

Mortazavi, A., Leeper Thompson, E. C., Garcia, S. T., Myers, R. M., & Wold, B. (2006). Comparative genomics modeling of the NRSF/REST repressor network: from single conserved sites to genome-wide repertoire. Genome research, 16(10), 1208-1221.

Mukhida, K., Mendez, I., McLeod, M., Kobayashi, N., Haughn, C., Milne, B., et al. (2007). Spinal GABAergic transplants attenuate mechanical allodynia in a rat model of neuropathic pain. Stem cells, 25(11), 2874-2885.

Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., et al. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature biotechnology, 26(1), 101-106.

Nakamura, K., Hioki, H., Fujiyama, F., & Kaneko, T. (2005). Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrain. The Journal of comparative neurology, 492(3), 263-288.

Namihira, M., Kohyama, J., Semi, K., Sanosaka, T., Deneen, B., Taga, T., et al. (2009). Committed neuronal precursors confer astrocytic potential on residual neural precursor cells. Dev Cell, 16(2), 245-255.

Narlikar, G. J., Fan, H. Y., & Kingston, R. E. (2002). Cooperation between complexes that regulate chromatin structure and transcription. Cell, 108(4), 475-487.

Noble, M., Proschel, C., & Mayer-Proschel, M. (2004). Getting a GR(i)P on oligodendrocyte development. Dev Biol, 265(1), 33-52.

Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., & Kriegstein, A. R. (2004). Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nature neuroscience, 7(2), 136-144.

Norton, V. G., Imai, B. S., Yau, P., & Bradbury, E. M. (1989). Histone acetylation reduces nucleosome core particle linking number change. Cell, 57(3), 449-457.

Nunzi, M. G., Russo, M., & Mugnaini, E. (2003). Vesicular glutamate transporters VGLUT1 and VGLUT2 define two subsets of unipolar brush cells in organotypic cultures of mouse vestibulocerebellum. Neuroscience, 122(2), 359-371.

Ohi, Y., Qin, H., Hong, C., Blouin, L., Polo, J. M., Guo, T., et al. (2011). Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells. Nature cell biology, 13(5), 541-549.

Packer, A. N., Xing, Y., Harper, S. Q., Jones, L., & Davidson, B. L. (2008). The bifunctional microRNA miR-9/miR-9* regulates REST and CoREST and is downregulated in Huntington's disease. The Journal of neuroscience : the official journal of the Society for Neuroscience, 28(53), 14341-14346.

Pan, G., Tian, S., Nie, J., Yang, C., Ruotti, V., Wei, H., et al. (2007). Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell stem cell, 1(3), 299-312.

171

Panchision, D. M., & McKay, R. D. (2002). The control of neural stem cells by morphogenic signals. Current opinion in genetics & development, 12(4), 478-487.

Panganiban, G., & Rubenstein, J. L. (2002). Developmental functions of the Distal- less/Dlx homeobox genes. Development, 129(19), 4371-4386.

Parras, C. M., Schuurmans, C., Scardigli, R., Kim, J., Anderson, D. J., & Guillemot, F. (2002). Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity. Genes & development, 16(3), 324-338.

Peljto, M., & Wichterle, H. (2011). Programming embryonic stem cells to neuronal subtypes. Current opinion in neurobiology, 21(1), 43-51.

Pickford, A. S., & Cogoni, C. (2003). RNA-mediated gene silencing. Cellular and molecular life sciences : CMLS, 60(5), 871-882.

Placantonakis, D. G., Tomishima, M. J., Lafaille, F., Desbordes, S. C., Jia, F., Socci, N. D., et al. (2009). Enriched motor neuron populations derived from bacterial artificial chromosome-transgenic human embryonic stem cells. Clinical neurosurgery, 56, 125- 132.

Poitras, L., Ghanem, N., Hatch, G., & Ekker, M. (2007). The proneural determinant MASH1 regulates forebrain Dlx1/2 expression through the I12b intergenic enhancer. Development, 134(9), 1755-1765.

Pollard, S. M., & Conti, L. (2007). Investigating radial glia in vitro. Progress in neurobiology, 83(1), 53-67.

Popovich, P. G., Wei, P., & Stokes, B. T. (1997). Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats. The Journal of comparative neurology, 377(3), 443-464.

Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., et al. (2000). Genome-wide location and function of DNA binding proteins. Science, 290(5500), 2306- 2309.

Richardson, W. D., Smith, H. K., Sun, T., Pringle, N. P., Hall, A., & Woodruff, R. (2000). Oligodendrocyte lineage and the motor neuron connection. Glia, 29(2), 136-142.

Ringner, M. (2008). What is principal component analysis? Nature biotechnology, 26(3), 303-304.

Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao, Y., Zeng, T., et al. (2007). Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature methods, 4(8), 651-657.

Ross, S. E., Greenberg, M. E., & Stiles, C. D. (2003). Basic helix-loop-helix factors in cortical development. Neuron, 39(1), 13-25.

Ryder, E. F., Snyder, E. Y., & Cepko, C. L. (1990). Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. Journal of neurobiology, 21(2), 356-375.

172

Scheffler, B., Walton, N. M., Lin, D. D., Goetz, A. K., Enikolopov, G., Roper, S. N., et al. (2005). Phenotypic and functional characterization of adult brain neuropoiesis. Proceedings of the National Academy of Sciences of the United States of America, 102(26), 9353-9358.

Schones, D. E., & Zhao, K. (2008). Genome-wide approaches to studying chromatin modifications. Nat Rev Genet, 9(3), 179-191.

Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B., & Cavalli, G. (2007). Genome regulation by polycomb and trithorax proteins. Cell, 128(4), 735-745.

Segal, R. A., & Greenberg, M. E. (1996). Intracellular signaling pathways activated by neurotrophic factors. Annual review of neuroscience, 19, 463-489.

Shivaswamy, S., & Iyer, V. R. (2007). Genome-wide analysis of chromatin status using tiling microarrays. Methods, 41(3), 304-311.

Snyder, E. Y., Deitcher, D. L., Walsh, C., Arnold-Aldea, S., Hartwieg, E. A., & Cepko, C. L. (1992). Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell, 68(1), 33-51.

Solomon, M. J., Larsen, P. L., & Varshavsky, A. (1988). Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell, 53(6), 937-947.

Song, L., & Tuan, R. S. (2006). MicroRNAs and cell differentiation in mammalian development. Birth defects research. Part C, Embryo today : reviews, 78(2), 140-149.

Song, M. R., & Ghosh, A. (2004). FGF2-induced chromatin remodeling regulates CNTF- mediated gene expression and astrocyte differentiation. Nat Neurosci, 7(3), 229-235.

Song, P. P., Hu, Y., Liu, C. M., Yan, M. J., Song, G., Cui, Y., et al. (2011). Embryonic ectoderm development protein is regulated by microRNAs in human neural tube defects. American journal of obstetrics and gynecology, 204(6), 544 e549-517.

Stadtfeld, M., Maherali, N., Breault, D. T., & Hochedlinger, K. (2008). Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell, 2(3), 230-240.

Stahl, N., & Yancopoulos, G. D. (1994). The tripartite CNTF receptor complex: activation and signaling involves components shared with other cytokines. Journal of neurobiology, 25(11), 1454-1466.

Stolt, C. C., Lommes, P., Sock, E., Chaboissier, M. C., Schedl, A., & Wegner, M. (2003). The Sox9 transcription factor determines glial fate choice in the developing spinal cord. Genes Dev, 17(13), 1677-1689.

Struhl, K. (1998). Histone acetylation and transcriptional regulatory mechanisms. Genes & development, 12(5), 599-606.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861-872.

173

Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663-676.

Takizawa, T., Nakashima, K., Namihira, M., Ochiai, W., Uemura, A., Yanagisawa, M., et al. (2001). DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain. Developmental cell, 1(6), 749-758.

Tate, P. H., & Bird, A. P. (1993). Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev, 3(2), 226-231.

Temple, S. (2001). The development of neural stem cells. Nature, 414(6859), 112-117.

Thomas, E. D., Lochte, H. L., Jr., Lu, W. C., & Ferrebee, J. W. (1957). Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. The New England journal of medicine, 257(11), 491-496.

Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282(5391), 1145-1147.

Tokunaga, A., Kohyama, J., Yoshida, T., Nakao, K., Sawamoto, K., & Okano, H. (2004). Mapping spatio-temporal activation of Notch signaling during neurogenesis and gliogenesis in the developing mouse brain. J Neurochem, 90(1), 142-154.

Tursun, B., Patel, T., Kratsios, P., & Hobert, O. (2011). Direct conversion of C. elegans germ cells into specific neuron types. Science, 331(6015), 304-308.

Vasudevan, S., Tong, Y., & Steitz, J. A. (2007). Switching from repression to activation: microRNAs can up-regulate translation. Science, 318(5858), 1931-1934.

Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Sudhof, T. C., & Wernig, M. (2010). Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 463(7284), 1035-1041.

Villa, A., Snyder, E. Y., Vescovi, A., & Martinez-Serrano, A. (2000). Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS. Exp Neurol, 161(1), 67-84.

Vitalis, T., & Rossier, J. (2010). New insights into cortical interneurons development and classification: Contribution of developmental studies. Developmental neurobiology.

Waddington, C. H. (1952). The epigenetics of birds. Cambridge Eng.: University Press.

Wiese, C., Rolletschek, A., Kania, G., Blyszczuk, P., Tarasov, K. V., Tarasova, Y., et al. (2004). Nestin expression--a property of multi-lineage progenitor cells? Cellular and molecular life sciences : CMLS, 61(19-20), 2510-2522.

Woo, N. H., & Lu, B. (2006). Regulation of cortical interneurons by neurotrophins: from development to cognitive disorders. The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry, 12(1), 43-56.

Wu, H., & Sun, Y. E. (2006). Epigenetic regulation of stem cell differentiation. Pediatr Res, 59(4 Pt 2), 21R-25R.

174

Wu, L., Fan, J., & Belasco, J. G. (2006). MicroRNAs direct rapid deadenylation of mRNA. [Research Support, N.I.H., Extramural]. Proceedings of the National Academy of Sciences of the United States of America, 103(11), 4034-4039.

Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., et al. (1999). Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet, 8(5), 723-730.

Yamanaka, S. (2009). A fresh look at iPS cells. Cell, 137(1), 13-17.

Yekta, S., Shih, I. H., & Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science, 304(5670), 594-596.

Yu, I. T., Park, J. Y., Kim, S. H., Lee, J. S., Kim, Y. S., & Son, H. (2009). Valproic acid promotes neuronal differentiation by induction of proneural factors in association with H4 acetylation. Neuropharmacology, 56(2), 473-480.

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318(5858), 1917-1920.

Yu, J. Y., Chung, K. H., Deo, M., Thompson, R. C., & Turner, D. L. (2008). MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation. Experimental cell research, 314(14), 2618-2633.

Yu, M., Xi, Y., Pollack, J., Debiais-Thibaud, M., Macdonald, R. B., & Ekker, M. (2011). Activity of dlx5a/dlx6a regulatory elements during zebrafish GABAergic neuron development. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience.

Zeng, Y., & Cullen, B. R. (2004). Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic acids research, 32(16), 4776-4785.

Zeng, Y., Yi, R., & Cullen, B. R. (2005). Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. The EMBO journal, 24(1), 138- 148.

Zhang, R. L., Chopp, M., Roberts, C., Jia, L., Wei, M., Lu, M., et al. (2011). Ascl1 lineage cells contribute to ischemia-induced neurogenesis and oligodendrogenesis. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 31(2), 614-625.

Zhang, X., Guo, C., Chen, Y., Shulha, H. P., Schnetz, M. P., LaFramboise, T., et al. (2008). Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies. Nature methods, 5(2), 163-165.

Zhou, Q., Wang, S., & Anderson, D. J. (2000). Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron, 25(2), 331-343.

Zhu, L. J., Gazin, C., Lawson, N. D., Pages, H., Lin, S. M., Lapointe, D. S., et al. (2010). ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC bioinformatics, 11, 237.