Molecular Mechanisms Regulating Mammalian Forebrain Development

David Chun Cheong Tsui

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Institute of Medical Science

University of Toronto

Title: Molecular Mechanisms Regulating Mammalian Forebrain Development Name: David Chun Cheong Tsui Degree: Doctor of Philosophy, 2013 Department: Institute of Medical Sciences, University of Toronto

ABSTRACT

While the extrinsic factors regulating neurogenesis in the developing forebrain have been widely studied, the mechanisms downstream of the various signaling pathways are relatively ill-defined.

In particular, we focused on proteins that have been implicated in cognitive dysfunction. Here, we ask what role two cell intrinsic factors play in the development of two different neurogenic compartments in the forebrain. In the first part of the thesis, the transcription factor FoxP2, which is mutated in individuals who have specific language deficits, was identified to regulate neurogenesis in the developing cortex, in part by regulating the transition from the radial precursors to the transit amplifying intermediate progenitors. Moreover, we found that ectopic expression of the human homologue of the protein promotes neurogenesis in the murine cortex, thereby acting as a gain-of-function isoform. In the second part of the thesis, the histone acetyltransferase CREB-binding protein (CBP) was identified as regulating the generation of neurons from medial ganglionic eminence precursors, similar to its role in the developing cortex.

But CBP also plays a more substantial role in the expression of late interneuron markers, suggesting that it is continuously required for the various stages of neurogenesis at least in the ventral neurogenic niche. Finally, similar to cortical precursors, the function of CBP in the ventral forebrain precursors is also dependent on histone acetylation. Together, these studies shed light on some of the key intrinsic players that regulate the differentiation of neural precursors in the embryonic murine forebrain, and they also suggest potential mechanisms for the pathogenesis of various cognitive dysfunctions.

ACKNOWLEDGEMENTS

I am grateful to my family and everyone who supported me during my PhD training. In particular, I thank Freda and David for teaching me how to focus on important scientific questions, critically think about experimental results and to be scientifically creative. In addition, they helped me to significantly improve my presentation and writing skills. Their guidance and support were valuable for my training experience and I will always be grateful for them pushing me to reach success. I also thank Derek van der Kooy, my committee member and also my previous research supervisor, for introducing me to the world of science and also pushing me to think outside the box. My experience in Derek’s laboratory had a great influence on my decision to pursue a scientific career.

Many of my colleagues in the lab have been crucial role models and people to exchange ideas with during my journey. In particular, I am grateful to Sarah Burns for teaching me the skills of in utero electroporation and also helping me to carry out some of the in utero electroporations in my projects; Jing Wang for teaching me the skills of cell culturing and critical thinking; John Vessey and Hideaki Tomita for assisting me in some of my experiments as well as their intellectual input. I’d also like to thank Joseph Anthony, Jeff Biernaskie, Sagar Dugani,

Masashi Fujitani, Denis Gallagher, Andree Gauthier-Fisher, Hiroyuki Jinno, Adam Johnston,

Andreea Norman, Milijana Vojvodic, Ian Weaver, and Mark Zander for their discussions, and the rest of the Miller and Kaplan labs.

Finally, I thank the MD/PhD office for their advice and support over the years: Director

Dr. Norman Rosenblum, Sandy McGugan, and past-director Dr. Mel Silverman. I’d also like to thank my MD/PhD classmates for their input and discussion during the seminars.

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PUBLICATION AND AUTHOR CONTRIBUTIONS

Chapter 4 was accepted for publication in the Journal of Neuroscience. David Tsui performed all experiments of the chapter except for the experiments listed below, and co-wrote the manuscript with Freda Miller and David Kaplan. John Vessey performed and analyzed experiments in figure 4.4F, 4.4J, 4.4K and 4.4L. Sarah Burns performed in utero electroporations for some of the in vivo experiements. Both John Vessey and Hideaki Tomita provided advice.

Freda Miller and David Kaplan helped design experiments, provided support, advice and guidance. This work was supported by Canadian Institutes of Health Research (CIHR) Grant

MOP 13958 from the CIHR to Freda Miller and David Kaplan. David Tsui was supported by a

CIHR CGS-D scholarship.

David Tsui performed all the experiments in Chapter 5.

TABLE OF CONTENTS

ABSTRACT ii ACKNOWLEDGEMENTS iii PUBLICATION AND AUTHOR CONTRIBUTIONS iv TABLE OF CONTENTS v LIST OF FIGURES viii LIST OF ABBREVATIONS ix

CHAPTER 1: INTRODUCTION 1

1.1 Neurogenesis in the developing cortex 2

1.1.1 Overview of murine cortical neurogenesis 4

A) Transition from neuroepithelial stem cells to radial precursor cells 4

B) Direct neurogenesis from radial precursor cells 6

C) Indirect neurogenesis via generation of basal progenitors 10

1.1.2 Extrinsic and intrinsic factors regulating neurogenesis 14

A) Extracellular signaling – ligands and receptors 14

B) Signaling cascades 25

C) Transcription factors 26

D) Epigenetic factors 32

1.2 Neurogenesis in the developing ventral forebrain 34

1.2.1 Overview of ventral forebrain neurogenesis 34

A) Generation of interneurons and basal forebrain cholinergic neurons 35

B) Tangential migration of cortical interneurons 38

C) Origin of interneuron subtypes 39

1.2.2 Extrinsic and intrinsic factors regulating neurogenesis 40

A) Regulation of interneuron generation 40

1.3 CREB binding protein (CBP) 44

1.3.1 CBP and Rubinstein-Taybi syndrome 44

1.3.2 Role of CBP in muscle and hematopoetic differentiation 45

1.3.2 Role of CBP in neural differentiation 46

1.4 Forkhead box protein P2 (FoxP2) 46

1.4.1 FoxP2 and specific language impairment 46

1.4.2 Mouse models of FoxP2 mutants 47

CHAPTER 2: OVERALL HYPOTHESIS AND AIMS 57

CHAPTER 3: MATERIALS AND EXPERIMENTAL PROCEDURES 59

Animals 59

Primers and Plasmids 59

Cortical precursor cell cultures 60

Medial ganglionic eminence precursor cell cultures 61

In utero electroporation 62

Immunocytochemistry and histological analysis 62

Western blot analysis 63

Flow cytometry 64

HEK-293 experiments 65

RT-PCR 65

Microscopy and quantification 65

Statistics 67

CHAPTER 4: FOXP2 PROMOTES NEUROGENESIS IN THE MAMMALIAN CORTEX

Abstract 68

Introduction 69

Results 70

Figures 83

Discussion 102

CHAPTER 5: CBP REGULATES INTERNEURON DIFFERENTIATION AND

MATURATION 107

Abstract 107

Introduction 108

Results 110

Figures 117

Discussion 127

CHAPTER 6: DISCUSSION and FUTURE DIRECTIONS 131

CHAPTER 7: REFERENCES 145

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LIST OF FIGURES

Figure 1.1: Evolution of cortical development 49 Figure 1.2: Receptor tyrosine kinase (RTK) activation of four different pathways 51 Figure 1.3: Wnt binding activates transcription of genes that mediate precursor maintenance 53 Figure 1.4: Parallel pathways regulate neurogenesis in the medial ganglionic eminence 55

Figure 4.1: Expression of FoxP2 in embryonic cortical precursors 83 Figure 4.2: FoxP2 knockdown decreases neurogenesis in the embryonic cortex in vivo 86 Figure 4.3: FoxP2 knockdown increases radial precursors at the expense of intermediate progenitors in the embryonic cortex 89 Figure 4.4: FoxP2 knockdown increases radial precursors at the expense of intermediate progenitors and neurons, and this is reversed by coincident expression of human FoxP2 92 Figure 4.5: Ectopic expression of human but not murine FoxP2 in the murine embryonic cortex enhances the genesis of intermediate progenitors and neurons 95 Figure 4.6: Ectopic expression of human but not mouse FoxP2 promotes genesis of intermediate progenitors and neurons 98 Figure 4.7: The KE family FoxP2 mutation acts as a dominant-negative with regard to embryonic cortical neurogenesis 100

Figure 5.1: Expression of CBP in medial ganglionic eminence (MGE) precursors 117 Figure 5.2: CBP knockdown in MGE precursors reduces neurogenesis without affecting precursor survival or proliferation 119 Figure 5.3: CBP knockdown in MGE precursors reduces the percentage of neurons expressing mature interneuron markers 121 Figure 5.4: CBP knockdown in MGE precursors reduces the percentage of neurons expressing mature interneuron markers 123 Figure 5.5. The interneuron development deficit caused by CBP knockdown is rescued by WT-CBP and the HDAC inhibitor Trichostatin A (TSA) 125

Figure 6.1: Schematic of the proposed pathway of FoxP2 in cortical neurogenesis 141 Figure 6.2: Schematic of the protein structure of CBP and p300 143

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LIST OF ABBREVIATIONS

AF-6 ALL-1 fusion partner from chromosome 6 APC Adenomatous polyposis coli aPKC Atypical protein kinase C Arx Aristaless-related homeobox protein ASPM Abnormal spindle-like microcephaly-associated protein BDNF Brain-derived neurotrophic factor BLBP Brain-lipid-binding protein BMP Bone morphogenetic protein BrdU Bromodeoxyuridine BSA Bovine serum albumin C/EBP CCAAT/enhancer-binding proteins CADM1 Cell adhesion molecule 1 CBF1 C repeat binding factor 1 CBP CREB binding protein CC3 Cleaved caspase 3 CGE Caudal ganglionic eminence ChAT Choline acetyltransferase ChIP Chromatin immunoprecipitation CMV Cytomegalovirus CNTF Cilliary neurotrophic factor CNTNAP2 Contactin associated protein-like 2 CP Cortical plate CRE cAMP responsive element CREB cAMP-response element binding protein CTBP1 C-terminal-binding protein 1 Ctip2 COUP-TF-interacting protein 2 Cux1 Cut-like homeobox 1 Cux2 Cut-like homeobox 2 DBH Dopamine-beta-hydroxylase Dbx1 Developing brain homeobox protein 1 Dcx Doublecortin DISC1 Disrupted in schizophrenia 1 Dll Delta-like1 Dlx Distal-less Dlx6as Distal-less homeobox 6, antisense DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl sulfoxide Dsh Disheveled E12 Embryonic day 12

ECL Enhanced chemiluminescence EGFP Enhanced green fluorescent protein Emx2 Empty spiracles homeobox 2 ERK Extracellular signal regulated kinase EV Empty vector Evf1/2 Embryonic ventral forebrain-1/2 FBS Fetal bovine serum FGF Fibroblast growth factors FGFR Fibroblast growth factor receptors FoxG1 Forkhead box G1 FoxP2 Forkhead box protein P2 GABA γ-aminobutyric acid Gad67 Glutamic acid decarboxylase-67 GCN5 General control of amino-acid synthesis 5 GFAP Glial fibrillary acidic protein GFP Green fluorescent protein GLAST Glutamate aspartate transporter GSK3β Glycogen synthase kinase 3 β HATs Histone acetyltransferases HBSS Hank's Balanced Salt Solution HDACs Histone deacetylases HEK-293 Human embryonic kidney 293 HRP Horseradish peroxidase HuD Hu antigen D ILK Integrin-linked kinase ISVZ Inner subventricular zone ITR Inverted tandem repeats IZ Intermediate zone JAK Janus activated kinase LEF Lymphocyte enhancer factor LGE Lateral ganglionic eminence Lhx6 LIM homeobox 6 LMO4 LIM domain only protein 4 LPR LDL receptor-related protein MAPK Mitogen activated protein kinase MCK M creatine kinase MEK Mitogen activated Erk kinase mFz Members of the Frizzled family MGE Medial ganglionic eminence MHC Myosin heavy chain Mib Mind bomb

x mInsc Mouse Inscuetable mRNA Messenger RNA MZ Mantle zone N-CoR Nuclear receptor copressor NeuN Neuron-specific nuclear protein NGF Nerve growth factor Ngn2/Ngn2 Neurogenin 1/Neurogenin 2 NICD Notch intracellular domain Nkx2.1 NK2 homeobox 1 NLI Nuclear LIM interactor NT-3/NT-4 Neurotrophin-3/neurotrophin-4 OSVZ Outer subventricular zone Otx1 Orthodenticle homeobox 1 P3 Postnatal day 3 p75NTR p75 neurotrophin receptors Pax6 Paired box gene 6 PB PiggyBac PBS Phosphate-buffered saline PCAF P300/CBP-associated factor PDGF Platelet-derived growth factor PDK1 Phosphoinositide-dependent protein kinase 1 PFA Paraformaldehyde Phox2a Paired-Like Homeobox 2a PI3K Phosphatidylinositol-3 kinase Pins Partner of Inscuteable PIP2 Phosphatidylinositol (4,5)-bisphosphate PIP3 Phosphatidylinositol (3,4,5)-trisphosphate PLAP Placental alkaline phosphatase PLC γ Phospholipase C γ PMSF Phenylmethanesulfonyl fluoride PVE Pseudostratified ventricular epithelium R26R ROSA26 reporter RBPJ Recombination Signal-Binding Protein 1 for J-Kappa RPM Rounds per minute Rsk Ribosomal S6 kinases RTK Receptor tyrosine kinase RTS Rubinstein-Taybi syndrome Satb2 Special AT-rich sequence-binding protein 2 SEM Standard error of the mean SFRP Secreted Frizzled Related Protein Shh Sonic hedgehog

SHP-2 Src homology domain protein tyrosine phosphatase-2 shRNA Short hairpin RNA siRNA Small interfering RNA SMRT Silencing mediator for retinoid or thyroid-hormone receptors Sox2 SRY-related HMG-box 2 SP Subplate SPP Secondary proliferative population SRC-2 Steroid receptor coactivator 2 SST Somatostatin STAT Signal transducer and activator of transcription Svet1 Subventricular zone expressed transcript 1 SVZ Subventricular zone TAF250 TATA box binding protein (TBP)-associated factor, 250kDa Tbr1/Tbr2 T-brain gene-1/T-brain gene-2 TBST Tris-buffered saline with Tween TCF T-cell factor TH Tyrosine hydroxylase TRPC6 Transient receptor potential cation channel, subfamily C, member 6 TSA Trichostatin-A vGlut Vesicular glutamate transporter 1 VZ Ventricular zone WT Wild-type XNGN-1 Xenopus Ngn-1

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CHAPTER 1: INTRODUCTION

All developmental neurobiologists ask one question: how does our nervous system begin as a sheet of neuroepithelium and end up with the complex network that underlies everything from homeostasis to higher cognition? Because of its involvement in higher cognition, the cerebral cortex has been a major focus in the study of neural development. We now know that there is an estimated 86 billion neurons in an adult male brain, of which 19%, or approximately

16 billion, reside in the cerebral cortex (Azevedo et al., 2009). What are the cellular and molecular players that contribute to the formation of this complex structure? Specifically, how are different regions of the brain specified? Is there a general program for cellular differentiation in the different precursor niches, or do they follow different developmental programs? How do environmental signals integrate to influence the fate of the cells? Downstream of the signaling cascade, what are the intracellular factors that regulate cell fate determination? And once a cell fate is determined, what factors are required for the terminal differentiation of the cells so that they can form the correct synapses and adopt the correct biochemistry and structure to carry out their function? My thesis will address some of these fundamental questions of neural development, specifically by using molecular biology techniques to study the role of intracellular factors in the differentiation of two different neuronal cell types of the cerebral cortex – projection neurons and interneurons. However, my thesis will not address gliogenesis, which occurs at later stages in cortical development.

I would like to first define several terms as they will be used in this thesis. The cortical germinal epithelium that I study is heterogeneous and contains several different types of precursors that may have different differentiation potential and self-renewal capacity. I therefore use the term “cortical precursors” to describe the mixture of cells isolated from embryonic day

12-13 cortices, which is the system of my study. A second ambiguity involves the terms cell fate determination and cell differentiation. Determination refers to the process in which a developmental choice is made among various options by a cell at a timepoint when the cell has not acquired its ultimate properties. Differentiation, on the other hand, refers to the process in which the cell acquires the actual biochemical, structural and functional changes that result in the specific cell type. While clearly defined, sometimes these possibilities are not clearly distinguished due to reasons such as the lack of prospective markers at earlier developmental stages, or the difficulty of coincidentally examining an alternative cell fate. Throughout the text, these terms are used as closely as they fit the above definitions but may be interchangeable at times when the evidence provided allows for ambiguous interpretation.

1.1 Neurogenesis in the developing cortex

Perhaps one of the most studied neurogenic niches in the developing central nervous system is the cerebral cortex. The mammalian cerebral cortex has six layers and each layer contains neurons that share a characteristic morphology, connection and gene expression pattern.

While the precursors that generate neurons are situated adjacent to the ventricles on the apical side of the cortex, neurons are generated in an inside-out fashion, meaning that the layers that are eventually closest to the precursors are generated first and in such a way, later born neurons must migrate past the earlier born neurons to get to their final destination. It was proposed that there is an intrinsic program encoded within individual precursors that drives the production of the different neuronal layer specific subtypes in an orderly fashion (Shen et al., 2006). However, it is clear that environment also plays a role. This is exemplified by a study by Mizutani et al

(Mizutani and Saito, 2005). Using electroporation, they first expressed a constitutively activated

2 form of Notch at embryonic day 13.5 when deep layer neurons are generated. This inhibited the precursors from generating neurons and kept them in the radial precursor state. They then force- expressed a Cre vector in the same region to excise the constitutively activated form of Notch at embryonic day 15.5, thereby removing Notch signaling in these precursors. Interestingly, they found that not only do these precursors resume neurogenesis, but they skipped the generation of deep layer neurons and now generated upper layer neurons. This pair of studies shows that neuronal subtype production depends on both intrinsic and extrinsic factors, and this interplay between the two is a recurrent theme in neural development.

On a cellular level, how are neurons generated from early stem cells? Early in development, just like the rest of the central nervous system, the cortex is composed of a single layer of proliferating neuroepithelial cells. At around embryonic day 10 to 12 during mouse development, these neuroepithelial cells gradually convert into radial precursor cells, and over the course of the next 7 days until the birth of these animals, these radial precursors will generate virtually all neurons from layer II to layer IV of the cortex (Takahashi et al., 1996). Early on, neurons are generated at a relatively slow rate as these radial precursors divide symmetrically to expand their pool. As neurogenesis proceeds, increasing numbers of asymmetrical divisions occur where radial precursors divide to form a daughter radial precursor and at the same time give rise to either a neuron or a transit amplifying cell called an intermediate progenitor (also called basal progenitors) (Noctor et al., 2004). The intermediate progenitors then divide at least once more to eventually give rise to two neurons. At late stages, approximately 90% of intermediate progenitor divisions result in 2 neurons, while in the other 10% of the cases, the progeny will divide again in the subventricular zone. Finally, towards the end of the neurogenic phase, some of these radial precursors may undergo terminal differentiation, meaning that they

3 divide symmetrically to give rise to two neurons (Haydar et al., 2003). However, most of the radial precursors give rise to, or transform into, glia after the neurogenic phase (Misson et al.,

1988; Noctor et al., 2004). Some of these precursors may also stay behind and eventually contribute to the pool of adult forebrain stem cells (Bonfanti and Peretto, 2007).

1.1.1 Overview of murine cortical neurogenesis

A) Transition from neuroepithelial stem cells to radial precursor cells

Neuroepithelial stem cells are the ectodermal cells that committed to a neural fate. These dividing neuroepithelial stem cells comprise the entire neuroepithelium at embryonic days 8-9.

They are bipolar, with an apical and a basal process contacting the ventricular and pial surfaces respectively. They also express the SRY-related HMG-box transcription factor Sox1, which when overexpressed transiently in the P19 cell line or embryonic stem cells induced neural fate

(Pevny et al., 1998; Zhao et al., 2004). In a process called interkinetic nuclear migration, which was first described by Sauer in 1935, the nuclei of these neuroepithelial stem cells migrate up and down the apical-basal axis during the cell cycle, thereby forming a pseudostratified neuroepithelium (Gotz and Huttner, 2005). The apical-basal polarity depends on the tight junction protein ALL-1 fusion partner from chromosome 6 (AF-6) as prominin (or CD133) localization is dramatically affected in AF-6 mutants (Zhadanov et al., 1999). Interestingly, at least in the in vitro setting, AF-6 is able to interact with H-Ras (Van Aelst et al., 1994; Kuriyama et al., 1996) and overexpression of Ras resulted in perturbation of cell-cell contact in conjunction with reduction in AF-6 and the tight junction protein ZO-1 on the cell surface (Yamamoto et al.,

1997). This is one of the many aspects of neural precursor behaviour in which the MAP kinase pathway is involved (see Signaling cascades section).

Between embryonic day 10 to embryonic day 12, these neuroepithelial stem cells undergo a transition to become radial precursor cells, which express the transcription factor Pax6

(Mori et al., 2005). Several extrinsic and intrinsic factors have been found to be important for this transition to take place. For extrinsic factors, a number of lines of evidence indicate that

Notch1 and Fgf signaling play a crucial role in the transition from neuroepithelial stem cells to radial precursors. First, overexpression of activated Notch by viral transduction in embryonic day

9.5 telencephalic vesicles resulted in induced expression of radial precursor markers such as

RC2, Nestin and brain-lipid-binding protein (BLBP) (Gaiano et al., 2000). Second, Blbp expression is abolished in a conditional knockout of Notch1 and Notch3 using the telencephalic driver Foxg1 (Anthony et al., 2005). More recently, it was found that Fgf10 is transiently expressed between embryonic day 9.5 and day 11.5 and regulates the transition from neuroepithelial stem cells to radial precursor cells (Sahara and O'Leary, 2009). When this transition is delayed in Fgf10 knockout brains, the expansion phase of the cortex is lengthened, thereby resulting in thicker cortices. In terms of intrinsic factors, overexpression of Sox1 not only promotes the neuroepithelial stem cell fate, but it also maintains them in the neuroepithelial stage (Suter et al., 2009). Moreover, knockdown of Sox1 resulted in exit from the neuroepithelial stage. Conversely, overexpression of Pax6 drives the transition of neuroepithelial cells to radial precursor cells, while knockdown of Pax6 represses this commitment to radial precursors. These results suggest that Sox1 is important in both promoting and maintaining neuroepithelial stem cell fate, while Pax6 promotes the transition of neuroepithelial stem cells to radial precursors, and they achieve these functions by either directly or indirectly regulating each other. In addition, Sox1 may also maintain neuroepithelial stem cell fate by repressing Prox1, which promotes cell cycle exit and neurogenesis (Elkouris et al., 2011).

Several changes occur as neuroepithelial stem cells are being converted into radial precursors. First, tight junction markers such as occludin are lost while adherens junction markers such as ZO-1 and N-cadherin, which were already present in neuroepithelial cells, are upregulated (Aaku-Saraste et al., 1996). Second, these radial precursors now start to express glial markers such as the glutamate aspartate transporter (GLAST), BLBP and S100β (Mori et al.,

2005). Finally, while the nuclei in neuroepithelial stem cells migrate through the entire cytoplasm in interkinetic nuclear migration, their migration is confined only to the ventricular/subventricular zone when they become radial precursors (Gotz and Huttner, 2005).

B) Direct neurogenesis from radial precursor cells

Radial precursor cells are also bipolar like neuroepithelial stem cells, with an apical endfoot in contact with the ventricle and a long radial fiber extending to the pia. They were once thought to be a type of astrocyte, because they express astrocytic markers, and one of their functions is to guide radial migration of newborn neurons in the cortex (Rakic, 1972); hence, they were called radial glia. It was only about a decade ago when these cells were found to be the precursors of various cell types of the cortex. Time lapse imaging of retrovirally- labelled clones in the cortex showed that these radial precursors divide and give rise to neurons that migrate along their radial fibers (Noctor et al., 2001). Similar results have also been shown in culture when DiI was placed on the surface of the embryonic day 14 cortex, thereby selectively labelling those cells that have processes in contact with the pial surface, 15% of DiI-sorted cells express one or more radial precursor markers (Malatesta et al., 2000). Most importantly, when examined in culture for 7 days, these radial precursors give rise to neurons in the clones they generate, supporting the idea that they themselves are the precursors for cortical neurons.

After neurons are born in the ventricular/subventricular zone, they migrate to the cortical plate where they eventually reside and form synapses and participate in neuronal circuits. Two types of migration have been proposed for the newborn neurons to reach their final destination – locomotion, in which the entire cell migrates along the radial fibers, and somal translocation, in which the nucleus of the cell translocates along the leading process that the cell extends in the direction of its migration, a process that does not require the guidance of radial fibers (Nadarajah et al., 2001). It is generally agreed that the radial fiber is retained during all phases of cell division (Miyata et al., 2001; Weissman et al., 2003), but specifically which of the daughters inherit the radial fiber has been an issue of debate. Miyata et al. has shown that in the majority of cases, the neuron daughter is the one that inherits the radial fiber, which may be responsible for somal translocation. On the other hand, Weissman et al., has shown that only 12.8% of the cells contacting the pia surface have morphology of translocating neurons, while 87.2% of the cells exhibit radial precursor morphology. Nevertheless, these different modes of translocation may explain the differential genetic contributions to neuronal migration in the developing cortex.

Just like neuroepithelial stem cells, radial precursor cells also undergo interkinetic nuclear migration with nuclear positions corresponding to their status in cell cycle. In this process, the nuclei of radial precursor cells migrate up and down the apical-basal axis of the ventricular/subventricular zone. At the apical surface, the nucleus goes through M phase and generates two progeny. The nucleus then migrates up as it goes through G1 phase to the basal side where it undergoes S phase. Finally the nucleus travels back down to the apical surface as it goes through G2 phase. (Del Bene, 2011). The time it takes to go through the cell cycle is not constant, however, as the cell cycle progressively slows down over the course of neurogenesis.

Specifically, cell cycle duration increases from 8 hours at the beginning of neurogenesis to

7 almost 20 hours towards the end of the neurogenic period (Takahashi et al., 1995; Kornack and

Rakic, 1998). This is mainly attributable to the fact that the length of the G1 phase of cortical precursors increases progressively over the neurogenic period (Caviness et al., 1995).

Perhaps the most studied phase of the cell cycle is the M phase, with specific regard to the relationship between the way radial precursors divide and the fate of their daughter cells.

During metaphase of the cell cycle, the mitotic spindles of radial precursors (which will ultimately determine the plane of cleavage and hence the mode of division) either exhibit little or no rotation or show wide oscillations prior to getting “tuned” to a certain final orientation at the onset of anaphase (Haydar et al., 2003). The correlation between cleavage plane orientation and the resulting daughter cell fates was first proposed by Chenn and McConnell in 1995 (Chenn and

McConnell, 1995). In that study, they suggested that daughters of divisions with vertical cleavage planes will continue to proliferate while daughters of divisions with horizontal cleavage planes will eventually migrate away and become neurons. This idea was challenged by the observation that towards the end of neurogenic phase, the cleavage plane reverts back to vertical at a time when most of the divisions are neurogenic (Haydar et al., 2003). It now appears that it is not the plane of cleavage that determines the fate of the progeny of radial precursor divisions, since the majority of the planes of cleavage are nearly vertical and very few of the planes of cleavage are horizontal (Kosodo et al., 2004). Rather, it is the asymmetric distribution of the apical plasma membrane that is the ultimate factor in determining the fate of the daughter cells.

It is estimated that the apical plasma membrane of radial precursor cells only constitutes 1-2% of the total plasma membrane of the cell (Kosodo et al., 2004). 80% of divisions with equal distribution of apical plasma membrane resulted in daughter cells that did not express the neurogenic marker Tis21, suggesting that the division is proliferative. On the other hand, 90% of

8 cells with unequal distribution of apical plasma membrane expressed Tis21, which is suggestive of neurogenic divisions. These results suggest that proliferative versus differentiative divisions are linked to equal versus unequal distribution of apical membrane.

How do differences in distribution of the apical membrane result in different cell fates?

The most likely explanation is that with the asymmetric distribution of apical membrane, the daughters also differentially inherit molecular determinants in the apical membrane that are important for cell fates. Indeed, Par3, which is associated with the apical membrane complex

(Manabe et al., 2002), is differentially inherited by the daughter cells when the apical membrane is unequally distributed (Kosodo et al., 2004). Interestingly, Par3 loss of function resulted in premature cell cycle exit while overexpression of Par3 or another member of the Par complex,

Par6, promotes the generation of Pax6+ self-renewing progenitors (Costa et al., 2008). This data supports the idea that cleavage plane orientation, or distribution of apical membrane constituents, is a crucial determinant of daughter cell fates. Several mechanisms have been implicated in the regulation of cleavage plane orientation. First, Emx2, which along with Pax6 is one of the earliest markers of dorsal regions of the telencephalon, promotes vertical cleavage-plane orientation as well as symmetric, proliferative divisions of radial precursors (Heins et al., 2001).

A second example is Lfc, which is a Rho-specific guanine nucleotide exchange factor that interacts with the mitotic spindle. Together with its negative regulator Tctex, it is also involved in regulating cortical neurogenesis and is required for determining the orientation of the mitotic spindle of radial precursors (Gauthier-Fisher et al., 2009). Third, knockdown of the abnormal spindle-like microcephaly-associated (Aspm) protein, which when mutated in humans causes microcephaly (Bond et al., 2002), resulted in a robust reduction in the percentage of dividing cells that adopt a vertical cleavage plane and a concomitant increase in the percentage of cells

9 that adopt a neuronal fate (Fish et al., 2006). This suggests that Aspm maintains symmetric proliferative divisions by maintaining a vertical cleavage plane of dividing cells. Finally, a recent study showed that there is an apically-localized RNA granule comprised of the RNA- binding proteins Staufen2 and Pumilio2 that likely acts to repress translation of associated mRNAs, and that this granule, which is asymmetrically-segregated during radial precursor mitoses, plays an important role in cortical precursor maintenance (Vessey et al., 2012).

Together, these examples show that mitotic spindle and cleavage plane orientation plays an important role in the consequences of the resultant precursor division and suggests that this effect of cleavage plane orientation is mediated via a change in the distribution of cell fate determinants associated with the apical membrane.

C) Indirect neurogenesis via generation of intermediate progenitors

It is estimated that approximately 11 precursor cell cycles contribute to the genesis of rodent cortical neurons during the neurogenic phase of cortical development, but it is the final 4 divisions between embryonic day 14 to day 17 that generate the majority of the neurons

(Caviness et al., 1995). How do the last 4 cycles generate more neurons than the preceding 7 cycles? One possible answer to this question lies in the existence of a second, transit amplifying pool of progenitors. In addition to the direct genesis of neurons by dividing radial precursors at the apical surface of the developing cortex, a second population of progenitors divide in the basal region of the proliferative zone and give rise to two neurons (Haubensak et al., 2004). These progenitors, termed intermediate or basal progenitors, exist in both the ventricular and subventricular zones and appear either as short radial cells with or without ventricular contact, or as multipolar cells that are situated in the subventricular zone (Kowalczyk et al., 2009). These

10 progenitors express the non-coding RNA Svet1 (Tarabykin et al., 2001), and the transcription factors Tbr2 (Englund et al., 2005), Cux1 and Cux2 (Nieto et al., 2004).

Intermediate progenitors are generated from divisions of radial precursors at the surface of the ventricle (Noctor et al., 2004). The transcription factor Tbr2 is a marker for intermediate progenitors that functions to drive the conversion from radial precursors to intermediate progenitors (Sessa et al., 2008). The zinc-finger transcription factor Insm1 also promotes generation of intermediate progenitors (Farkas et al., 2008). Finally, a loss- and gain-of-function study showed that mouse Inscuteable (mInsc), which is an adaptor that links the Par-aPKC complex with the Pins complex (Schober et al., 1999; Wodarz et al., 1999; Schaefer et al., 2000;

Yu et al., 2000), is necessary and sufficient for generation of intermediate progenitors, respectively, and subsequently the proper formation of adult cortical layers (Postiglione et al.,

2011). Interestingly, this study also showed that mInsc controls mitotic spindle orientation of radial precursors, suggesting that mitotic spindle orientation may be a deciding factor for the generation of intermediate progenitors. Consistent with this idea, it was discovered that the exon junction complex component Magoh is required for generation and/or maintenance of intermediate progenitors, and also controls mitotic spindle orientation by regulating Lis1 expression (Silver et al., 2010).

What is the relative contribution of direct neurogenesis from radial precursors and indirect neurogenesis via these intermediate progenitors? It has been postulated that intermediate progenitors only generate upper layer neurons. This hypothesis is based on the observation that markers of upper layer neurons (Cux1, Cux2 and Svet1) are also expressed in the subventricular zone at or prior to the onset of upper layer neurogenesis (Tarabykin et al., 2001; Nieto et al.,

2004). However, thus far there is no evidence that directly shows that intermediate progenitors

11 preferentially contribute to upper rather than lower layer neurons. In contrast, one study estimates that intermediate progenitors contribute 100% and 32-44% of cortical neurons at embryonic days 13 and 14, a period which mostly generates deep layer neurons (Miyata et al.,

2004). In fact, another study using a Tis21-GFP transgenic line, showed that from embryonic day 10.5 to day 18.5, the neurogenic fraction from intermediate progenitors was 40% to 80% while the apical neurogenic fraction remained at 10-20% (Kowalczyk et al., 2009). Based on this observation, they estimated that more than 80% of all cortical neurons are generated via intermediate progenitors. Finally, the fact that perturbations that affect the generation and/or maintenance of intermediate progenitors also cause profound deficits in the generation of all cortical layers (Sessa et al., 2008; Silver et al., 2010; Postiglione et al., 2011) supports the idea that this pool of transit amplifying progenitors is indeed required for the generation of all cortical layers. However, this conclusion is contradicted by a time lapse imaging study by Noctor et al.

(2004), which found that towards the end of neurogenesis from embryonic day 17 to day 19,

65.8% of radial precursor divisions are of the neurogenic type, while only 7.3% of radial precursor divisions generate a non-radial-precursor progeny that divides again, which is presumably an intermediate progenitor. Future studies are required to resolve these views but intermediate progenitors are clearly important for the generation of a major portion of neurons in the cerebral cortex.

What is the function of intermediate progenitors? Over the course of evolution, the cerebral cortex evolved from a three-layer structure in chicken and reptiles to a six-layer structure in mammals such as mice (reviewed in Striedter, 2005) (Fig. 1.1). Furthermore, convolutions called sulci and gyri evolved to accommodate the increase in cortical surface area.

Interestingly, the proportion of abventricular divisions, which occur at a distance to the

12 ventricular surface and are likely divisions of intermediate progenitors to generate two neurons, increased from the three-layered turtle cortex to the sixed-layered rat cortex, and further increased in the gyrencephalic six-layered ferret cortex (Martinez-Cerdeno et al., 2006). Because intermediate progenitors add mitotic cycles away from the ventricular surface, thereby increasing the available pool of neurogenic precursors, it is postulated that they contribute to the expansion of the cerebral cortex via an increase in cortical surface area and/or cortical thickness.

Perturbations of genes that increase or decrease the number of intermediate progenitors suggest that their abundance is associated with cortical thickness and not cortical surface area (Arnold et al., 2008; Farkas et al., 2008; Sessa et al., 2008; Postiglione et al., 2011).

Recently, a new type of cortical precursor has been characterized and is thought to also contribute to the expansion of the cortex. These cells were first called outer radial glia because they were first discovered to populate the outer subventricular zone of the developing human cortex (Hansen et al., 2010). However, later studies changed the name to intermediate or basal radial glia because these precursors are not confined to the outer subventricular zone but also exist in the inner subventricular zone of the human cortex (Reillo et al., 2011). Perhaps one could argue that another reason for the name change is because a similar cell type was discovered in the developing mouse cortex, which lacks the distinction of outer and inner subventricular zone

(Wang et al., 2011b). Similar to the “traditional” radial precursors, these basal radial glia, which have been characterized independently by three labs (Fietz et al., 2010; Hansen et al., 2010;

Reillo et al., 2011), express Pax6 and Sox2, but not Tbr2. However, unlike radial precursors, these basal radial glia do not have an apical process, but retain a basal process. In mice, this population of cells represent only 2-9% of total mitotic precursors in the developing cortex depending on age and region (Wang et al., 2011b). In humans, proliferating cells in the outer

13 subventricular zone alone account for 40-75% of total proliferating cells (Hansen et al., 2010).

While this population includes both basal radial glia and intermediate progenitors, it does emphasize the extensive contribution of these populations in human cortical neurogenesis. In mice, basal radial glia are derived from radial precursors, and they divide to self-renew and to generate neurons (Wang et al., 2011b). In humans, these basal radial glia are also capable of generating intermediate progenitors (Hansen et al., 2010). Interestingly, these basal radial glia undergo mitotic somal translocation instead of the interkinetic nuclear migration exhibited by radial precursors (Wang et al., 2011b). During somal translocation, the centrosome of basal radial glia move into the basal processes during interphase, followed by saltatory movements of the nuclei. Similar to radial precursors, basal radial glia also require Notch signaling to remain undifferentiated (Wang et al., 2011b).

1.1.2 Extrinsic and intrinsic factors regulating neurogenesis

A) Extracellular signaling – ligands and receptors

As with all other developmental systems, cortical development is strongly influenced by the major extracellular signaling pathways of Notch, FGF, Sonic Hedgehog and Wnt. While each of these signaling pathways have distinct cellular targets, the fate of the neural precursor cells depend on the integration of these extracellular signaling mechanisms. I will briefly touch on the influence of each of these signaling pathways and how they interact with each other during cortical development. In addition, I will also write about the role of neurotrophins which are particularly important for neural precursor proliferation and neurogenesis in the context of cortical development.

Notch

Perhaps the most studied signaling pathway in the context of neural development is the

Notch pathway. The gene that encodes the Notch receptor was discovered in 1919 in Drosophila and the name was coined because haploinsufficiency of the gene resulted in “notches” at the wing margin (Artavanis-Tsakonas et al., 1999). Notch signaling is activated when the membrane-bound Notch ligands such as Delta and Jagged on one cell bind the Notch receptor on the neighbouring cell. This activation causes the Notch intracellular domain (NICD) to be cleaved by a γ-secretase complex. Once cleaved, the NICD translocates to the nucleus and, in the context of the developing mammalian nervous system, it interacts with CBF1/RBP-J to activate target genes such as the inhibitory bHLH transcription factors Hes1 and Hes5 (Ohtsuka et al.,

1999) and Blbp (Anthony et al., 2005). In the absence of Notch signaling, the SMRT/N-CoR complex interacts with RBP-J and suppresses RBP-J mediated transcription by competing with the NICD and by recruiting histone deacetylases (Kao et al., 1998; Zhou and Hayward, 2001).

While Notch receptors are ubiquitously expressed in the ventricular/subventricular zone, they are only activated in a subset of precursors. Specifically, immunostaining showed that activated Notch (NICD) is expressed in Nestin+ radial precursors but not in Ngn2+ intermediate progenitors (Tokunaga et al., 2004). Another study used a transgenic reporter of Notch signaling in which EGFP is expressed under the control of the CBF1/RBP-J-response element, and showed that EGFP is expressed in Nestin+ and CD133+ radial precursors (Mizutani et al., 2007b).

However, even cells that expressed low/no EGFP immunostained for activated Notch, suggesting that not all cells activate CBF1/RBP-J in response to Notch activation. Finally, Hes reporters were used to more definitively show that Notch signaling is predominant in radial precursors but not in intermediate progenitors (Kohyama et al., 2005; Mizutani et al., 2007b). However, a more

15 recent study adds additional complexity to this issue. Using real-time imaging of dissociated precursor cultures and slice cultures, Kageyama’s group discovered that expression of Hes1 as well as the Notch ligand Delta-like1 (Dll) dynamically oscillates within single precursor cells in the cortex (Shimojo et al., 2008). Interestingly, they found that Hes1 is downregulated at early

G1 phase in all cells but is expressed again at various levels during S and G2 phases, and remains absent in neurons and neuronal progenitors.

Numerous studies have examined the effects of perturbing various components of the

Notch pathway including Notch receptors (de la Pompa et al., 1997; Hitoshi et al., 2002), Notch ligands (Grandbarbe et al., 2003), CBF1/RBP-J (de la Pompa et al., 1997; Hitoshi et al., 2002;

Imayoshi et al., 2010) and target genes such as hes family members (Ishibashi et al., 1995;

Ohtsuka et al., 1999; Nakamura et al., 2000; Hatakeyama et al., 2004), and found that in general

Notch signaling is required for maintenance of neural precursors in culture and in vivo. On the other hand, activation of Notch signaling leads to suppressed neurogenesis and maintenance of radial precursor identity (Gaiano et al., 2000; Mizutani and Saito, 2005). It is unclear, however, what effect constitutive Notch activation has on precursor proliferation. When the NICD was introduced by viral infection into the embryonic forebrain and assessed by BrdU labelling, it was found that some infected clusters were proliferatively active, while others were quiescent

(Gaiano et al., 2000). Indeed, one study performed by Kageyama’s group suggested that it may be the oscillation in Notch signaling that is crucial for precursor proliferation. They found that persistent high level of Hes expression not only inhibited neuronal differentiation by repressing neurogenic genes, but also led to G1 phase retardation by repressing cell cycle regulators

(Shimojo et al., 2008). These results further support the idea that oscillations in Notch activation are required for sustained cell cycle progression and maintenance of neural precursors. At the

16 precursor level, Notch signaling is also required for the conversion between radial precursors and intermediate progenitors. Whereas knock down of CBF1/RBP-J leads to conversion of neural precursors to intermediate progenitors, activation of CBF1/RBP-J is sufficient to convert intermediate progenitors back into neural precursors (Mizutani et al., 2007b). Finally, numerous studies have shown that ectopic expression of the intracellular domain of Notch promotes generation of astrocytes during embryonic stages (Gaiano et al., 2000; Chambers et al., 2001).

So where is the source of Notch ligand in the developing cortex? In the Drosophila nervous system, Notch and its ligand Delta are expressed in all progenitors and it is the unequal segregation of Notch regulators such as Numb and Neuralized that ensures Notch signaling is activated in only one of the siblings, thereby leading to binary cell fate decision (Bardin et al.,

2004). In the mammalian telencephalon, Notch ligands Dll1 and Dll3 are not expressed in the precursors themselves but are expressed instead by intermediate progenitors and new-born neurons (Campos et al., 2001). This compartmentalization of Notch receptor and ligand expression suggest that in the mammalian telencephalon, Notch signaling serves as a negative feedback mechanism to regulate the balance between precursor maintenance and neurogenesis.

In support of this idea, it was found that an E3 ubiquitin ligase named Mind bomb (Mib), which regulates the endocytosis and thereby availability of Notch ligands, is expressed in intermediate progenitors and newborn neurons but not in radial precursor cells (Yoon et al., 2008). In this study, they showed that conditional ablation of Mib in the developing forebrain leads to decreased radial precursor maintenance and premature differentiation into intermediate progenitors and neurons.

FGFs

While Notch signaling is required for maintenance and self-renewal of neural precursors,

FGFs determine neural cell number by regulating neural precursor expansion. In mammals, there are 22 members of the FGF ligand family and 4 receptors – FGFR1-4 (Itoh and Ornitz, 2011).

Fgf2 transcripts are present in the cortex as early as embryonic day 9, while Fgf1 transcripts are first detected at embryonic day 11 (Nurcombe et al., 1993). In terms of receptors, FGFR1-3 are expressed at least as early as embryonic day 10, while FGFR4 is not expressed in the developing cortex (Qian et al., 1997; Kang et al., 2009). Members of the FGF family bind their receptors with varying affinity. For example, FGF1 binds all four FGFRs while FGF2 binds FGFR1,

FGFR2 and FGFR4 with high affinity (Dionne et al., 1990; Ornitz et al., 1992; Werner et al.,

1992). Importantly, the interaction of heparan sulfate proteoglycans with FGFs is a requirement for stable high affinity binding of FGFs with their receptors (Itoh and Ornitz, 2011). Binding of

FGFs to their receptors causes receptor dimerization, autophosphorylation and activation of four key signaling pathways: the Ras/MEK/ERK mitogen activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI-3K)/AKT pathway, the JAK/STAT pathway and the PLC γ pathway (Fig. 1.2)

It has been known for more than two decades that FGF2 is a mitogen for neural precursors. First, FGF2 was shown to stimulate proliferation of embryonic day 14 cortical precursor cells culture in mice (Ghosh and Greenberg, 1995) as well as embryonic day 13 forebrain precursors in the rat (Gensburger et al., 1987). Similar results were observed for embryonic day 16 hippocampal precursors, under both direct culture conditions as well as after being passaged (Vicario-Abejon et al., 1995). Finally, FGF2 was shown to elicit neural stem cell proliferation in neurospheres derived from the dissected neurogenic niche or embryonic stem cells (Tropepe et al., 1999; Hitoshi et al., 2004).

The in vitro finding that FGF2 promotes neural precursor proliferation is also supported by in vivo studies. For example, Fgf2 knockout mice show reduced cortical precursor proliferation early in neurogenesis and decreased numbers of cortical neurons at the end of neurogenesis (Vaccarino et al., 1999; Raballo et al., 2000). Importantly, the reduction in cortical precursors is not due to change in cell survival. Conversely, injection of FGF2 into the ventricles during early neurogenesis increases the proportion of proliferating precursors without affecting cell-cycle length and the total number of cortical neurons in adults (Vaccarino et al., 1999). On the receptor side, when FGFR1-3 are ablated in radial precursors using a GFAP-Cre driver, radial precursors are depleted as they commit to intermediate progenitors, suggesting that one of the key functions of FGF signaling is to inhibit the transition from radial precursors to intermediate progenitors (Kang et al., 2009). Also, expression of a dominant-negative tyrosine kinase-deficient FGFR1 under the regulation of the Otx1 promoter, which drives expression in the precursors of pyramidal neurons as well as pyramidal neurons during neurogenesis, decreased proliferative cells in the early cortex and the number of pyramidal neurons (Shin et al.,

2004). On the other hand, mice carrying a constitutively active Fgfr3 allele showed increased precursor cell proliferation and increased cortical neuron numbers (Inglis-Broadgate et al., 2005).

Finally while coactivation of Fgfr1 and Fgfr3 promoted symmetric division of neural precursors, transient knockdown of either caused asymmetric division, increased neurogenesis and a concomitant decrease in proliferation (Maric et al., 2007). Together there is ample evidence showing that FGF signaling promotes neural precursor proliferation and prevents neuronal differentiation, with the end result of expansion of the precursor pool and ultimately, an increase in cortical neuron numbers.

Interestingly, in addition to its proliferative effects, FGF2 also regulates the fate of neural progeny generated from cortical precursors. This is demonstrated by a study in which cortical precursors were cultured at varying concentrations of FGF2 during the differentiation phase of the experiment and it was discovered that at increasing concentrations of FGF2, more neuron- glia mixed clones were generated at the expanse of neuron-only clones, suggesting that FGF2 stimulates multipotent cortical precursors to generate glial progeny (Qian et al., 1997).

Wnt

Wnt signaling is also another important player in the regulation of cortical precursor maintenance. Wnt signaling is defined by the stabilization of β-catenin in response to a cascade triggered by Wnt binding to its receptor (Chenn, 2008). In the absence of Wnt signalling, the cytoplasmic protein Axin coordinates the formation of protein complex together with

Adenomatous Polyposis Coli (APC) and GSK3β (Fig. 1.3). This enables the phosphorylation of

β-catenin by GSK3β, which is then ubiquitinated and degraded by the cell. When Wnt binds to the Frizzled receptor and LRP co-receptor, this phosphorylates and activates Disheveled (Dsh), which binds to Axin and inhibits formation of the degradation complex. This series of events results in the “stabilization” of β-catenin, that prevents it from ubiquitination and degradation, and allows it totranslocate into the nucleus, interact with DNA-binding proteins of the TCF/LEF family and activates transcription of genes that drive proliferation such as cyclin D1 and c-myc

(He et al., 1998; Shtutman et al., 1999).

In the developing cortex, various Wnt ligands are expressed in the cortical hem, which is a transient structure located in the dorso-medial area of the embryonic telencephalon between the prospective hippocampus and the choroid plexus epithelium, including Wnt2b, Wnt3a, Wnt5a,

Wnt7b and Wnt8b, while Wnt7a is expressed in the ventricular zone of the cortex (Grove et al.,

1998). With respect to the relevant receptors, mFz-5 and mFz-8 are expressed in the ventricular zone of the cortex, as is SFRP-1 of the Secreted Frizzled Related Protein (SFRP) family of Wnt inhibitors (Kim et al., 2001). Perhaps the most well-known study demonstrating the role of Wnt signaling in cortical precursors is the study by Chenn and Walsh (2002) which showed that mice carrying a stabilized version of β-catenin developed enlarged brains with increased cortical surface area and the surprising formation of sulci and gyri-like structures (Chenn and Walsh,

2002). Transgenic animals expressing lower levels of the same stabilized version of β-catenin have enlarged forebrains characterized by cerebral cortices with increased surface area, variable lamination and neuronal heterotopias (Chenn and Walsh, 2003). Conversely, inhibition of Wnt-

β-catenin signaling by overexpression of a dominant-negative form of TCF in cortical precursors resulted in decreased production of Pax6-positive radial precursor pairs and an increased production of Tbr2-positive intermediate precursor pairs in precursor cell divisions (Noles and

Chenn, 2007). Conditional ablation of GSK3b in the developing nervous system of a GSK3a null background resulted in maintenance of radial precursor fate and suppression of the genesis of intermediate progenitors and neurons (Kim et al., 2009). While these results are consistent with a role of canonical Wnt signaling in cortical precursor proliferation, it is important to note that in addition to the Wnt signaling pathway, GSK3β is also downstream of NGF and Notch.

Furthermore, this conclusion is complicated by a recent study suggesting that β-catenin is not required for maintenance or differentiation of neural precursors, but rather regulates their adhesion and survival (Holowacz et al., 2011). Finally, in addition to its role in radial precursor biology, Wnt signaling is also required for differentiation of intermediate progenitors into

21 neurons (Munji et al., 2011). Ectopic overexpression of Wnt3a promoted expansion of radial precursors and differentiation of intermediate progenitors, as well as neuronal heterotopias.

Sonic Hedgehog

Although not as extensively studied, Shh signaling also plays important roles in cortical precursor maintenance. Shh signaling is triggered by Shh binding to Patched, which is a 12-pass transmembrane glycoprotein (Wicking et al., 1999). Smoothened is the key Shh signal transducer whose 7-pass transmembrane structure resembles that of G-protein coupled receptor, and is responsible for triggering intracellular signalling and the subsequent activation of target genes. In the absence of Shh, Patched interacts at the membrane with Smoothened, rendering it inactive.

Binding of Shh to Patched relieves the inhibition of Patched on Smoothened, thus activating downstream transcription complex including Gli family members, Fused and Sufu, ultimately resulting in transcription of downstream targets.

In the cerebral cortex, Shh is weakly expressed in the precursors, Cajal-Retzius cells and

GABAergic interneurons (Komada et al., 2008), while the downstream target of Shh hedgehog, the transcription factors Gli1, Gli2 and Gli3, are expressed in the developing cortex as early as embryonic day 13.5 (Hui et al., 1994). Due to the dorso-ventral patterning defects of Shh knockout animals, which showed fused telencephalic vesicles and loss of ventral forebrain structures (Chiang et al., 1996), it was not possible to study the role of Shh signaling during the neurogenic stage of cortical development until conditional knockout mice were developed.

Condition ablation of Shh and Smo in the cortex led to a reduction in cortical precursor proliferation (Komada et al., 2008). On the other hand, conditional inactivation of Patched1 in nestin-positive radial precursors caused an increase in the number of symmetric proliferative

22 divisions of radial precursors (Dave et al., 2011). Finally, conditional ablation of the downstream transcription factor Gli3 in Nestin-positive precursors caused a reduction in intermediate progenitors as a result of both reduced radial precursor proliferation and premature intermediate progenitor differentiation (Wang et al., 2011a). All these results suggest that Shh signaling plays an important role in promoting the maintenance of precursors in the developing cortex.

Interaction between the four major pathways

While each of the four major pathways are individually shown to play important roles in cortical development, they interact with each other and it is this integration that ultimately determines the kinetics of cortical precursor proliferation and neurogenesis. At least in the context of ventral forebrain precursors, constitutive activation of CBF1-RBP-J has been shown to increase neurosphere frequency in the presence of FGF2 but not EGF, suggesting that Notch signaling interacts with FGF2 in the maintenance of telencephalic neural precursors (Yoon et al.,

2004). The level of NICD and hes gene expression is increased in GSK3 mutants, and either inhibition of Notch signaling or overexpression of dominant negative Hes block the enhanced proliferation in these mutants (Kim et al., 2009). Finally, it was shown that the NICD interacts with β-catenin, and the complex binds to and transactivates expression of the hes-1 gene, thereby repressing neurogenesis (Shimizu et al., 2008). In peripheral neurons, NGF activates GSK3β via the integrin-linked kinase (ILK) and regulates dendritic initiation and growth (Naska et al.,

2006). Together, these studies point to a model in which FGF2, Wnt and Notch pathways interact with one another to regulate precursor maintenance and suppression of neurogenesis, and GSK3 is a likely hub of convergence for these interactions.

Neurotrophins

Neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor

(BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and play important roles in many aspects of neural development at various stages including cell fate decisions, axon growth, dendrite pruning, the patterning of innervation and the expression of neurotransmitters and ion channels (Huang and Reichardt, 2001). Neurotrophins bind to two different types of receptors, the Trk tyrosine kinase receptors and the p75 neurotrophin receptors (p75NTR). In the developing forebrain, NT-3 is expressed in immature regions where precursor proliferation, differentiation and migration are ongoing and its expression decreases over time, while BDNF is increasingly expressed as these regions mature (Maisonpierre et al., 1990; Barnabe-Heider and

Miller, 2003). In terms of receptors, both TrkB and TrkC are expressed in cortical precursors, while TrkA is not (Barnabe-Heider and Miller, 2003).

One key function of neurotrophins in the developing central nervous system is cellular survival. Loss of TrkB and/or TrkC cause neuronal death in the cortex (Alcantara et al., 1997;

Xu et al., 2000), the hippocampus (Minichiello and Klein, 1996; Alcantara et al., 1997) and cerebellum (Minichiello and Klein, 1996). In the context of cortical precursors, BDNF and NT-3 are produced by cortical precursors and are required for the survival of these precursors in culture (Barnabe-Heider and Miller, 2003). These studies suggest that neurotrophins are required for survival of both cortical precursors and also postmitotic neurons in various brain regions including the cortex. However, this cell death phenotype appears to be masking other functions of the neurotrophins. Knock down of TrkB and/or TrkC in cortical precursors by in utero electroporation did not result in cell death, but rather led to a decrease in precursor proliferation and neurogenesis (Bartkowska et al., 2007). A similar phenotype of neuronal differentiation and

24 migration deficit is observed when TrkB is ablated in neural precursors using the Nestin promoter (Medina et al., 2004). Conversely, overexpression of BDNF (Bartkowska et al., 2007) and injection of NT-3 (Ohtsuka et al., 2009) both resulted in increased proliferation and subsequently neurogenesis in the cortex. Altogether, these results suggest that neurotrophins play important roles in various aspects of cortical precursor cell biology, namely survival, proliferation and differentiation of cortical precursors.

B) Signaling cascades

One key signaling cascade downstream of the neurotrophin pathway, which is perhaps the best characterized signaling cascade involved in cortical neurogenesis, is the Ras/MEK/ERK mitogen activated protein kinase (MAPK) pathway. Upon neurotrophin or other ligand binding to tyrosine kinase receptors such as TrkB, the receptors dimerize and this leads to activation of the tyrosine kinase domain and reciprocal autophosphorylation of tyrosines in the catalytic domain (Thiele et al., 2009). These phospho-tyrosine residues serve as docking sites for adaptor proteins, whose recruitment to the receptors leads to activation of the Ras GTPase, which in turn triggers the activation of the downstream kinases Raf, MEK and ERK.

Each of the kinases in this serial phosphorylation cascade has been examined with respect to cortical precursor biology. Overexpression of a constitutively activated form of H-Ras increases proliferation and decreases neuronal differentiation in cortical precursors (Paquin et al.,

2009). Downstream of Ras, B-Raf is required for cortical precursor survival, proliferation, neuronal differentiation and migration (Camarero et al., 2006). Inactivation of MEK both by pharmacological inhibitors and expression of a dominant-negative form of MEK resulted in a decrease in neurogenesis and in certain cases, increase in the proportion of proliferating

25 precursors (Menard et al., 2002; Paquin et al., 2005). Conversely, overexpression of an activated form of MEK resulted in an increase in neurogenesis (Menard et al., 2002). Conditional ablation of ERK2 in neural precursor cells caused a reduction in neurogenesis and an increase in the number of astrocytes populating the neonatal and mature cortex, suggesting that ERK2 promotes neurogenesis while suppressing gliogenesis (Samuels et al., 2008). Finally, while infection of a dominant-negative form of ERK5 reduced the generation of neurons in rat cortical precursors, infection of a constitutively active form of ERK5 enhanced neurogenesis (Liu et al., 2006).

Together, these studies show that the MAPK pathway is important for generation of neurons in cortical precursors and inhibition of the various components of the pathway caused neural precursors to remain in the undifferentiated state.

Recently, a key regulator of the MAPK pathway was also found to be important for cortical neurogenesis. SHP-2 is a growth-factor regulated protein tyrosine phosphatase and a genetic mutation in this protein that results in constitutive activation results in a disorder called

Noonan syndrome, which causes heart problems and, in many individuals, some degree of cognitive dysfunction. Interestingly, knockdown of SHP-2 resulted in reduced neurogenesis and at the same time enhanced precocious generation of astrocytes (Gauthier et al., 2007).

Conversely, overexpression of a SHP-2 allele that carries the Noonan syndrome mutation promoted neurogenesis while inhibiting generation of astrocytes. These results suggest that as a regulator of the MAPK pathway in response to growth factor stimuli, SHP-2 acts as a switch to bias the generation of neurons over astrocytes during the neurogenic period in the cortex.

C) Transcription factors

C/EBP

CCAAT/enhancer-binding proteins (C/EBP) are a family of basic leucine zipper transcription factors that binds to specific DNA sequences as dimers, which includes six family members (C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ,. C/EBPε and C/EBPζ). These factors are phosphorylation targets for both ERK and Ribosomal S6 kinases (Rsk); the latter is implicated in

Coffin-Lowry syndrome and is required for neurogenesis in cortical precursors (Dugani et al.,

2010). Overexpression of a dominant-negative form of C/EBPβ resulted in reduced neurogenesis and increased astrocyte generation, and conversely, overexpression of a phosphomimetic form of

C/EBPβ enhanced neurogenesis at the expanse of astrocyte formation (Menard et al., 2002;

Paquin et al., 2005). It is likely that phosphorylation of C/EBPs acts to promote neurogenesis by directly transactivating neuronal genes, including the genes for tα1-α-tubulin and some neurogenic bHLHs (Menard et al., 2002; Uittenbogaard et al., 2007). It was shown that p300, a close homologue of CBP, synergizes with Myb and C/EBP to activate transcription of myelomonocyte-specific genes (Mink et al., 1997). Based on the similarity between CBP and p300, it was speculated that CBP also binds to C/EBP. A later study has confirmed this hypothesis, and showed that the E2F transcription factors, which regulate cell cycle progression, recruit CBP via C/EBP to enhance target gene transcription (Wang et al., 2007). It is intriguing that C/EBP may interact with CBP, which itself is also required for neuronal differentiation

(discussed below), to regulate neurogenesis in the developing cortex.

Pax6

Pax6 is perhaps the most characteristic marker for cortical radial precursors (Mori et al.,

2005). It is a transcription factor that belongs to the Pax family of transcription factors and contains two DNA-binding domains – a paired domain and a paired type homeodomain (Singh et

27 al., 2000). In humans, mutation in Pax6 is believed to account for most cases of aniridia, which is a congenital malformation of the eye (Hanson et al., 1993). A mouse model for aniridia named

Smalleye fails to develop eyes or a nose and dies soon after birth (Hogan et al., 1986); the

Smalleye mutation was later mapped to the gene encoding Pax6 (Hill et al., 1991). In the developing central nervous system, Pax6 is expressed as early as embryonic day 8.5, and is expressed throughout development in the cortex and thalamus (Walther and Gruss, 1991;

Mansouri et al., 1994; Stoykova and Gruss, 1994). Within the cortex, Pax6 is specifically expressed in radial precursors that also express RC2 (Gotz et al., 1998).

Early in development, Pax6 plays an important role in regional specification of forebrain neural precursors. In Pax6 null cortices, the Dlx1 expression domain, which is normally limited to the ventral forebrain and stops at the pallio-subpallial boundary, extends into the dorsal cortex

(Stoykova et al., 1996). This regional specification deficit is further exacerbated in the Emx2-/-;

Pax6-/- double mutants, in which the presumptive cortex loses any cortical specification and displays features that are a hybrid of the adjacent lateral ganglionic eminence and cortical hem

(Muzio and Mallamaci, 2003). Perhaps as a consequence of this or by a different process, Pax6 also specifies cortical neuronal identity and suppresses a subcortical GABAergic differentiation program during mid-late neurogenesis, either independently or in synergy with Tlx (Schuurmans et al., 2004).

During the neurogenic period, Pax6 has been implicated in radial precursor biology, influencing their cell cycle, mode of division and neurogenic potential. First, RC2-positive radial precursors from the Pax6 deficient cortex display a different morphology compared to radial precursors from the wild-type cortex in culture (Gotz et al., 1998). Specifically, while normal radial precursors are bipolar and possess long processes, Pax6 deficient radial precursors are

28 multipolar and possess short processes. Both Gotz et al. and Estivill-Torrus et al. found that Pax6 deficient radial precursors exhibit a shortened cell cycle in early neurogenesis which is supported by increased BrdU labelling both in culture and in vivo (Gotz et al., 1998; Estivill-Torrus et al.,

2002). However, Estivill-Torrus et al. observed a longer cell cycle in these same animals in late neurogenesis due to a significant lengthening of S phase compared to controls (Estivill-Torrus et al., 2002) while Gotz et al. only saw the initial cell cycle shortening diminish over time (Gotz et al., 1998). Interestingly, in both studies, a disruption of interkinetic nuclear migration was observed. Altogether, these studies showed that Pax6 plays an important role in regulation of cell cycle kinetics. One might speculate since the cell cycle length and mode of precursor division is tightly coupled (see section “Direct neurogenesis from radial precursor cells” for details), changes in cell cycle in Pax6 deficient precursors would have an impact on the mode of division.

Indeed, Pax6 controls the rate of progression from symmetric division, which predominates in early neurogenesis, to asymmetric division, which increases in proportion towards late neurogenesis. Specifically, there is an increase in the proportion of divisions with non-vertical cleavage planes in the Pax6 null cortex (Estivill-Torrus et al., 2002; Asami et al., 2011).

Interestingly, both constitutive Pax6 null mutation (Estivill-Torrus et al., 2002) and acute Pax6 deletion (Asami et al., 2011) resulted in ectopically dividing precursors that retain the hallmark of radial precursors. These studies suggest that Pax6 functions to prevent the switch from symmetric to asymmetric precursor division and consequently prevents precocious neuronal differentiation. Paradoxically, cortical precursors isolated from Pax6 null animals have reduced neurogenic potential, and Pax6 null cortices have only half the number of neurons compared to control (Heins et al., 2002). Conversely, Pax6 transduction in embryonic day 14 cortical precursors enhances neuronal differentiation, suggesting that Pax6 promotes neuronal

29 differentiation. How do we reconcile these two different roles of Pax6, namely preventing precocious neuronal differentiation on the one hand and promoting neuronal differentiation on the other hand? Quinn et al. (2007) proposed a plausible explanation. They showed that in the absence of Pax6, cortical precursors exit the cell cycle and differentiate precociously early in neurogenesis, therefore reducing the neurogenic precursor pool (Quinn et al., 2007). In addition, less Tbr2-positive intermediate progenitors are generated, which further reduces the production of neurons in the Pax6 mutants. This is supported by a different study which shows that Svet1- positive cells are abolished in Pax6 null mutants despite the subventricular zone being expanded

(Tarabykin et al., 2001). Altogether, the most plausible theory is that Pax6 prevents early cell cycle exit and neuronal differentiation, and promotes the formation of intermediate progenitors, resulting in both a sustained radial precursor population as well as intermediate progenitor population and ultimately increasing neuronal production.

bHLH Transcription Factors

Since the role of the negative bHLH proteins, the Hes family, was covered in the Notch signaling section, then this section will only focus on positively-acting bHLHs. In this regard, the most well-known neurogenic transcription factors are the bHLH transcription factors, which include Neurogenins, NeuroD and Mash. Of these, Neurogenin-2 (Ngn2) is a downstream target of Pax6. This is supported by the observation that both Ngn2 transcripts and protein are reduced in the Pax6 null cortex (Stoykova et al., 2000; Toresson et al., 2000; Heins et al., 2002).

Moreover, transduction of Pax6 into embryonic day 14 cortical precursors increased the percentage of Ngn2-positive cells and reduced the percentage of Mash1-positive cells (Heins et al., 2002). Early in development, Neurogenins are required for specification of dorsal precursor

30 cell fate, while suppressing the alternative ventral precursor cell fate, which expresses the ventral bHLH Mash1 (Fode et al., 2000). However, no obvious defect in proliferative properties, neuronal numbers and cortical organization was observed in Ngn2 single mutant cortices. This is perhaps due to the compensation by the ventral bHLH Mash1 when Ngn2 is lost. Indeed, in

Ngn2/Mash1 double mutants, the cortices are thinner and are disorganized, with reduced cell density (Nieto et al., 2001). When cultured, Ngn2/Mash1 double mutant cortical precursors give rise to clones that are three times larger than those of wild-type precursors, suggesting that these bHLH factors plays a role in cell cycle exit, thereby promoting precursor cell differentiation.

Moreover, both knockout and overexpression studies show that Neurogenins promote neurogenesis and at the same time inhibit glial fate in neural precursor cells.

How do Neurogenins promote neurogenesis on a molecular level? Neurogenins dimerize with ubiquitous bHLH proteins such as E12 or E47, and as heterodimers, they bind to DNA sequences that contain the E-box consensus motif (CANNTG) (Gradwohl et al., 1996). The ability of Ngns to bind to the E-box motif is abolished in the AQ mutant, which contains a two amino acid substitution mutation in the C terminus of the basic region (Farah et al., 2000; Sun et al., 2001). Importantly, the AQ-Ngn1 mutant also failed to promote neurogenesis, suggesting that E-box binding is required for Ngn1 to promote neurogenesis (Sun et al., 2001). Interestingly, similar to AQ-Ngn1, while overexpression of the AQ-Ngn2 mutant had no effect on neurogenesis, it is capable of promoting neuronal migration (Ge et al., 2006). In contrast, overexpression of Ngn2 with a mutation in a potential phosphorylation site (Y241A-Ngn2) is capable of promoting neurogenesis, but fails to promote neuronal migration. These data suggest that Ngn2 regulates both processes, but that its effects on neurogenesis and neuronal migration can be uncoupled. Exogenous expression of Neurogenin-1 (Ngn1) in embryonic day 14 rat

31 cortical precursors also showed that Ngn1 inhibits astrocyte differentiation by mechanisms that are distinct from the neurogenic role of Ngn1 (Sun et al., 2001). Altogether, these studies show that the Neurogenins serve as a molecular hub that coordinates and mediates many aspects of neural precursor biology via distinct downstream mechanisms.

Neurogenins are also implicated in the generation of intermediate progenitors. Tbr2 expression levels are decreased in Ngn2 mutant cortices and even more severely reduced in

Ngn1/Ngn2 double mutants (Schuurmans et al., 2004). In animals that are null for the LIM domain transcription factor LMO4, which along with its binding partner NLI, are co-activators of Ngn2, the number of Tbr2-positive intermediate progenitors is also reduced in the subventricular zone and more strikingly in the ventricular zone (Asprer et al., 2011).

D) Epigenetic factors

While transcription factors can interact with the basal transcription apparatus and facilitate transcriptional events necessary for neural development, the vast majority of eukaryotic genomic DNA is wrapped around histone complexes and alternates between relaxed and condensed states (Lee and Lee, 2010). Moreover, enzymes are present to regulate the accessibility of DNA to transcription factors, which adds another layer of complexity to the regulation of gene expression. Here I will briefly discuss the role of epigenetic regulation in cortical development, and will focus more on histone modification which is more relevant to my own work.

The tails of core histones are lysine rich which means that they are normally positively charged and therefore associate tightly with the negatively charged phosphate backbone of DNA

(Lee and Lee, 2010). This interaction can be relieved by addition of acetyl groups to the lysine

32 residues and this reaction is modulated by histone acetyltransferases (HATs) which add acetyl groups, and histone deacetylases (HDACs) which remove acetyl groups. There are four major protein families that have been shown to possess histone acetyltransferase activity: the CBP/p300 family, the PCAF/GCN5 family, the TAF250 family and the SRC-2/ACTR family (Kouzarides,

1999). The functions of many of these genes have been studied in the context of brain development. For example, homozygous Cbp-deficient mice have neural tube closure defects and exhibit massive hemorrhages caused by defective blood vessel formation in the central nervous system (Tanaka et al., 2000). Similar defects in neurulation are also observed in p300- deficient mutants (Yao et al., 1998). Homozygous Gnc5 mutants also demonstrate neural tube closure defects and exencephaly (Bu et al., 2007). These studies suggest that HATs are important for early neural tube formation. However, these early neural tube defects prevented the analysis of the role of HATs during later stages of neural development. This is circumvented by the use of conditional mutants and transient expression via in utero electroporation. For example, using in utero electroporation, CBP was shown to be important for neurogenesis in both the spinal cord and the developing cortex (Lee et al., 2009; Wang et al., 2010a). More details on the role of CBP in neurogenesis will be discussed in the section dedicated to CBP.

Histone deacetyltransferases (HDACs) have also been shown to play a role in neural development. First, treatment of hippocampal precursors with the HDAC inhibitor valproic acid inhibits proliferation and induces neuronal differentiation (Yu et al., 2009). This is accompanied by increased expression of proneural transcription factors and increased histone acetylation associated with their promoters. In contrast to this, ablation of both HDAC1 and HDAC2 in neuronal precursors resulted in a deficit in neuronal differentiation and excess cell death

(Montgomery et al., 2009). Thus, while it appears that HDACs have some function in neuronal

33 differentiation, it is unclear what their precise role is with regard to neural precursor function in vivo.

1.2 Neurogenesis in the developing ventral forebrain

1.2.1 Overview of ventral forebrain neurogenesis

I will now shift to the discussion of the generation of the interneurons, which compose approximately 20% of the total cortical neurons and play a major role in setting the excitatory tone as well as synaptic integration. Unlike cortical projection neurons, the majority of cortical interneurons are not generated from the pallial ventricular/subventricular zone. Emx1-Cre/Dbx1-

Cre/R26R-GFP reporter mice showed that GFP labelled cells are mutually exclusive from any interneuron marker (Fogarty et al., 2007). Instead they are generated from the ventral forebrain and migrate tangentially through the striato-pallial border into the cortex. The ventral forebrain is composed of three proliferative zones: lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) (Nery et al., 2002). About 70% of all cortical interneurons are generated from the MGE, while the remaining interneurons are generated from the LGE/CGE (Fogarty et al., 2007).

First I will briefly discuss the early steps in specification of the ventral forebrain. In contrast to the dorsal forebrain, which is specified by BMP signalling, the ventral forebrain is primarily specified by nodal and sonic-hedgehog signalling, and inhibition of BMP signalling. In particular, sonic hedgehog plays a crucial role in ventralizing the neural tube. Mice that are homozygous for a disrupted Shh gene have a single fused telencephalic vesicle where the dorsal telencephalic marker Emx-1 is expressed throughout (Chiang et al., 1996). Ablation of

Smoothened, which is a transducer of Shh signalling, in telencephalic precursors using the

FoxG1-Cre driver also resulted in expansion of the dorsal forebrain genes Emx2 and Ngn2 into the ventral forebrain, at the expanse of ventral genes of the interneuron lineage such as Dlx2,

Lhx6 and Gad67 (Fuccillo et al., 2004). On the other hand, ectopic overexpression of Shh via viral infection in the dorsolateral telencephalon at E8.5 induced the expression of various ventral markers such as the MGE-specific Nkx2.1 and Dlx2 (Gaiano et al., 1999). Interestingly, the role of Shh in the specification of ventral forebrain fate is conserved in chick (Gunhaga et al., 2000) and in zebrafish (Barth and Wilson, 1995). Finally, humans carrying a mutation in Shh have a condition called holoprosencephaly, presenting with a fused telencephalic vesicle, loss of ventral forebrain structures and cyclopia, phenotypes very similar to mice carrying the Shh mutation

(Muenke and Beachy, 2000). These studies provide strong evidence that Shh signalling is a highly conserved mechanism for the specification of the ventral forebrain.

A) Generation of cortical interneurons and oligodendrocytes

Once the ventral forebrain identity is determined, the precursors then have to make the decision of generating a specific neural lineage and in the case of neurons, a neuronal subtype. In contrast to cortical precursors which are thought to be able to generate all three neural lineages

(Malatesta et al., 2000), a subset of precursors of the MGE are thought to be bipotent, with restricted potential to generate neurons and oligodendrocytes only. MGE precursors transplanted into both developing embryonic (Wichterle et al., 2001) and postnatal forebrain (Alvarez-Dolado et al., 2006) rarely give rise to astrocytes. This restricted potential of MGE precursors is also demonstrated indirectly by the observation that Shh exposure of cortical precursors resulted in the restriction of their potential to generate only neurons and oligodendrocytes, at the expense of multipotent precursors (Yung et al., 2002). Because the ventral forebrain is exposed to a high

Shh signal at an early embryonic stage, it has been postulated that this restricts their potential so that the MGE precursors only generate neurons and oligodendrocytes. Finally, neurospheres generated from PDGF-responsive precursors, which mainly reside in the MGE, generate both neurons and oligodendrocytes but not astrocytes after 2 days in vitro in 1% FBS (Chojnacki and

Weiss, 2004). However, this limited potential is challenged by the observation that these PDGF- responsive precursors are able to give rise to astrocytes in the presence of BMP-2 and CNTF.

Lineage tracing of single precursors in the MGE revealed that similar to cortical precursors, MGE precursors and their progenies are also organized in radial clonal units originating from a radial precursor-like cell dividing at the ventricular surface (Brown et al.,

2011). Neurons generated in each division initially migrate along the radial fiber of the precursor cell. They then adopt a bipolar morphology and eventually migrate tangentially towards the cerebral cortex. Despite the lack of direct evidence, it is thought that the MGE precursors display cell cycle behaviour similar to precursors in other niches, such as those in the LGE, given the similarities in transcription factors driving neurogenesis. Study of MGE cytokinetics has shown that, like the cortex, the ventral germinal niche is composed of two proliferative compartments, called the pseudostratified ventricular epithelium (PVE) and the secondary proliferative population (SPP) (Sheth and Bhide, 1997). Analogous to the radial precursors in the developing cortex, the PVE precursors undergo interkinetic nuclear migration and go through S phase away from the ventricle and M phase adjacent to the ventricle (Bhide, 1996). On the other hand, SSP progenitors go through all phases of the cell cycle away from the ventricle, which is analogous to the intermediate progenitors of the cortex. It is estimated that at E11, the cellular outputs are equivalent between the PVE and the SSP (Sheth and Bhide, 1997).

A number of lines of evidence indicate that the interneuron subtypes that are generated by

MGE precursors depend upon which of the two proliferative compartments they come from. I will focus more on interneuron subtypes in the section below, but in general, there are three non- overlapping types of cortical interneurons, as defined by marker expression; parvalbumin- positive interneurons, somatostatin-positive interneurons and calretinin-positive interneurons.

Mutation of the cell cycle regulator cyclin D2 caused a 30% reduction in parvalbumin-positive interneurons in the cortex, while the number of somatostatin-positive interneurons was unchanged (Glickstein et al., 2007b). Interestingly, cyclin D2 is more prevalent in the MGE subventricular zone than in the ventricular zone, while cyclin D1 is more prevalent in the ventricular zone (Glickstein et al., 2007a). Cyclin D2 is also more prominent in the ventral MGE compared to the dorsal MGE. This is also consistent with the observation that parvalbumin- positive interneurons are preferentially generated from the ventral MGE, while somatostatin- positive interneurons are preferentially generated from the dorsal MGE (Flames et al., 2007).

These studies suggest that parvalbumin-positive interneurons are generated from divisions of the subventricular zone of the MGE.

Oligodendrocytes are another major neural cell types that are generated from the ventral forebrain. There are three major source of oligodendrocytes in the developing brain and the earliest source of oligodendrocytes come from the MGE (Kessaris et al., 2006). As discussed above, these oligodendrocytes are derived from the same precursors that give rise to interneurons. Moreover, competing transcription factor pathways regulate the cell fate decision to make an interneuron versus an oligodendrocyte. In particular, Dlx1/2 promotes the generation of interneurons and inhibits the genesis of oligodendrocytes, potentially by repressing the

37 transcription factor Olig2. On the other hand, Mash1 promotes the generation of oligodendrocytes at least in part by repression of Dlx1/2 (Petryniak et al., 2007).

B) Tangential migration of cortical interneurons

Despite being a critical part of cortical interneuron development, I will only briefly discuss interneuron migration, since this thesis is mainly focused on cell fate specification. After interneurons are born in the ganglionic eminences, they migrate tangentially into the cortex. This migration begins at approximately E12.5 of mouse development and is dependent on the Dlx genes (Anderson et al., 1997). Importantly, in Dlx1/2 mutants, interneuron migration is defective, and the newly-born interneurons are arrested in the subventricular zone in these mutants. In contrast, the radial migration of projection neurons is unaffected. One of the key downstream targets of Dlx with regard to interneuron migration is the Aristaless-related homeobox transcriptional repressor Arx, which when mutated in human leads to a variety of neuropathological conditions. Dlx2 directly binds to the mUAS3 element of Arx and drives its expression, and overexpression of Arx is capable of rescuing the tangential migration deficit of

Dlx1/2 mutants (Colasante et al., 2008). On the other hand, the ability of ectopic Dlx2 expression to induce GABAergic gene expression is retained in Arx mutants, suggesting Arx is downstream of Dlx2 for tangential migration but not interneuron specification. Dlx2 may also regulate interneuron migration via Dlx5/6. This was shown by studying Dlx5/6 mutants, where both the superficial and deep migratory streams showed progressive retardation in comparison to controls

(Wang et al., 2010b). However, in contrast to the Dlx1/2 mutants, which result in a complete block of tangential migration, interneuron migration in Dlx5/6 mutants is only delayed.

Finally, as mentioned in the previous section of interneuron generation, Lhx6 is required for interneuron migration. Specifically, transgenic mice that have the placental alkaline phosphatase (PLAP) gene knocked into the Lhx6 locus to create a loss-of-function mutant showed that these PLAP labelled cells are delayed in their tangential migration into the cortex

(Zhao et al., 2008). These results were also confirmed by another group using a mouse carrying both mutated Lhx6 alleles and a Gad67-GFP transgene that marks interneurons (Liodis et al.,

2007).

C) Origin of interneuron subtypes

Cortical interneurons can be categorized by molecular features, morphology and electrophysiology. By neurochemical markers, there are three major types of cortical interneurons; parvalbumin-positive, somatostatin-positive and calretinin-positive interneurons

(Gonchar and Burkhalter, 1997). Approximately half of the interneurons are parvalbumin- positive, and somatostatin-positive and calretinin-positive interneurons each account for approximately 17% of total interneurons. Because these markers are largely non-overlapping, the three markers combined account for approximately 85% of total interneurons in the cortex.

Transplantation studies have shown that the MGE (and perhaps the LGE) is the major source of parvalbumin-positive and somatostatin-positive interneurons, while the CGE mainly gives rise to calretinin-positive interneurons (Xu et al., 2004).

In contrast to projection neurons, cortical interneurons of different neuropeptide phenotypes are distributed throughout the cortex thickness. In addition, a single precursor in the

MGE is capable of generating interneuron progeny of different peptidergic phenotypes. This was demonstrated by lineage tracing experiments showing that a single clone derived from an

MGE precursor can give rise to both parvalbumin-positive and somatostatin-positive interneurons (Brown et al., 2011).

1.2.2 Extrinsic and intrinsic factors regulating neurogenesis

A) Regulation of interneuron generation

While it is important for the initial specification of the ventral forebrain, Shh is also important for the subsequent maintenance of MGE precursor identity. Ablation of Shh from forebrain precursors using a Nestin-Cre driver, as well as treatment of wild-type brain slices with cyclopamine, a Shh inhibitor, resulted in a robust loss of Nkx2.1-positive cells, and subsequently somatostatin-positive cortical interneurons (Xu et al., 2005). This was not due to changes in proliferation or survival of MGE precursors because in both types of experiments, BrdU-positive cells and cleaved caspase-3 labeling were largely unchanged. Moreover, addition of Shh could rescue the reduction in Nkx2.1-positive and somatostatin-positive cells in the conditional knockout.

Like cortical neurogenesis, multiple pathways are involved in generation of interneurons.

Nkx2.1 mutants showed a 50% reduction in Dlx2-positive and a 40% reduction in GABA- positive cortical interneurons (Sussel et al., 1999). One month old Dlx1 mutant animals showed a

22% reduction in Gad67-positive interneurons (Cobos et al., 2005). However, Dlx1 and Dlx2 double mutants showed an 80% reduction of cortical interneurons (Anderson et al., 1997). This apparent redundancy may occur because interneurons are crucial for normal functioning of the animal, and multiple pathways have evolved to compensate for each other in the event that one goes awry. Alternatively, these different pathways may not be completely redundant, but may instead serve slightly different functions, for example, generating different types of interneurons.

Is there any evidence to support this latter possibility, that different pathways generate different subtypes of interneurons? This appears to be case. For example, even though there is only a 40% reduction in GABAergic interneurons in Nkx2.1 mutants, somatostatin-positive interneurons are undetectable (Anderson et al., 2001). In contrast, somatostatin-positive interneurons are reduced but nevertheless present in Dlx1/2 mutant cortex. Mice lacking only

Dlx1 also showed a reduction in somatostatin-positive and calretinin-positive interneurons

(Cobos et al., 2005). In a similar vein, both parvalbumin-positive and somatostatin-positive interneurons are absent in primary cultures of dissociated E18.5 cortices from Nkx2.1 mutants, while calretinin-positive interneurons are present (Xu et al., 2004). On the other hand, calretinin- positive interneurons are absent in similar cultures of Dlx1/2 mutants. These results suggest that while both Nkx2.1 and Dlx1/2 are required for interneuron generation, they are specifically regulating generation of distinct interneuron subtypes.

Similar to the cortex, there is also a transcriptional cascade associated with the generation of interneurons. However, it appears that negative regulation plays a more prominent role in ventral neurogenesis with regard to neuronal subtypes and the precise role that each transcription factor plays appears to depend on developmental stage. For example, Mash1 directly binds the I12b intergenic enhancer element that regulates Dlx1/2 expression (Ghanem et al., 2007; Poitras et al., 2007). While neurogenesis is reduced at E10.5 in Mash1 mutants (Yun et al., 2002), premature expression of neuronal markers is observed in the ventricular zone of these same mutants at E11.5 (Casarosa et al., 1999; Horton et al., 1999; Yun et al., 2002), suggesting that Mash1 is required for the generation of early neurons, and then for maintenance of precursors at later stages. And perhaps as a result of the loss of precursors, cortical interneurons are nearly absent in E18.5 Mash1 mutants (Casarosa et al., 1999). In the wild-type forebrain,

Dlx1 and Dlx2 are expressed by a subset of cells in the ventricular zone and by most cells in the subventricular zone (Porteus et al., 1994). In Mash1 mutants, however, Dlx1 and Dlx2 are expressed by almost all cells in the ventricular zone (Casarosa et al., 1999; Yun et al., 2002), suggesting that Mash1 represses the expression of Dlx genes. Interestingly, in Dlx1/2 mutants, there is elevated expression of Mash1 in the subventricular zone (Yun et al., 2002), suggesting that while Mash1 represses expression of Dlx genes, Dlx genes also repress the expression of

Mash1. This mutual regulation of Mash1 and Dlx genes appears to be important not only for the specification of interneurons, but also for controlling the timing of interneuron generation such that there is an optimal balance for interneuron generation and precursor maintenance. Any alteration in this tightly controlled system would result in deficit in interneuron generation or depletion of precursors.

Once Dlx2 is activated, it activates a cascade of transcription of other Dlx genes.

Interestingly, not only do the Dlx genes share a similar expression pattern, but they have a unique genetic organization in the genome. The forebrain Dlx genes are arranged in pairs in the genome – Dlx1 and Dlx2, Dlx5 and Dlx6 (Simeone et al., 1994; McGuinness et al., 1996). Both pairs are arranged in a tail-to-tail organization and have a short intergenic region between them, and because of the similar expression pattern of the genes in each pair, it is thought that their expression is driven by shared enhancer elements. Consistent with this idea, enhancer elements have been identified that are shared between the two genes in each pair; i12a and i12b (Ghanem et al., 2003; Park et al., 2004) drive expression of Dlx1 and Dlx2, and i56i and i56ii (Zerucha et al., 2000) drive expression of Dlx5 and Dlx6. Studies have shown that in the forebrain. Dlx1 and

Dlx2 directly bind to the i56i enhancer and activate transcription of Dlx5 and Dlx6 (Zerucha et al., 2000; Zhou et al., 2004a). This is further confirmed by expressing Dlx2 together with a LacZ

42 reporter downstream of the intergenic i56i enhancer element in the cerebral cortex, which resulted in strong expression of β-galactosidase in the cortex (Stuhmer et al., 2002).Interestingly,

Dlx2 can also bind to its own i12b enhancer elements, and in so doing drive its own expression

(Poitras et al., 2007). Finally, Dlx5/6 double mutants are deficient in the differentiation of parvalbumin-positive interneurons, although this is amid a slew of abnormalities such as exencephaly and an interneuron migration deficit (Wang et al., 2010b). Interestingly, the authors also observed the deficit in parvalbumin-positive interneurons when they transplanted Dlx5/6 mutant cells into wild-type brains, suggesting a cell-autonomous role for Dlx5 and Dlx6 in the development of parvalbumin-positive interneurons, as well as ruling out the alternative explanation that the defects may be secondary to the aforementioned abnormalities such as exencephaly. Together, these studies showed that the Dlx genes can both auto-regulate and also cross-regulate other Dlx genes, and subsequently regulate different phases of interneuron development.

In contrast to Dlx’s role in specification of the general interneuron phenotype, the LIM homeobox transcription factor Lhx6 appears to play a role in the specification of interneuron subtypes. Blocking Lhx6 in E13.5 MGE cultures did not interfere with the expression of GABA and GABA synthesizing enzymes Gad65/67 (Alifragis et al., 2004). Also, normal numbers of

GABAergic interneurons were observed in the cortex of Lhx6 mutants, despite their abnormal distribution in different layers (Liodis et al., 2007). However, Lhx6 loss of function resulted in a robust reduction in the percentage of parvalbumin-positive and somatostatin-positive interneurons in the cortex, and a less severe reduction in the percentage of calretinin-positive interneurons (Zhao et al., 2004; Liodis et al., 2007). In this regard, the MGE-specific transcription factor Nkx2.1 is upstream of Lhx6 in this pathway to specify interneuron subtype.

This is neatly demonstrated by a study that showed overexpression of Lhx6 can rescue the interneuron specification defects in Nkx2.1 mutants while knockdown of Lhx6 blocks the ability of overexpressed Nkx2.1 to rescue the deficits in parvalbumin-positive and somatostatin-positive cells in Nkx2.1 mutants (Du et al., 2008). However, as discussed above, Nkx2.1 mutants resulted in a significant reduction in total GABAergic interneurons. Therefore, there must be additional factors downstream of the Nkx2.1 pathway that regulate this function of specification of general interneuron phenotype (Fig. 1.4).

1.3 CREB binding protein (CBP)

1.3.1 CBP and Rubinstein-Taybi syndrome

CREB binding protein (CBP) is a transcriptional adaptor and histone acetyltransferase that binds to and cooperates with hundreds of different transcription factors. Moreover, in addition to its own HAT activity, CBP binds to and acts as an adaptor for other HATs such as

P/CAF and SRC-1 (Vo and Goodman, 2001). It is thought that CBP has dual roles in regulating transcription. First, CBP is a large protein that contains multiple protein-protein interaction domains and interacts with both promoter-bound transcription factors and with the transcriptional machinery (Chan and La Thangue, 2001). Therefore, it can act as a bridging molecule to mediate interactions between sequence-specific transcription factors and the transcriptional apparatus. Second, while CBP binds to HATs, it also has an intrinsic HAT activity (Bannister and Kouzarides, 1996). Therefore binding of CBP to transcription factor activation domains renders it in a good position to open up the chromatin for transcription of the target gene. Mutation in the gene encoding CBP accounts for approximately half of the cases of

Rubinstein-Taybi syndrome (see Introduction section of Chapter 4).

1.3.2 Role of CBP in muscle and hematopoietic differentiation

The developmental function of CBP has been studied in the nematode C. elegans; knockdown of cbp-1 in early embryos resulted in the absence of mesodermal, endodermal and hypodermal cells (Shi and Mello, 1998). However, neuronal differentiation appeared to be spared, suggesting that CBP is required for differentiation of all non-neuronal somatic lineages.

Similarly, in Drosophila, CBP is important for regulating midgut development by inhibiting hedgehog signalling through acetylation of Drosophila TCF (Waltzer and Bienz, 1998). CBP also plays an important role in wing vein formation (Akimaru et al., 1997) and eye development

(Kumar et al., 2004) in Drosophila. These studies are just a few examples of how CBP plays important roles in the development of many different tissues in invertebrates.

CBP also plays a role in tissue differentiation in mammals, with the best characterized examples in myogenic and hematopoietic differentiation. Using a mouse myoblastic cell line, it was shown that CBP/p300 is required for myotube fusion and expression of late but not early muscle marker genes (Polesskaya et al., 2001). In the hematopoietic system, CBP plays an important role in the maintenance of hematopoietic precursors and their subsequent differentiation into various lineages. In one study, it was shown that CBP null animals had a multilineage deficit in hematopoietic differentiation (Kung et al., 2000). In a different study, it was shown that it is p300, but not CBP, that is required for hematopoietic differentiation, while

CBP, but not p300, is required for self-renewal of hematopoietic stem cells (Rebel et al., 2002).

The most likely explanation to reconcile these differences is that perhaps CBP is required to maintain both self-renewal and multipotency of hematopoietic stem cells.

1.3.3 Role of CBP in neural differentiation

The role of CBP in the development of nervous system is best documented in vertebrates.

In Xenopus, CBP is recruited by XNGN-1 (Xenopus Ngn-1) to activate genes that are required for neuronal differentiation such as NeuroD (Koyano-Nakagawa et al., 1999). In chick and mice spinal cord, retinoic acid induces the recruitment of CBP to the retinoic acid receptor/Ngn2 (a bHLH transcription factor) complex, facilitating transcription of motor neuron genes and thereby promoting motor neuron specification (Lee et al., 2009). Recently, using both an siRNA knockdown approach and CBP+/- mutant animals, our lab showed that CBP promotes the differentiation of neural precursors into neurons, astrocytes and oligodendrocytes in the developing cortex both in vivo and in vitro by binding to and acetylating histones at promoters of neural differentiation genes (Wang et al., 2010a). Importantly, neonatal CBP+/- mutant animals exhibited defective ultrasonic vocalization, suggesting that these differentiation defects have behavioural consequences. Finally, it has also been shown that Ngn2 promotes radial migration of cortical neurons, potentially by recruiting CBP to the doublecortin (Dcx) promoter and displacing CBP from the RhoA promoter (Ge et al., 2006). Therefore, building on the theme that

CBP is a ubiquitous HAT and cofactor, these studies show that CBP plays an important role in many stages of neural development.

1.4 Forkhead box protein P2 (FoxP2)

1.4.1 FoxP2 and specific language impairment

FoxP2 is a transcription factor that belongs to the forkhead box family and has both a leucine zipper and a zinc finger domain. While it has the potential to be both a transcriptional activator and a repressor, depending on the protein partners it binds with, FOXP2 has mainly

46 been shown to be a transcriptional repressor, interacting with proteins such as NKX2.1 (Zhou et al., 2008) and CTBP1 (Li et al., 2004a).

Rare mutations involving FOXP2 are associated with impairments in the learning and production of sequences of oral movements that lead to speech. Thus far, it is the only gene implicated in a Mendelian form (in the KE family) of human speech and language dysfunction

(Fisher et al., 1998). Moreover, because the FOXP2 gene resides at the chromosomal locus 7q31, which is a region implicated in autism, FOXP2 is also considered as a potential susceptibility locus for language deficits in autism (Feuk et al., 2006). It is still unclear how FOXP2 mutations disturb speech and language development.

1.4.2 Mouse models of FoxP2 mutants

While language is unique to humans, since FoxP2 is highly conserved in many vertebrates and shows comparable neural expression patterns to humans, its function can be assessed in animal models and could be similar to humans on a cellular/systems level. A transgenic mouse strain carrying a null mutation in the FoxP2 gene (Shu et al., 2005) and another strain carrying the R522H substitution (Fujita et al., 2008), which is homologous to the mutation in the KE family, have been developed and both exhibited developmental abnormalities in the cerebellum. However, heterozygous animals, which would best model the affected members of the KE family who carry a single copy of the mutant allele, showed no or only modest abnormalities. In particular, mice carrying the null mutation exhibited misalignment of Purkinje cells and impairment of granule cell migration, while mice carrying the R522H substitution exhibited sparse (but not misaligned) Purkinje cells and normal granule cell migration. Recently, it has also been discovered that FoxP2 together with FoxP4 regulates neurogenesis in the

47 developing spinal cord, by regulating the expression of N-cadherin and Sox2 (Rousso et al.,

2012). Thus FoxP2 plays important roles in regulating neural development in at least two niches, the cerebellum and the spinal cord.

Figure 1.1

Figure 1.1. Evolution of cortical development. The cerebral cortex evolved from a three-layer architecture (left panel) to a smooth (lissencephalic) six-layered structure (middle) and eventually developed sulci and gyri to accommodate the expansion of cortical volume (right panel). The change in the mature cortical architecture (illustration on the right of each panel) is accompanied by, and may be attributed to, the development of more complex primordial cortical layers (illustration on the left of each panel). Specifically, in the developing cortex of the turtle, there is only one germinal layer, the ventricular zone (VZ). Neurons are generated from the VZ and migrate through the intermediate zone (IZ) into the cortical plate (CP), which eventually became a mature three layer cortex (layers I-III). While still debatable, it is thought that the subplate (SP) emerged in mammals to support development of cortico-cortical connectivity, and there is no obvious SP homolog in the reptilian cortex (Montiel et al., 2011). In addition, a subventricular zone (SVZ) also emerged concomitantly with the expansion in the number of intermediate progenitors. These changes eventually led to the development of a six-layer cortex

(layers I-VI) in the mature rodent brain. Finally, the SVZ in primates has a massively expanded outer region called the outer subventricular zone (OSVZ), which is separated from the inner subventricular zone (ISVZ) by a thin fiber layer. The development of an expanded SVZ is accompanied by the expansion of the number of outer or basal radial glia, which are also present in small numbers in the rodent developing cortex. It is thought that these basal radial glia further amplify the precursor pool in the developing cortex, and contribute to the expansion of cortex volume in primates.

Figure 1.2

Figure 1.2. Receptor tyrosine kinase (RTK) activation of four different pathways. Receptor tyrosine kinases activate components of the MAPK, PI3K, JAK-STAT and PLC-γ pathways to mediate cell survival, proliferation and differentiation. Blue circles with “P” denote phosphorylation.

Figure 1.3

In the absence of ligand Upon ligand binding

Figure 1.3. Wnt binding activates transcription of genes that mediate precursor maintenance.

In the absence of ligand binding (left panel), Axin coordinates the formation of a protein complex with APC and GSK3β. This leads to phosphorylation of β-catenin by GSK3β, and β- catenin undergoes ubiquitination and degradation. When Wnt binds to its receptor Frizzled and coreceptor LRP, Dishevelled (Dsh) is phosphorylated, which prevents the formation of the protein complex and the subsequent degradation of β-catenin. Thus, β-catenin is “stabilized” and translocates into the nucleus of the cell, where it interacts with TCF to activate genes that mediate precursor maintenance.

Figure 1.4

Figure 1.4. Parallel pathways regulate neurogenesis in the medial ganglionic eminence

(MGE). Two major pathways regulate neurogenesis from MGE precursors. The Dlx proteins are widely expressed in the ventricular zone (VZ, yellow), subventricular zone (SVZ, green) and mantle zone (MZ, red) of the MGE, with Dlx2 primarily expressed in undifferentiated precursors and Dlx6 primarily expressed in differentiated cells in the MZ. Dlx1 and Dlx5 are expressed in an intermediate pattern. The Dlx pathway is comprised of a signaling cascade where one Dlx transcription factor activates a downstream Dlx transcription factor, in the order of: Dlx2 →

Dlx1 → Dlx5 → Dlx6. In a parallel fashion, Nkx2.1 also regulates the generation of both striatal and cortical interneurons by activating downstream LIM homeobox factors (Lhx6 and Lhx7).

Downregulation of Nkx2.1 is required for interneurons that migrate into the cortex, while striatal interneurons retain Nkx2.1 expression.

CHAPTER 2: OVERALL HYPOTHESIS AND RESEARCH AIMS

Relatively little is known about how the signals from the precursor cell’s environment are integrated with each other and how they interact with intrinsic factors to coordinate the formation of the different cell types and circuitries in our nervous system. The primary objective of my thesis was to examine the role of two different intrinsic factors – a transcription factor, FoxP2, and an epigenetic regulator, CBP – in the development of neural precursors in the mouse forebrain.

FoxP2 is an interesting gene because it is one of the rare mono-genetic causes of impairment in a speech. However, the underlying biological cause for this impairment is still unclear. As discussed in the introduction, while FoxP2 is known to play a role in the development of the cerebellum and the spinal cord, its role in embryonic cortical development is not known. The one exception is an fMRI study that indirectly provided evidence that FoxP2 is critically involved in the development of the cerebral cortex. In this study, members of the KE family who have language specific impairment and unaffected family members (as controls) were asked to carry out a verb generation task while subjected to fMRI analysis (Liegeois et al.,

2003). During the task, unaffected members showed a left-hemisphere-dominant activation involving Broca’s area, but affected members showed a bilateral pattern of activation in the cortex, suggesting impaired cortical function in response to this task. A second line of evidence that individuals with FoxP2 mutations have affected cortical development is that there are abnormally low levels of grey matter in Broca’s area of affected KE family members (Vargha-

Khadem et al., 2005). Finally, FoxP2 null and FoxP2-R522H mutant pups demonstrated impairments in an innate behaviour called ultrasonic vocalization (Shu et al., 2005; Fujita et al.,

2008), which is impaired in mice with perturbed cortical development (Wang et al., 2010a).

Together, these findings led us to hypothesize that FoxP2 plays an essential role in regulating neurogenesis in the mouse embryonic cortex. The experiments described in Chapter 4 are aimed at defining the role of FoxP2 in cortical development and asking whether there is any functional difference between mouse and human FoxP2, a question that is particularly important for the evolution of human speech.

As discussed in the introduction, Rubinstein-Taybi syndrome is caused by mutation in the gene encoding the HAT CBP. Previous work in the lab showed that CBP is required for differentiation of cortical precursors into the three neural lineages – neurons, astrocytes and oligodendrocytes, and that both the HAT activity and phosphorylation by aPKC are critical for

CBP’s function in this regard (Wang et al., 2010a). Interestingly, one of the clinical features of

Rubinstein-Taybi syndrome is an abnormally high rate of epileptic activities and presentation of seizures. We therefore hypothesized that CBP also plays an essential role in regulating neuronal differentiation and/or maturation of interneurons via its histone acetyltransferase activity. The experiments addressing this hypothesis are described in Chapter 5 and they aim to define the role of CBP in interneuron differentiation and maturation.

My work will shed light on some aspects of the downstream components of signaling pathways that may be responsible for driving the execution steps of neurogenesis such as neuronal delamination and expression of neuronal specific genes and adoption of neurotransmitter phenotype.

CHAPTER 3: MATERIALS AND EXPERIMENTAL PROCEDURES

Animals: All animal use was approved by the Animal Care Committee of the Hospital for Sick

Children in accordance with the Canadian Council of Animal Care policies. CD1 mice were used for all experiments except the cell sorting, and were obtained from the Charles River Laboratory.

Sox2:EGFP mice (Ellis et al., 2004) were genotyped and maintained as described (Biernaskie et al., 2009). Embryos and neonatal pups of both sexes were used.

Primers and Plasmids: Two primer sets were used for FoxP2 mRNA. The first was FoxP2 forward (5’-CCAAACCATCTCCCAAACCT-3’), FoxP2 reverse (5’-

TCTGAATGTCGCCTTCGTATG-3’), and the second was FoxP2 forward (5’-

GCTAAGTAACCCTGGACTGATC-3’), FoxP2 reverse (5’-TCTTCATCCTCTGCAATCACG-

3’). Two primer sets were also used for Cbp mRNA. The first was Cbp forward (5’-

CTGAGCCTGAACCTACTGAATC-3’), Cbp reverse (5’-AGGAGATGTTGATTGTGAGGC-

3’), and the second was Cbp forward (5’-AGCAAATGGAGAGGTTCGAG-3’), Cbp reverse

(5’-CTTAAGGAAGTGGCATTCTGTTG-3’). The nuclear EGFP expression plasmid was driven from the Ef1α (Eef1a - Mouse Genome Informatics) promoter (pEF-EGFP) and has been previously described (Barnabe-Heider et al., 2005). The PB-EGFP construct contains an EGFP reporter driven by the CAG promoter and flanked by inverted tandem repeats (ITRs), and is recognized by the PB transposase (Nagy et al., 2011). The FoxP2 shRNAs and the negative control were purchased from EZBiolab (Westfiled, IN) and made in the pGPU6-neo backbone.

The targeted sequence was 5’-GCACTTTAAGCAGCCAATTAG-3’ for shFoxP2#1, and 5’-

GCAAACCAGTGGATTGAAATC-3’ for shFoxP2#2. The sequence for the shRNA negative control was 5’-TTCTCCGAACGTGTCACGT-3’. All plasmids for the FoxP2 overexpression

59 studies were cloned into pCMV-Tag4A and include pCMV-Tag4A-mouse FoxP2 cDNA

(mFoxP2), pCMV-Tag4A-human FoxP2 cDNA (hFoxP2) or pCMV-Tag4A-human FoxP2-

R553H (hFoxP2-R553H). The pCMV-Tag4A empty vector, human FoxP2 and human FoxP2-

R553H were kindly provided by Dr. Genevieve Konopka (UT Southwestern Medical Center;

(Konopka et al., 2009). The mouse FoxP2 construct was subcloned into the pCMV-Tag4A empty vector from an E13/14 mouse cortex cDNA library. FoxP1 plasmids in the pCMV-Tag2A backbone and FoxP4 plasmids in the pCMV-Tag3B backbone were kindly provided by Dr.

Edward Morrissey (University of Pennsylvania School of Medicine; (Li et al., 2004a). The overexpression plasmid for mouse FoxP2 used for experiments in Figures 1B and 1G was kindly provided by Dr. Zea Borok (University of South California; (Zhou et al., 2008). For CBP knockdown, the ON-TARGETplus SMARTpool Crebbp siRNAs (proprietary mixture,

Dharmacon) and the ON-TARGETplus Non-targeting pool siRNA (Dharmacon) and were previously characterized in a study from our lab (Wang et al., 2010a).

Cortical precursor cell cultures: Cortical precursor cells were cultured as previously described

(Barnabe-Heider et al., 2005). Briefly, cerebral cortices were dissected from embryonic day 12

CD1 mouse embryos in ice-cold HBSS (Invitrogen) and transferred to Neurobasal medium

(Invitrogen) containing 500 µM L-glutamine (Cambrex Biosciences), 2% B27 supplement

(Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 40 ng/ml FGF2 (BD Biosciences).

The tissue was mechanically triturated with a plastic pipette and plated onto four-well chamber slides (Nunc) precoated with 2% laminin (BD Biosciences) and 1% poly-D-lysine (Sigma). Cells were plated at a density of 200,000 cells/well for four-well chamber slides. Culture medium was not changed for the duration of the experiment. For transfections, 1–2 h after plating, 1 μg total

DNA (comprised of 0.25 μg of pEF-EGFP plasmid plus 0.75 μg of shRNA for knockdown experiments; 0.1 μg of pEF-EGFP plasmid plus 0.9 μg of pCMV-Tag4A empty vector, human

FoxP2, mouse FoxP2 or human FoxP2-R553H plasmids for overexpression experiments) or 1.4

μg of total DNA (comprised of 0.1 μg of PB transposase plasmid plus 0.1 μg of PB-EGFP plasmid plus 0.3 μg of control or FoxP2 shRNA plasmid plus 0.9 μg of pCMV-Tag4A empty vector or human FoxP2 plasmid) and 2 μl of Fugene 6.0 (Roche) or 1 μl of Lipofectamine 2000

(Invitrogen) were mixed with 100 μl of Opti-MEM (Invitrogen), incubated at room temperature for 1 hour and added to the cultures. This resulted in a transfection of, at most, 1-3% of cells.

Medial ganglionic eminence precursor cell cultures: Neural precursor cells from the medial ganglionic eminence (MGE) were cultured in a similar manner as cortical precursor cell cultures as previously described (Barnabe-Heider et al., 2005). Briefly, MGE precursors were dissected from embryonic day 12 CD1 mouse embryos in ice-cold HBSS (Invitrogen) and transferred to

Neurobasal medium (Invitrogen) containing 500 µM L-glutamine (Cambrex Biosciences), 2%

B27 supplement (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and 40 ng/ml FGF2 (BD

Biosciences). The tissue was mechanically triturated with a plastic pipette and plated onto either two-well or four-well chamber slides (Nunc) precoated with 2% laminin (BD Biosciences) and

1% poly-D-lysine (Sigma). Cells were plated at the density of 200,000 cells/well for four-well chamber slides or 400,000 cells/well for two-well chamber slides. For transfections, 1–2 h after plating, 1 μg of pEF-EGFP plasmid and 80 pmol siRNA (for two-well chamber slides) were mixed with 1 μl of Lipofectamine 2000 (Invitrogen) and 100 μl of Opti-MEM (Invitrogen), incubated at room temperature for 1 hour and added to the cultures. 0.5 μg of pEF-EGFP plasmid

61 and 40 pmol siRNA were used for four-well chamber slides. This resulted in a transfection of, at most, 1-3% of cells.

In utero electroporation: In utero electroporation was performed as described (Barnabé-Heider et al., 2005; Gauthier et al., 2007) with E13/14 CD1 mice, injecting a 1:3 ratio of the nuclear

EGFP plasmid with the shRNA or overexpression plasmids (total of 4 µg DNA) and 0.5% trypan blue as a tracer. For the rescue experiments, DNA was mixed at a ratio of 1 µg pEF-EGFP:1 µg

FoxP2 shRNA: 3 µg human FoxP2 for a total of 5 μg DNA per embryo. For knockdown experiments in which P3 animals were analyzed, DNA was mixed at a ratio of 1 µg PBase:1 µg

PB-EGFP: 3 µg FoxP2 shRNA for a total of 5 μg DNA per embryo. The square electroporator

CUY21 EDIT (TR Tech, Japan) was used to deliver five 50 ms pulses of 40-50 V with 950 ms intervals per embryo. Except for analysis of neonatal animals, all brains were dissected 3 days post transfection in ice-cold HBSS. For analysis of neonatal animals, embryos were electroporated from separate litters but pups were born on the same day and brains were harvested at postnatal day 3. All electroporated brains were fixed in 4% paraformaldehyde at 4ºC overnight or until they sank to the bottom, cryoprotected and cryosectioned coronally at 16 m.

Immunocytochemistry and histological analysis: Immunocytochemistry was performed as previously described (Barnabe-Heider et al., 2005). Briefly, sections were fixed in 4% paraformaldehyde for 10 minutes, and then permeabilized and blocked with 5% BSA and 0.3%

Triton-X in phosphate-buffered saline (PBS) for 1 hour. Primary antibodies were diluted in the same permeabilization and blocking buffer and incubated either at 4ºC overnight or room temperature for 3 hours. Secondary antibodies and Hoechst were diluted in PBS. Sections were

62 washed three times for five minutes with PBS between each step. For FoxP2 immunostaining, an antigen retrieval step was added post-fixation that involved boiling slides at 92ºC in sodium citrate buffer (pH6) for 20 minutes. For cultures, four-well chambers were fixed for 10 minutes in paraformaldehyde and then permeabilized with 0.2% NP40 in PBS. Cells were then blocked with 0.5% BSA and 6% normal goat serum or normal donkey serum diluted in PBS. Primary antibodies were diluted in 0.25% BSA and 3% normal goat serum or normal donkey serum in

PBS, and incubated overnight. Secondary antibodies were diluted in the same blocking solution as primary antibodies and incubated for one hour. Hoechst was diluted in PBS. The wells were washed with PBS in between each step. The primary antibodies used were mouse anti-GFP

(1:1000; Invitrogen), rabbit anti-GFP (1:5000; Abcam), chicken anti-GFP (1:2000; Abcam), mouse anti-βIII-tubulin (1:1000; Covance), rabbit anti-Pax6 (1:1000; Covance), mouse anti-Pax6

(1:100; Development Studies Hybridoma Bank, The University of Iowa), rabbit anti-Tbr2

(1:1000; Abcam), mouse anti-Satb2 (1:400; Abcam), rabbit anti-cleaved caspase 3 (1:200;

Chemicon), mouse anti-Flag (1:1000; Sigma), goat anti-FoxP2 (1:100; Santa Cruz sc-21069). rat anti-Ctip2 (1:400; Abcam), mouse anti-HuD (1:100; Life Tech) and rabbit anti-Tbr1 (1:1000;

Abcam). The secondary antibodies used were Alexa Fluor 555-, Alexa Fluor 488- and Alexa

Fluor 647- conjugated goat or donkey antibodies to mouse, rabbit or goat IgG (1:1000 for

488/555, 1:500 for 647; Invitrogen). Nuclear staining was performed with Hoechst 33258

(Sigma). In some cases, immunostaining was performed using the MOM (Mouse on Mouse) Kit

(Vector Labs), as per manufacturer’s instructions.

Western blot analysis: Western blots were performed as described previously (Barnabe-Heider et al., 2005). Briefly, tissue or cells were harvested and incubated for 30 minutes at 4ºC in RIPA

63 lysis buffer supplemented with 1 mM of PMSF, 1 mM Na3VO4, 10 µg/mL aprotenin and 10

µg/mL leupeptin. Lysate was then centrifuge at 13000 RPM for 10 minutes and the supernatant was collected. Protein concentration was measured with the BCA Protein Assay Kit (Thermo

Scientific). For most experiments, 50-100 µg of lysate was loaded onto each lane of a 10% SDS gel. Proteins were then transferred to nitrocellulose membranes (Bio-Rad) at 250 mA at 4ºC overnight (Bio-Rad). Membranes were blocked with 5% skim milk in TBST for 1 hour, and incubated with primary antibodies diluted in 5% skim milk in TBST overnight at 4ºC overnight.

Secondary antibodies were also diluted in 5% skim milk in TBST and incubated for 1 hour at room temperature. Three 5 minute washes were applied between every step. To develop the blots, membranes were incubated in either ECL or ECL-plus (GE Healthcare) for 5 minutes and then exposed for varying periods of time. The primary antibodies used were rabbit anti-FoxP2

(Ab #1) (1:1000; Abcam), rabbit anti-FoxP2 (Ab #2) (1:1000; Sigma), goat anti-FoxP2 (Ab #3)

(1:1000; Santa Cruz), mouse anti-Flag (1:5000; Sigma), mouse anti-c-myc (1:1000; Santa Cruz), and rabbit anti-ERK1 K23 (1:5000; Santa Cruz Biotechnology). Secondary antibodies were

HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:5000; Boehringer Mannheim).

Flow cytometry: E13 littermate embryos were harvested from crosses between Sox2-EGFP and wild-type B6 mice. EGFP-positive and negative animals were separated, and cortical cells were harvested as described for the culture experiments, dissociated to single cells in 1% BSA and sorted for EGFP expression on a MoFlo fluorescent activated cell sorter (Dako) with viable cells identified by propidium iodide exclusion as previously described (Biernaskie et al., 2009). Gates were set using cells isolated from the EGFP-negative cortices.

Cell culture and transfection for HEK-293 cells: HEK-293 cells were cotransfected with mouse FoxP1, FoxP2, or FoxP4 cDNA and, for the knockdown experiments, either the negative control shRNA or shRNAs against mouse FoxP2. Briefly, cells were plated in 2 ml DMEM

(Lonza) supplemented with 10% FBS, 2 μM L-glutamine (Cambrex Biosciences) and 1% penicillin-streptomycin (Invitrogen), in 6-well polystyrene multiwell plates (BD Falcon). The next day, 500 μl of Opti-MEM (Invitrogen) and 5 μl of Lipofectamine 2000 (Invitrogen) were mixed with the DNA and incubated at room temperature for 30 minutes. The medium was replaced with 1.5 mL Opti-MEM, and 500 μL of the transfection reaction mixture was added.

The medium was replaced the next day with DMEM, and cells were harvested one or two days later for analysis.

RT-PCR: Cortices were dissected from embryos and either directly subjected to RNA isolation, or plated and cultured as described above for the indicated number of days prior to RNA isolation. Trizol (Invitrogen) was used to isolate RNA according to the manufacturer's protocol.

RNA was treated with DNase (Fermentas) to remove any contaminating genomic DNA. Reverse transcription was performed using RevertAid H Minus M-MuLV Reverse Transcriptase

(Fermentas) primed with random hexamers, according to manufacturer's instructions. All reactions were subjected to the following PCR protocol: 94°C for 2 minutes, 35 cycles of 94°C for 15 seconds, 58°C for 30 seconds and 72°C for 30 seconds, and a final elongation at 72°C for

2 minutes. Products were then resolved on a 2% agarose gel.

Microscopy and quantification: For quantification of cell culture experiments, depending on the transfection efficiency, 44-820 cells per condition per experiment were counted and analyzed

65 using the Zeiss Axioplan2 upright microscope equipped with fluorescence optics. In clonal studies, 28-210 clones were analyzed per condition per experiment. Clones were defined as groups of EGFP-positive cells in close proximity to each other that were well separated from any other EGFP-positive clusters but which were not part of distinct unlabeled clusters. Digital image acquisition was performed with Northern Eclipse software (Empix, Mississauga, Ontario,

Canada) using a Sony (Tokyo, Japan) XC-75CE CCD video camera. For quantification of tissue sections, cortical sections were chosen that showed a similar anatomical distribution and amount of EGFP-positive cells for comparison. Brains were sectioned at 16 µm at E16/17, three to four brain sections at the same anatomical level per embryo were analyzed using a Zeiss

(Oberkochen, Germany) Pascal confocal microscope and the manufacturer's software. A mean of two single optical sections taken with a 40x objective were computed for each image. A total of

3–4 sections were analyzed per embryo by taking up to three 8–10-mm pictures to cover the electroporated ventricular zone, SVZ and cortical plate of each coronal section with a 40X objective and comparing them with equivalent sections in littermate, control counterparts. For some experiments, sections were instead analyzed using a similar approach with an Olympus

IX81 inverted fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned

EM-CCD camera and Yokogawa CSU X1 spinning disk confocal scan head (with Spectral

Aurora Borealis upgrade), using Volocity (Perkin Elmer) software. Signals were considered to be positive when they were more intense than background labeling, and showed the predicted cellular localization. For measurement of cell location in electroporated cortices, image acquisition was performed using NorthernEclipse Software (Empix) with a Sony-XC-75CE CCD video camera. Ventricular, subventricular and cortical plate layers were delineated using Hoechst staining. For quantification of FoxP2 overexpression, confocal images were taken as described

66 above. ImageJ was used to quantify immunofluorescent intensity as described in a published study (Burgess et al., 2010). Briefly, the corrected fluorescence was obtained by subtracting the integrated density by the measured area multiplied by the mean fluorescence background reading, which was determined as an average of three background readings taken of the region adjacent to the cells of interest. Approximately 50 cells from three electroporated embryos each for human versus mouse FoxP2 were measured and the mean fluorescence determined.

Statistics: All data were expressed as the mean plus or minus the standard error of the mean

(S.E.M.), and were tested for statistical significance with two-tailed unpaired Student’s t-tests unless otherwise indicated, in which case they were analyzed with a Student Neuman-Keuls post-hoc ANOVA. Differences were considered significant if p < 0.05.

CHAPTER 4: FOXP2 PROMOTES NEUROGENESIS IN THE MAMMALIAN CORTEX

Abstract

The transcription factor FoxP2 has been associated with the development of human speech but the underlying cellular function of FoxP2 is still unclear. Here we provide evidence that FoxP2 regulates genesis of some intermediate progenitors and neurons in the mammalian cortex, one of the key centers for human speech. Specifically, knockdown of FoxP2 in embryonic cortical precursors inhibits neurogenesis, at least in part by inhibiting the transition from radial glial precursors to neurogenic intermediate progenitors. Moreover, overexpression of human, but not mouse, FoxP2 enhances the genesis of intermediate progenitors and neurons. In contrast, expression of a human FoxP2 mutant that causes vocalization deficits decreases neurogenesis, suggesting that in the murine system human FoxP2 acts as a gain-of-function protein, while a human FoxP2 mutant acts as a dominant-inhibitory protein. These results support the idea that

FoxP2 regulates the transition from neural precursors to transit-amplifying progenitors and ultimately neurons, and shed light upon the molecular changes that might contribute to evolution of the mammalian cortex.

Introduction

Speech and language disorders in humans have a substantial genetic component and likely involve many genes. The forkhead box transcription factor FoxP2 has been widely studied in this regard since it is mutated and autosomal dominant in developmental verbal dyspraxia

(Fisher et al., 1998), a syndrome involving significant speech and language deficits, as well as cognitive dysfunction such as autism spectrum disorder (Feuk et al., 2006). FoxP2 is highly conserved in vertebrates (Enard et al., 2002), and the mouse and human orthologues differ by three amino acid changes and an extra glutamine in the human poly-glutamine tract. One approach for understanding how FoxP2 might regulate speech and language involves modeling

FoxP2 function in animals with defined verbal behaviors such as songbirds and mice. In this regard, mice carrying a null mutation in FoxP2 (Shu et al., 2005) and knock-in mice carrying either a humanized FoxP2 allele (Enard et al., 2009) or human FoxP2 mutations (Fujita et al.,

2008) have been generated. Interestingly, mice where FoxP2 is perturbed displayed abnormalities in neonatal ultrasonic vocalization, a stereotypic behavior that is studied as a model for development of human speech and social cognition. However, these mice were also developmentally delayed and exhibited abnormalities in cerebellar development and motor function. It is not yet clear whether these neural abnormalities are the cause of the ultrasonic vocalization deficits and/or how these abnormalities relate to the impaired language development seen in humans.

These studies indicate that FoxP2 plays an important role in neural development, which is perhaps not surprising given the developmental roles played by other members of the same family. For example, FoxP1 defines columnar identity in the spinal cord (Rousso et al., 2008), and regulates outflow tract septation and myocardial proliferation during cardiac development

(Wang et al., 2004). FoxP4 is required for cardiac development, regulating fusion of the bilateral cardiac primordia and subsequent formation of the primitive heart tube (Li et al., 2004b). In addition, FoxP1 and FoxP2 cooperatively regulate lung airway morphogenesis and differentiation, and esophageal muscle development (Shu et al., 2007). Moreover, FoxP2 and

FoxP4 were recently found to regulate neurogenesis in the chick and mouse spinal cord (Rousso et al., 2012). Thus, the FoxP family regulates development in part by regulating embryonic precursors. In this regard, several studies have shown that FoxP2 mRNA is expressed in the precursor zones of the developing brain (Shu et al., 2001; Ferland et al., 2003). Since we have previously shown that perturbations in embryonic neural precursor development likely occur in genetic syndromes that cause cognitive dysfunction (Gauthier et al., 2007; Wang et al., 2010a), we hypothesized that FoxP2 mutations may, at least in part, cause developmental verbal dyspraxia by dysregulating embryonic neurogenesis. Here, we provide evidence for this idea, showing that in the developing mouse cortex, FoxP2 knockdown inhibits the transition from radial precursors to intermediate progenitors and neurons, and that a human FoxP2 mutant that causes verbal dyspraxia also inhibits neurogenesis, acting as a dominant-negative protein.

Results

FoxP2 is expressed in neural precursors in the embryonic murine cortex

To determine whether FoxP2 is expressed in neural precursors of the embryonic cortex, as suggested by previous in situ hybridization studies (Shu et al., 2001; Ferland et al., 2003; genepaint.org; set ID EH3230), we initially analyzed expression of FoxP2 mRNA. RT-PCR of

RNA isolated from the murine cortex at embryonic day 12 (E12) demonstrated that FoxP2 mRNA is expressed at this timepoint (Fig. 4.1A), when the cortex is predominantly comprised of precursors. To ask whether the protein is also expressed, we performed western blot analysis.

To do this, we first characterized three different FoxP2 antibodies for their specificity. HEK-

293 cells were transfected with myc- or flag-tagged expression constructs for FoxP1, FoxP2, and

FoxP4, all of which are expressed in the embryonic cortex (Shu et al., 2001; Lu et al., 2002).

Western blots of these lysates were then probed with three different FoxP2 antibodies and with antibodies for flag or myc, as relevant. This analysis (Fig. 4.1B) demonstrated that all three

FoxP2 antibodies recognized overexpressed FoxP2, but that two of them also recognized other

FoxP family members. We therefore utilized the third, FoxP2-specific antibody to probe western blots of the embryonic cortex; FoxP2 protein was present at E11, when the cortex does not contain neurons or glial cells, and this expression was maintained throughout embryogenesis, into early postnatal life (Fig. 4.1C).

To ask whether FoxP2 is expressed in cortical precursors, as suggested by its early embryonic expression, we performed two additional experiments. First, we analyzed E12 cortical precursor cultures which, when plated, are almost entirely comprised of proliferating radial precursors that go on to generate transit-amplifying intermediate progenitors and newly- born neurons. RT-PCR and western blot analysis demonstrated that FoxP2 mRNA and protein

71 were both expressed in these cultures one day post-plating (Fig. 4.1A,C). Second, we used flow cytometry to isolate neural precursors from the embryonic cortex, taking advantage of mice where EGFP is knocked-in to the Sox2 locus (Ellis et al., 2004). In these mice, embryonic radial precursors and intermediate progenitors can be sorted from other cells by virtue of their expression of EGFP (Hutton and Pevny, 2011). RT-PCR and western blot analysis of

Sox2:EGFP-positive cells isolated from the E13 cortex demonstrated that cortical precursors expressed both FoxP2 mRNA and FoxP2 protein (Fig. 4.1A,C).

Two classes of precursors are present in the embryonic cortex, radial glial precursors and intermediate progenitors. To ask which of these two populations express FoxP2, we immunostained the E12/13 cortex with the FoxP2-specific antibody. At this stage, low levels of nuclear FoxP2 were present in most Pax6-positive radial precursors (Fig. 4.1D). Tbr2-positive intermediate progenitors also expressed nuclear FoxP2, although this expression was more heterogeneous, with some cells expressing low levels and some higher levels (Fig. 4.1E).

Similar results were obtained with E12 precursors cultured for one day; the most robust levels of nuclear FoxP2 were observed in Tbr2-positive intermediate progenitors, and lower levels were seen in Tbr2-negative radial precursors (Fig. 4.1F).

FoxP2 knockdown decreases neurogenesis in the embryonic cortex

To ask about a potential function for FoxP2 in these neural precursors, we generated two shRNAs to knock it down. Western blot analysis of HEK-293 cells cotransfected with a mouse

FoxP2 overexpression vector and each of the FoxP2 shRNAs showed that these shRNAs robustly decreased FoxP2 protein levels (Fig. 4.1G). In contrast, these FoxP2 shRNAs had no effect on the levels of FoxP1 or FoxP4 in similar cotransfection experiments (Fig. 4.1G), as

72 predicted by their sequences (FoxP2 shRNA #1 had 13/21 mismatches with FoxP1 mRNA and

12/21 mismatches with FoxP4 mRNA while FoxP2 shRNA #2 had 5/21 and 8/21 mismatches with FoxP1 and FoxP4 mRNAs, respectively). To ask whether these shRNAs were similarly efficacious in cortical precursors, we cotransfected either FoxP2 shRNA #2 or a control shRNA together with a plasmid expressing EGFP into E12 precursor cultures and immunostained these cultures three days later with the FoxP2-specific antibody. Quantification of three independent experiments demonstrated that the number of EGFP-positive cells that expressed bright FoxP2

(as seen in Fig. 4.1F) was significantly decreased (control shRNA, 8.0 +/- 1.7%, FoxP2 shRNA,

1.5 +/- 0.3%; p < 0.05; n = 3 independent experiments).

Having demonstrated their efficacy, we used these two FoxP2 shRNAs to ask about the biological role of FoxP2 in embryonic cortical precursors. To do this, we electroporated E13/14 cortices with plasmids encoding nuclear EGFP and one of the two shRNAs; this manipulation electroporates embryonic cortical radial precursors, many of which will generate neurons that migrate out of the precursor regions of the cortex (the ventricular and subventricular zones or

VZ/SVZ) into the cortical plate (CP) region. Some of these radial precursors will also generate intermediate progenitors that will then divide and differentiate into neurons. Analysis of cortices at E16/17, three days following electroporation, revealed that both of the FoxP2 shRNAs altered the location of EGFP-positive cells relative to a control shRNA (Fig. 4.2A,B). In particular, they caused a significant reduction in the percentage of EGFP-positive cells, presumably newly-born neurons, that were present in the CP, and a significant increase in the percentage that remained in the VZ/SVZ (Fig. 4.2A,B).

We asked whether this change in cellular distribution could be due to enhanced cell death by immunostaining sections for EGFP and the apoptotic marker cleaved caspase-3 (CC3) 2 and 3

73 days post-electroporation. Numbers were similar in control and experimental brains at both timepoints, averaging approximately 0 to 3 cells per section (at 3 days, p > 0.05 for the comparisons between control versus FoxP2 shRNA#1 and control versus FoxP2 shRNA#2, n = 3 embryos each, 5 sections per embryo). We then asked whether the changes were due to perturbed neurogenesis by immunostaining sections for the transcription factor Satb2. Satb2 is a marker for one subpopulation of cortical neurons (Alcamo et al., 2008), and, consistent with this, we found that at E16/17, no Satb2-positive cells coexpressed the intermediate progenitor marker

Tbr2 or the radial precursor marker Pax6 (Fig. 4.2C,D). Moreover, virtually all of the electroporated, EGFP-positive neurons in the cortical plate expressed Satb2 when cortices were electroporated at E13/14 (Fig. 4.2E,F). Quantification of electroporated cortical sections immunostained with Satb2 demonstrated that FoxP2 knockdown caused a decrease in the number of neurons that were generated (Fig. 4.2G), consistent with the decrease in EGFP- positive cells in the cortical plate (Fig. 4.2B). A similar decrease was observed when sections were immunostained for the panneuronal marker HuD (Fig. 4.2G).

To ask whether cortical precursors were also altered by FoxP2 knockdown, we immunostained sections for Pax6 and Tbr2, markers for radial precursors and intermediate progenitors, respectively, three days following electroporation of the two different FoxP2 shRNAs. This analysis demonstrated that FoxP2 knockdown caused an increase in the proportion of EGFP-positive, Pax6-positive radial precursors (Fig. 4.3A,B) and a decrease in the proportion of EGFP-positive, Tbr2-positive intermediate progenitors (Fig. 4.3C,D).

These results indicate that the transition from radial precursors to intermediate progenitors and neurons is perturbed by FoxP2 knockdown. To ask whether this perturbation has long-lasting consequences for cortical neurogenesis, we utilized the piggybac (PB)

74 transposon; in this system, a plasmid encoding the PB transposase is cotransfected with a plasmid encoding EGFP flanked by inverted terminal repeats, and when the transposase is expressed, this leads to genomic integration of the flanked EGFP sequence (Nagy et al., 2011), thereby allowing longterm EGFP expression in the progeny of electroporated radial precursors.

We coelectroporated the transiently-expressed FoxP2 shRNA together with plasmids encoding

PB transposase and the EGFP reporter at E13/14, and analyzed cortical neurons at postnatal day three. This analysis demonstrated that approximately 80% of EGFP-expressing progeny of electroporated precursors were Satb2-positive neurons, and that transient knockdown of FoxP2 during embryogenesis did not cause longterm alterations in this number (Fig. 4.3E,F).

Immunostaining for two other cortical neuron markers, Tbr1 and Ctip2, demonstrated that only 1 to 5% of the EGFP-positive cells expressed either of these markers, and that these were unaltered by FoxP2 knockdown (Fig. 4.3E,F). However, while the decrease in neuronal number that was observed at E16/17 was lost by P3, potentially due to the transient nature of the FoxP2 knockdown, the localization of these neurons was aberrant (Fig. 4.3G). In particular, in contrast to controls, in cortices electroporated with the FoxP2 shRNA, approximately 20% of the NeuN- positive, EGFP-positive neurons were located in layers V and VI (Fig. 4.3G,H), and most of these mislocalized neurons expressed Satb2.

These data indicate that FoxP2 knockdown during embryogenesis perturbs neurogenesis.

To ensure that these results are not due to off-target effects of the shRNA, we performed rescue experiments with human FoxP2, which is not targeted by the shRNAs. Specifically, E13/14 cortices were electroporated with plasmids encoding EGFP and FoxP2 shRNA #1 plus or minus a human FoxP2 overexpression vector. Analysis of these cortices three days later confirmed that coincident human FoxP2 expression largely reversed the decrease in EGFP-positive cells in the

75 cortical plate and the increase in the VZ/SVZ caused by FoxP2 knockdown (Fig. 4.4A,B).

Similarly, immunostaining demonstrated that coincident human FoxP2 expression completely reversed the decrease in Tbr2-positive intermediate progenitors and Satb2-positive neurons and the increase in Pax6-positive radial precursors seen with FoxP2 knockdown (Fig. 4.4C-E).

FoxP2 knockdown inhibits the genesis of neurogenic intermediate progenitors

These data indicate that FoxP2 knockdown delays the progression from radial precursors to intermediate progenitors and neurons in vivo. To characterize this phenotype further, we knocked-down FoxP2 in culture; radial precursors were isolated from the E12 cortex, and upon plating were cotransfected with a control shRNA or with FoxP2 shRNA #2 plus an EGFP expression construct. Initially we characterized these cultures for apoptosis and proliferation.

Immunostaining for CC3 demonstrated that FoxP2 knockdown had no significant effect on the low proportion of CC3-positive apoptotic cells (Fig. 4.4F). In contrast, immunostaining for the proliferation marker Ki67 demonstrated that FoxP2 knockdown caused a significant increase from approximately 60% to 80% in the proportion of proliferating cells (Fig. 4.4F).

To ask whether this increase in proliferation reflected an increase in self-renewing radial precursors, as suggested by the in vivo knockdown data, we performed clonal analysis using the

PB transposon. Specifically, E12 precursors were cotransfected with FoxP2 shRNA #2 plus or minus a human FoxP2 overexpression construct together with plasmids encoding PB transposase and the PB EGFP reporter construct, under conditions where only a small number of cells were transfected. We then analyzed the size and composition of EGFP-positive clones in these cultures (Fig. 4.4G). This analysis showed that FoxP2 knockdown caused a significant increase in the size of the EGFP-positive clones (Fig. 4.4H), with a decrease in single cell clones, and an

76 increase of almost 3-fold in the proportion of clones containing 9 or more cells (Fig. 4.4I).

Moreover, only a very small number of control clones contained more than 17 cells, but approximately 10% of the FoxP2 knockdown clones were 17 cells or larger (Fig. 4.4H).

Importantly, coexpression of human FoxP2 reversed this increase in clone size (Fig. 4.4H,I). We also performed an additional control for the single cell clonal analysis, transfecting cells with a control shRNA together with human FoxP2. This control group was statistically similar to the

FoxP2 shRNA plus human FoxP2 group (p > 0.05) and was significantly higher than the FoxP2 shRNA plus empty vector group (p < 0.01; n = 3 independent experiments for both comparisons).

We also asked about clone composition by immunostaining these cultures for βIII-tubulin and Tbr2 (Fig. 4.4J). FoxP2 knockdown increased by approximately 30% the proportion of

EGFP-positive clones that did not contain βIII-tubulin or Tbr2-positive cells, and this increase in radial precursor clones was significantly reversed by coincident expression of human FoxP2 (p <

0.01 for both comparisons, n = 3 independent experiments). Moreover, quantification showed that the FoxP2 but not control shRNA decreased the genesis of intermediate progenitors and neurons, and that these decreases were also reversed by coincident overexpression of human

FoxP2 (Fig. 4.4K,L). Thus, FoxP2 knockdown in culture enhanced radial precursors at the expense of intermediate progenitors and neurons.

Ectopic expression of human but not mouse FoxP2 promotes the radial precursor to intermediate progenitor transition in the mouse cortex

These data indicate that FoxP2 knockdown decreases genesis of intermediate progenitors from radial precursors. Since a number of groups have suggested that evolutionary changes in

77 mammalian FoxP2 might be important (Enard et al., 2002; Krause et al., 2007; Konopka et al.,

2009) we asked whether mouse and human FoxP2 proteins had similar or different activities when ectopically expressed in radial precursors. To do this, we utilized flag-tagged expression constructs for mouse and human FoxP2 (Konopka et al., 2009) that were identical except that one encoded mouse and the other human FoxP2 coding sequences (Fig. 4.5A). We verified that these constructs were expressed at similar levels by transfecting them into HEK-293 cells, and probing equal amounts of protein on western blots with either the FoxP2 antibody or with an antibody for the flag tag (Fig. 4.5B).

We then introduced these constructs into cortical precursors in vivo, electroporating them into the E13/14 cortex along with a nuclear EGFP expression plasmid. Three days later we demonstrated that both constructs were expressed by staining cortical sections either for the flag tag present on the tagged proteins or for FoxP2 itself. Flag-tagged murine and human FoxP2 were both detectably expressed in EGFP-positive cells (Fig. 4.5C). Moreover, image analysis of sections immunostained for FoxP2 (Fig. 4.5D) demonstrated that mouse and human FoxP2 were expressed at similar levels in electroporated precursors (mouse FoxP2 levels were set to 1.0; mouse FoxP2, 1.0 +/- 0.28, human FoxP2, 1.0 +/- 0.17; p = .99, n = 3 embryos each). Since both proteins were expressed in electroporated precursors, we asked whether they affected cortical precursor biology. Immunostaining for EGFP (Fig. 4.5E) demonstrated that the distribution of transfected cells was similar in cortices electroporated with the control vector and mouse FoxP2

(p > 0.05; n = 3 embryos each), but that electroporation with human FoxP2 caused a small but significant decrease in the proportion of electroporated cells in the VZ/SVZ relative to controls

(control vector, 31.6 +/- 2.6, human FoxP2, 26.0 +/- 1.0; p < 0.05, n = 4 embryos each). To ask if this reflected a change in precursor composition, we immunostained these sections for EGFP

78 and Pax6 or Tbr2. Quantification demonstrated that human FoxP2 overexpression decreased the proportion of Pax6-positive radial precursors and increased the proportion of Tbr2-positive intermediate progenitors (Fig. 4.5F) relative to controls. In contrast, mouse FoxP2 had no effect on the proportions of these two precursor types (for Pax6-positive cells, control vector, 20.3 +/-

0.8%, mouse FoxP2, 20.0 +/- 3.4%, p = 0.94; for Tbr2-positive cells, control vector, 16.8 +/-

0.8%, mouse FoxP2, 17.1 +/- 1.8%, p = 0.87; n = 3 embryos each).

To more definitively establish these differences between human and mouse FoxP2 proteins, we performed similar studies electroporating different embryos in the same mothers with human versus mouse FoxP2 constructs. We then analyzed these cortices 3 days later by immunostaining for Pax6 and Tbr2. Quantification showed that human and mouse FoxP2 overexpression had significantly different effects, with human FoxP2 causing a decrease in

Pax6-positive radial precursors and an increase in the percentage of Tbr2-positive intermediate progenitors relative to mouse FoxP2 (Fig. 4.5G).

Since human FoxP2 increased the number of neurogenic intermediate progenitors, we asked whether it also altered the number or phenotype of cortical neurons. To ask about neuron number, we analyzed cortices three days post-electroporation at E16/17. Immunostaining for

EGFP and HuD demonstrated a small but significant increase in the proportion of EGFP-positive cortical neurons (Fig. 4.6A). A similar small but significant increase was observed when sections were immunostained for Satb2 (control vector, 42.3 +/- 4.8, human FoxP2, 52.6 +/- 4.8; p = .03, two-tailed paired Student's t-test, n = 4 embryos each). To ask about neuronal phenotypes, we analyzed cortices postnatally, electroporating embryos at E13/14 with plasmids encoding EGFP and human FoxP2, and immunostaining them for different neuronal markers at postnatal day 3 (Fig. 4.6B,C). Quantification showed that human FoxP2 did not significantly

79 affect the proportion of Satb2-positive neurons at this longer timepoint nor did it affect the small number of Ctip2 or Tbr1-positive neurons (Fig. 4.6D).

To further explore the possibility that human, but not mouse, FoxP2 enhances the transition from radial precursors to intermediate progenitors when ectopically expressed in the mouse, we performed similar experiments in culture, cotransfecting mouse versus human FoxP2 constructs together with an EGFP expression plasmid into cultured radial precursors. Clonal analysis four days later showed that human FoxP2 caused a decrease in clone size, significantly increasing the proportion of one cell clones relative to either mouse FoxP2 or the empty vector

(Fig. 4.6E,F). Consistent with this, immunostaining showed that human, but not mouse FoxP2 increased the proportion of Tbr2-positive intermediate progenitors (Fig. 4.6G) and βIII tubulin- positive neurons (Fig. 4.6H) that were generated in these cultures. Thus, human FoxP2 apparently acts as a gain-of-function protein in murine cortical precursors, acting to promote the radial precursor to intermediate progenitor transition and enhance neurogenesis.

The human FoxP2 KE mutation acts like a dominant-inhibitoryFoxP2 protein

Since our data indicate that even the small differences between human and murine FoxP2 proteins can alter its ability to regulate neural precursor development, we asked about a mutant

FoxP2 protein that has been implicated in aberrant vocalization. To do this, we examined a

FoxP2 mutation from the KE family (Lai et al., 2001) using a flag-tagged expression construct identical to that used for the human versus mouse overexpression studies. This mutation results in a FoxP2 protein carrying a single amino acid switch in the forkhead DNA binding domain

(Fig. 4.5A). Cotransfection into HEK-293 cells demonstrated that the KE mutant was expressed at levels similar to the normal human FoxP2 protein (Fig. 4.5B). We then cotransfected cultured

80 cortical precursors with plasmids encoding nuclear-localized EGFP and the wild-type or mutant human FoxP2 or murine FoxP2, and immunostained them 1 day later for EGFP and the flag tag.

This analysis (Fig. 4.7A) demonstrated that all three proteins were expressed, but that their localization differed. In particular, the normal murine and human FoxP2 proteins were limited to nuclei, colocalizing closely with nuclear EGFP (Fig. 4.7A). In contrast, the human KE mutant was present in both the cytoplasm and nucleus (Fig. 4.7A). Quantification showed that more than 60% of the flag-positive cells transfected with the KE mutant expressed detectable FoxP2 in the cytoplasm (Fig. 4.7B).

These experiments indicate that the mutant human FoxP2 protein is expressed at readily detectable levels in cortical precursors, but that it is aberrantly localized. These data are consistent with previous reports expressing it in cell lines (Vernes et al., 2006; Mizutani et al.,

2007a), which showed that the KE mutant protein was present in the cytoplasm and that it even caused aberrant localization of wild-type FoxP2 protein in the cytoplasm. We therefore electroporated the E13/14 cortex with expression plasmids for the KE mutant protein and EGFP, and analyzed precursor development 3 days later. Immunostaining for EGFP demonstrated that location of transfected cells within the cortex was perturbed by expression of the mutant FoxP2, with significantly more cells present in the VZ/SVZ and significantly fewer in the cortical plate relative to the empty vector (Fig. 4.7C,D). Coincident with this change in location, there was an increase in the proportion of Pax6-positive radial precursors and a decrease in Tbr2-positive intermediate progenitors (Fig. 4.7E). There was also a trend towards a decrease in Satb2- positive neurons, but this did not reach significance (Fig. 4.7E). Thus, in contrast to human

FoxP2, the KE mutant FoxP2 protein decreased the radial precursor to intermediate progenitor

81 transition thereby phenocopying the FoxP2 knockdown phenotype, suggesting that it was functioning as a dominant-inhibitory protein.

To further explore the possibility that the KE mutant FoxP2 has a different phenotype from normal human FoxP2 protein, we overexpressed it in cultured cortical precursors for 4 days. Consistent with the idea that the KE mutant protein inhibited cortical precursor differentiation, clonal analysis demonstrated that it increased clone size relative to human and murine FoxP2 proteins (Fig. 4.6E,F). Moreover, immunostaining demonstrated that there were only approximately half as many Tbr2-positive intermediate progenitors (Fig. 4.6G) and βIII tubulin-positive neurons (Fig. 4.6H) generated from cells that expressed the mutant human

FoxP2 compared to normal human FoxP2. Overexpression of the KE mutant also caused a decrease in neurons relative to the empty vector and murine FoxP2 overexpression (Fig. 4.6H), thereby phenocopying the FoxP2 knockdown data (Fig. 4.4H). Together with the in vivo studies, these data provide support for the idea that the human KE mutant acts as a dominant-inhibitory

FoxP2 protein.

Figure 4.1

Figure 4.1: Expression of FoxP2 in embryonic cortical precursors. A, RT-PCR analysis for

FoxP2 mRNA in total RNA isolated from E12 murine cortex (E12; left panel), from E12 cortical precursor cultures 1 or 3 days after plating (1 or 3 DIV; left panel), or from Sox2:EGFP-positive precursors sorted from the E13 cortex (right panel). As controls, samples were generated in the absence of reverse transcriptase (RT-). Molecular weight markers are shown to the left of both panels. B, Western blot analysis of lysates of HEK-293 cells transfected with expression constructs for flag-tagged FoxP1, flag-tagged FoxP2, or myc-tagged FoxP4, probed with antibodies for flag, myc, the Erk proteins as a control, or one of three different FoxP2 antibodies

(Ab#1 is from Abcam, Ab#2 is from Sigma and Ab#3 is from Santa Cruz). Note that only

FoxP2 Ab#3 is specific for FoxP2. C, Western blot analysis of lysates of the embryonic cortex at various timepoints (top left panel), of E12 cortical precursors cultured for 1 day (1DIV; right top panel), and of Sox2:EGFP-positive precursors sorted from the E13 cortex (bottom right panel), all probed with FoxP2 Ab#3. The top right panel also includes flag-tagged FoxP2 that was overexpressed in HEK-293 cells (FoxP2, right lane), which runs at a slightly higher molecular weight due to the triple-flag tag. The blot shown on the top left panel was reprobed for total Erk protein as a loading control (bottom left panel). Molecular weight markers are shown to the left of the panels. D,E, Confocal micrographs of coronal sections through the

E12.5 cortex immunostained for FoxP2 (red) and Pax6 (D) or Tbr2 (E) (both in green; the right panels show the merged images). The bottom row of micrographs in each panel shows higher magnification images. In (D) arrows denote cells that are positive for Pax6 and FoxP2, while in

(E) they denote Tbr2-positive cells that express more robust levels of FoxP2. Scale bar, 50 μm for top panels, 20 μm for bottom panels. F, Fluorescence micrograph of E12 cortical precursors cultured for 1 day, immunostained for FoxP2 (red) and Tbr2 (green) and counterstained with

Hoechst 33258 (blue) to show cell nuclei. Arrows denote a Tbr2-positive intermediate progenitor that expresses higher levels of FoxP2, and arrowheads cells with low levels of nuclear FoxP2 that are negative for Tbr2. Scale bar, 20 μm. G, (Left panel) Western blot analysis of lysates of

HEK-293 cells cotransfected with a flag-tagged murine FoxP2 overexpression construct either alone (Con) or with one of two different shRNAs for murine FoxP2 (sh1 and sh2), probed with

FoxP2 Ab#3. (Middle and right panels) Western blot analysis of lysates of HEK-293 cells cotransfected with flag-tagged murine FoxP1, flag-tagged murine FoxP2, or myc-tagged murine

FoxP4 plus or minus FoxP2 shRNAs #1 or #2, and probed with antibodies for flag (middle panel) or myc (right panel). All blots were reprobed for total Erk as a loading control.

Molecular weights are shown to the left of the panels.

Figure 4.2

Figure 4.2: FoxP2 knockdown decreases neurogenesis in the embryonic cortex in vivo. A,

Fluorescence micrographs of coronal sections through E16/17 cortices that were coelectroporated at E13/14 with nuclear EGFP and control shRNA (Control) or FoxP2 shRNA

#2 (shFoxP2 #2) and immunostained for EGFP (green) and Tbr1 (red; blue is Hoechst 33258 nuclear counterstain). Tbr1 staining was used to mark the border between the intermediate zone

(IZ) and the cortical plate (CP). In each set of two panels, the left panel shows the merged images of Tbr1 and Hoechst 33258, and the right panel shows the EGFP immunostaining. The top white line demarcates the border between the CP and IZ, and the bottom white line the border between IZ and the VZ/SVZ. Scale bar, 100 μm. B, Quantification of sections similar to those in A for the proportion of EGFP-positive cells in the VZ/SVZ (left pair), IZ (middle pair) and CP (right pair). (***p < 0.001; n = 5 each for control and FoxP2 shRNA#1). C, Confocal micrographs of coronal E16/17 cortical sections immunostained for Satb2 (red) and Pax6

(middle panel, green) or Tbr2 (right panel, green). Scale bar, 50 μm. D, Higher magnification confocal images of sections similar to those shown in C. Arrows denote cells that are positive for

Satb2 (red) and arrowheads denote cells that are positive for Pax6 (green, top panel) or Tbr2

(green, bottom panel; right panels show the merges). Note the absence of colocalization between

Satb2-positive cells and Pax6-positive or Tbr2-positive cells. Scale bar, 20 μm. E, Confocal micrographs of coronal cortical sections from E16/17 brains three days after electroporation with

EGFP and control shRNA (Control) or FoxP2 shRNA #2 (shFoxP2), immunostained for EGFP

(green) and Satb2 (red). The white lines demarcate the different regions of the embryonic cortex. Scale bar, 50 μm. F, High magnification confocal images of sections similar to those in

E. The bottom panels show Satb2 (red) alone, and the top panels Satb2 plus EGFP (green).

Arrows denote EGFP-positive, Satb2-positive cells. Scale bar, 20 μm. G, Quantification of

87 sections similar to those in E,F for the percentage of EGFP-positive cells that are also positive for Satb2 or HuD, as indicated, three days after electroporation with control shRNA or with

FoxP2 shRNA#1 or FoxP2 shRNA#2. (*p < 0.05; **p < 0.01; ***p < 0.001; n = 3-5 embryos for each of the shRNAs). Error bars denote standard error of the mean (S.E.M.).

Figure 4.3

Figure 4.3: FoxP2 knockdown increases radial precursors at the expense of intermediate progenitors in the embryonic cortex. A-D, Murine cortices were electroporated at E13/14 with

EGFP and control shRNA (Control) or FoxP2 shRNAs #1 or #2 (shFoxP2) and analyzed three days later. A, Confocal micrographs of coronal cortical section from electroporated brains immunostained for EGFP (green) and Pax6 (red). The left panels are low magnification images of the VZ/SVZ of cortices electroporated with control shRNA (top) or FoxP2 shRNA #2

(bottom). The middle and right panels are high magnification images showing EGFP-positive cells that are positive (arrows) for Pax6, with the middle panels showing the merges and the right panels Pax6 immunostaining only. Scale bar, 50 μm. B, Quantification of sections similar to those in A for the percentage of EGFP-positive cells that are also positive for Pax6. (*p < 0.05;

***p < 0.001; n = 4 or 5 embryos each). C, Confocal micrographs of coronal cortical section from electroporated brains immunostained for EGFP (green) and Tbr2 (red). The left panels are low magnification images of the VZ/SVZ of cortices electroporated with control shRNA (top) or

FoxP2 shRNA #2 (bottom). The middle and right panels are high magnification images showing

EGFP-positive cells that are positive (arrows) for Tbr2, with the middle panels showing the merges and the right panels Tbr2 immunostaining only. Scale bar, 50 μm. D, Quantification of sections similar to those in C for the percentage of EGFP-positive cells that are also positive for

Tbr2. (*p < 0.05; ***p < 0.001; n = 4 or 5 embryos each). E-H, Murine cortices were electroporated at E13/14 with a PB transposon-based EGFP reporter and control shRNA

(Control) or FoxP2 shRNA #2 and analyzed at postnatal day 3. E, Confocal micrographs of coronal cortical sections immunostained for EGFP (green) and Satb2 (left panel, blue), Tbr1

(middle panel, red) or Ctip2 (right panel, red; the bottom panels show the merges). Arrows denote double positive cells. Scale bar, 20 μm. F, Quantification of sections similar to those in

E for the percentage of EGFP-positive cells that are also positive for Satb2, Tbr1 and Ctip2 (p >

0.05; n = 3 animals each). G, Confocal micrographs of coronal cortical sections from electroporated P3 brains immunostained for EGFP. The white lines distinguish the various cortical layers, in part as identified by immunostaining for Ctip2 (not shown) which labels layer

V cortical neurons. Scale bar, 400 μm. H, Quantification of sections similar to those in G for the percentage of EGFP-positive cells in the different cortical layers that also expressed NeuN. (*p

< 0.05; **p < 0.01; n = 3 animals each). Error bars denote S.E.M.

Figure 4.4

Figure 4.4: FoxP2 knockdown increases radial precursors at the expense of intermediate progenitors and neurons, and this is reversed by coincident expression of human FoxP2. A-E,

Murine cortices were electroporated at E13/14 with an EGFP plasmid and either control shRNA plus pCMV-Tag4A empty vector (shCon/EV), FoxP2 shRNA #1 plus pCMV-Tag4A empty vector (shFoxP2/EV), or FoxP2 shRNA #1 plus human FoxP2 in the pCMV-Tag4A backbone

(shFoxP2/hFoxP2), and analyzed three days later. A, Fluorescence micrographs of coronal cortical sections showing EGFP-positive cells (green). The white lines denote the different regions of the cortex. Scale bar, 100 μm. B, Quantification of sections similar to those in A for the number of EGFP-positive cells in the different cortical regions. (*p < 0.05; **p < 0.01; ***p

< 0.001, ANOVA with Student-Newman-Keuls post-hoc analysis; n = 3-7 embryos each). C-E,

Quantification of the percentage of EGFP-positive cells that were also positive for Pax6 (C),

Tbr2 (D), or Satb2 (E) in cortical sections similar to those in A that were immunostained for

EGFP and one of these three markers. (*p < 0.05; **p < 0.01; ***p < 0.001, ANOVA with

Student-Newman-Keuls post-hoc analysis; n = 3-5 embryos each). F, E12 cortical precursors were cotransfected with EGFP and control shRNA (Control) or FoxP2 shRNA #2 (shFoxP2 #2) and quantified for the percentage of transfected, Ki67-positive (left two bars) or CC3-positive

(right two bars) cells after 3 days. (**p < 0.01; n = 3 independent experiments). G-I, E12 cortical precursors were cotransfected with PB-EGFP and PBase, and control shRNA plus empty vector

(shCon/EV), FoxP2 shRNA plus pCMV-Tag4A empty vector (shFoxP2/EV), or FoxP2 shRNA plus a plasmid encoding hFoxP2 in the pCMV-Tag4A backbone (shFoxP2/hFoxP2). G,

Fluorescence micrographs of cortical precursor cultures cotransfected with plasmids encoding

PB-EGFP and PBase and either control shRNA (top) or FoxP2 shRNA#2 (bottom), immunostained 4 days later for EGFP (green). Scale bar, 50 μm. H, Quantification of clone size

93 in experiments similar to those shown in G. Results are pooled data from three independent experiments. I, Quantification of the percentage of transfected, EGFP-positive clones that contained only a single cell (left three bars) or nine or more cells (right three bars). The graphs show pooled data from three independent experiments. (*p < 0.05; **p < 0.01, ANOVA with

Student-Newman-Keuls post-hoc analysis). J-L, E12 cortical precursors were cotransfected with

PB-EGFP and PBase, and control shRNA plus empty vector (shCon/EV), or FoxP2 shRNA plus pCMV-Tag4A empty vector (shFoxP2/EV), control shRNA plus a plasmid encoding hFoxP2 in the pCMV-Tag4A backbone (shCon/hFoxP2), or FoxP2 shRNA plus hFoxP2

(shFoxP2/hFoxP2). J, Fluorescence micrographs of transfected cells immunostained 4 days after transfection for EGFP (green) and Tbr2 (top, red) or βIII-tubulin (bottom, red). Cells were counterstained with Hoechst 33258 (blue). The right panels show the merges. Scale bar, 20 μm.

K,L, Quantification of the percentage of transfected, Tbr2-positive (K) or βIII-tubulin-positive

(L) cells in experiments similar to those shown in J. Both graphs show pooled data from three independent experiments. (*p < 0.05; **p < 0.01, ANOVA with Student-Newman-Keuls post- hoc analysis). Error bars denote S.E.M.

Figure 4.5

Figure 4.5: Ectopic expression of human but not murine FoxP2 in the murine embryonic cortex enhances the genesis of intermediate progenitors and neurons. A, Schematics of the domain structure of human FoxP2 (hFoxP2), mouse FoxP2 (mFoxP2) and human FoxP2 carrying the KE mutation in the DNA binding domain (hFoxP2-R553H). Both of the human

FoxP2 proteins are 715 amino acids in length while mouse FoxP2 is 714. All amino acid substitutions indicated in the diagram are with reference to human FoxP2. B, Western blot analysis of lysates of HEK-293 cells transfected with pCMV-Tag4A empty vector (EV), or flag- tagged expression constructs for mouse FoxP2, wild-type human FoxP2 (WT), or human FoxP2 carrying the R553H KE family mutation (KE), all in the pCMV-Tag4A backbone, probed with antibodies against FoxP2 or the flag tag. Blots were also probed with an antibody against Erk as a control for equal protein loading. C-G, Murine cortices were electroporated at E13/14 with a nuclear EGFP expression construct and either the empty vector (Control), or the flag-tagged human FoxP2 (hFoxP2) or mouse FoxP2 (mFoxP2) expression constructs, and analyzed three days later. C, High magnification confocal micrographs of coronal cortical sections through the

VZ/SVZ of electroporated brains immunostained for EGFP (green) and Flag (red; right panels show the merges) three days after electroporation. Arrows indicate EGFP-positive cells that were also positive for the flag tag. Scale bar, 50 μm. D, High magnification confocal micrographs of coronal cortical sections through the VZ/SVZ of electroporated brains immunostained for Flag

(green) and FoxP2 (red; right panels show the merges) three days after electroporation. Arrows indicate flag-positive cells that were also positive for FoxP2. Scale bar, 50 μm. E, Fluorescence micrographs of coronal sections of electroporated brains three days after electroporation showing

EGFP-positive cells (green). The white lines demarcate the different cortical regions. Scale bar,

100 μm. F, Quantification of the percentage of double-labelled cells in sections similar to those

96 in E that were immunostained for EGFP and Pax6 or Tbr2 three days following electroporation with the empty vector (Control) or the expression construct for human FoxP2 (hFoxP2). (*p <

0.05; n = 4 embryos each). G, Quantification of the percentage of double-labelled cells in sections similar to those in E that were immunostained for EGFP and Pax6 or Tbr2 three days following electroporation with EGFP and the expression construct for either mouse FoxP2

(mFoxP2) or human FoxP2 (hFoxP2). (**p < 0.01; ***p < 0.001; n = 7 embryos each). Error bars denote S.E.M.

Figure 4.6

Figure 4.6: Ectopic expression of human but not mouse FoxP2 promotes genesis of intermediate progenitors and neurons. A-D, Murine cortices were electroporated at E13/14 with

EGFP and either the pCMV-Tag4A empty vector (Control) or a human FoxP2 expression construct in the same vector (hFoxP2), and analyzed at E16/17 (A) or at P3 (B-D). A, Confocal micrographs of coronal cortical sections of E16/17 electroporated brains immunostained for

EGFP (green) and HuD (red). The graph on the right shows quantification of similar sections for the percentage of EGFP-positive cells that express HuD. (*p < 0.05, two-tailed paired Student’s t-test; n = 4 embryos each). Scale bar, 100 μm. B, Confocal micrographs of coronal cortical sections of electroporated brains at P3 immunostained for EGFP (green), Tbr1 (red) and Satb2

(blue). Scale bar, 400 μm. C, Confocal micrographs of coronal cortical sections of electroporated brains at P3 immunostained for EGFP (green) and Ctip2 (red). Scale bar, 400 μm. D,

Quantification of sections similar to those in B and C for the percentage of EGFP-positive cells that were also positive for Satb2 (left two bars), Tbr1 (middle two bars) or Ctip2 (right two bars).

(p > 0.05; n = 3 animals each). E-H, E12 cortical precursor cultures were cotransfected with an

EGFP plasmid and expression constructs for mouse FoxP2 (mFoxP2), wild-type human FoxP2

(hFoxP2), or human FoxP2 carrying the KE mutation (hFoxP2-R553H or KE FoxP2). As a control, cultures were cotransfected with the empty vector (Control). E, Quantification of the sizes of transfected, EGFP-positive clones. F, Quantification of the percentage of transfected,

EGFP-positive clones that contain only a single cell, normalized to the empty vector control cultures. G,H, Quantification of the percentage of transfected, Tbr2-positive (G) or βIII-tubulin- positive (H) cells, normalized to the empty vector control cultures. (*p < 0.05; **p < 0.01; ***p

< 0.001, ANOVA with Student-Newman-Keuls post-hoc analysis; n = 3 or 4 experiments for each). Error bars denote S.E.M.

Figure 4.7

100

Figure 4.7: The KE family FoxP2 mutation acts as a dominant-negative with regard to embryonic cortical neurogenesis. A, Fluorescence micrographs of E12 cortical precursor cultures cotransfected with a nuclear EGFP plasmid and expression constructs for mouse FoxP2

(mFoxP2; left panels), wild-type human FoxP2 (hFoxP2; left center panels) or human FoxP2 carrying the KE mutation (hFoxP2-R553H; right center and right panels), immunostained one day later for EGFP (green) and Flag (red). Cells were counterstained with Hoechst 33258 (blue).

Dotted lines mark nuclear borders as indicated by Hoechst staining. Note the presence of cytoplasmic Flag-tagged hFoxP2-R553H. Scale bar, 20 μm. B, Quantification of the percentage of transfected cells in experiments as in A where Flag immunoreactivity was detectable in the cytoplasm. (***p < 0.001, ANOVA with Student-Newman-Keuls post-hoc analysis; n = 3 independent experiments). C-E, E13/14 murine cortices were electroporated with EGFP and pCMV-Tag4A empty vector (Control) or an expression construct for hFoxP2-R553H in the same vector. C, Fluorescence micrographs of coronal cortical sections three days post-electroporation, showing EGFP-positive cells (green). The white lines demarcate the different cortical regions.

Scale bar, 100 μm. D, Quantification of sections similar to those in C for the number of EGFP- positive cells in the VZ/SVZ (left pair), IZ (middle pair) and CP (right pair). (**p < 0.01; n = 7 animals for the control and 6 for hFoxP2-R553H). E, Quantification of sections similar to those in C for the percentage of EGFP-positive cells that were positive for Pax6 (left two bars), Tbr2

(middle two bars), or Satb2 (right two bars) three days after electroporation. (*p < 0.05; ***p <

0.001; n = 6 for control and 5 for hFoxP2-R553H). Error bars denote S.E.M.

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Discussion

Our results suggest a novel role for the FoxP2 transcription factor in developing neural precursors that may have implications for evolution of a larger mammalian cortex. In this regard, our findings support three major conclusions. First, our data indicate that FoxP2 is expressed in developing cortical precursors, and that when it is knocked-down, this perturbs the genesis of intermediate progenitors and neurons from multipotent radial precursors. Second, our data indicate that human and mouse FoxP2 proteins differ with regard to their ability to regulate the genesis of intermediate progenitors when ectopically expressed. When mouse FoxP2 is overexpressed either in vivo or in culture, this has no effect on cell genesis, indicating that mouse

FoxP2 is not sufficient to promote the genesis of intermediate progenitors, and suggesting that it is normally not limiting for neural precursors. However, when human FoxP2 is overexpressed in the same way, this enhances the genesis of intermediate progenitors and neurons, indicating that human FoxP2 acts as a gain-of-function protein relative to murine FoxP2. This is an intriguing result, given the high homology between these two orthologues. Third, our data indicate that the

KE mutant FoxP2, which acts as an autosomal dominant gene in humans, functions as a dominant-inhibitory protein with regard to neurogenesis. Specifically, we show that, as seen previously in cell lines, this mutant FoxP2 is localized to both the nucleus and cytoplasm in cortical precursors, and that it phenocopies FoxP2 knockdown when overexpressed, causing increased radial precursors and decreased intermediate progenitors and neurons. Together, these findings suggest a novel proneurogenic role for FoxP2 and, from a broader context, suggest that genetically-defined changes in proteins like FoxP2 may contribute to the evolution of the human cortex.

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The FoxP2 gene has garnered significant interest because it is mutated in a monogenic syndrome causing speech and language dysfunction (Lai et al., 2001). This relationship has led to speculation that small changes in FoxP2 sequence are causally related to the evolution of human speech (Enard et al., 2002; Zhang et al., 2002; Vernes et al., 2007). In support of this idea, human FoxP2 has transcriptional targets that are distinct from mouse and even chimpanzee

FoxP2 (Vernes et al., 2007; Konopka et al., 2009; Vernes et al., 2011), and cortico-basal circuitry is functionally different in a mouse where two of the human amino acid changes are knocked-in to the mouse FoxP2 allele (Enard et al., 2009; Reimers-Kipping et al., 2011). Thus, the few amino acid changes between the different FoxP2 proteins are clearly important and are likely somehow involved in functional neural differences between species. In this regard, though, it is still unclear whether FoxP2 is only important for speech and language and/or whether it plays a broader role in neural function.

Previous studies on FoxP2 have utilized the songbird (Haesler et al., 2007) and the mouse

(Shu et al., 2005; French et al., 2007; Fujita et al., 2008) as models for understanding how this gene might regulate vocalization. Work in both models has shown that FoxP2 is expressed in neurons, and that depletion of FoxP2 leads to changes in vocalization (Shu et al., 2005; French et al., 2007; Fujita et al., 2008) and neuronal morphology (Vernes et al., 2011). In this regard, knockout mice and mice homozygous for mutant human FoxP2 alleles are developmentally delayed, die within several weeks of birth, display perturbed ultrasonic vocalizations, and have smaller brains with a particularly small cerebellum. While these neural phenotypes are thought to be due to perturbed neuronal connectivity, the results presented here suggest that they are, in part, due to aberrant neurogenesis. Strong additional support for this conclusion comes from a recent study that placed FoxP2 and FoxP4 together in a proneurogenic pathway (Rousso et al.,

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2012). This study showed that Foxp2 is initially expressed throughout the neuroepithelium, and that inactivation of FoxP2 and FoxP4 function led to neuroepithelial disorganization and enhanced maintenance of neural precursors, a result consistent with the findings reported here.

How then does FoxP2 regulate neurogenesis? During cortical development, neurons are generated either directly from radial glial precursors, or indirectly via intermediate progenitors.

Intriguingly, the number of intermediate progenitors correlates with cortical expansion

(Martinez-Ceredeno et al., 2006) and the intermediate progenitor population is enhanced in humans relative to rodents (Hansen et al., 2010). Moreover, deletion of genes required for intermediate progenitors results in decreased cortical thickness and neuron number (Farkas et al.,

2008; Sessa et al., 2008), and humans carrying homozygous mutation in Tbr2 also present with anatomical anomalies including microcephaly, polymicrogyria and corpus callosum agenesis, and cognitive deficits and severe motor delay (Baala et al., 2007). In this regard, our knockdown data argue that FoxP2 promotes neurogenesis in part by enhancing the genesis of intermediate progenitors from radial precursors. Intriguingly, our expression data also show that while FoxP2 is expressed at low levels in radial precursors, its expression is robustly increased in at least a subset of intermediate progenitors. Thus, one interpretation of our data is that FoxP2 levels normally increase as part of a pro-intermediate progenitor gene expression program, and that when we knock it down in radial precursors, this prevents this induction and inhibits the transition. However, our data showing that overexpression of mouse FoxP2 in these same radial precursors does not cause them to prematurely generate intermediate progenitors argues that increased FoxP2 is necessary but not sufficient for this transition.

What are the molecular mechanisms that allow FoxP2 to regulate the radial precursor to intermediate progenitor transition? First, in the recent Rousso et al. (2012) paper, the authors

104 show that ectopic expression of FoxP2 downregulates expression of N-cadherin, a key component of apical adherens junctions. Since radial precursors, but not intermediate progenitors, are part of the apical epithelium, then perhaps a FoxP2-mediated delamination from this epithelium is a key part of the transition between these two cell types. Second, FoxP2 can directly inhibit transcription of the DISC1 gene (Walker et al., 2012), and suppression of DISC1 causes developing neural precursors to stop proliferating and to differentiate (Mao et al., 2009).

Third, FoxP2 regulates transcription of the autism-associated gene CNTNAP2, which encodes a member of the neurexin family of cell adhesion molecules. In mice, Cntnap2 is expressed in neural precursors, and regulates neuronal development (Penagarikano et al., 2011). Thus, FoxP2 may regulate these and other downstream targets to promote neurogenesis.

One intriguing result presented here is that human FoxP2 acts as a gain-of-function protein within the murine context. These data imply that the four amino acid changes that distinguish the human and mouse proteins enhance the proneurogenic actions of FoxP2. While we don’t yet know which amino acid change accounts for this difference, the amino acid switch at position 324 of mouse FoxP2 (homologous to position 325 of human FoxP2) introduces a consensus protein kinase C (PKC) phosphorylation site into the human FoxP2 protein. In this regard, we recently showed that atypical PKCs regulate cortical neurogenesis (Wang et al.,

2012), and PKCs are activated in neural precursors by neurotransmitters, peptide ligands, and growth factors (Canoll et al., 1996; Hansel et al., 2001; Song and Ghosh, 2004; Bartkowska et al., 2007; He et al., 2009). Moreover, this particular amino acid substitution is one of two that have been knocked-in to the mouse FoxP2 gene, and shown to result in alterations in cortico- basal ganglia circuits (Enard et al., 2009; Reimers-Kipping et al., 2011). Regardless of the underlying mechanism, these findings indicate that FoxP2 evolved an enhanced proneurogenic

105 function coincident with evolution of a larger cortex. Since recent work suggests that an increase in intermediate progenitors was important for evolution of a larger human cortex (Noctor et al.,

2007; Pontious et al., 2008), then this raises the possibility that a more active FoxP2 was perhaps somehow involved in facilitating this evolution.

One final conclusion arising from these studies is that the KE mutant FoxP2 acts as a dominant-inhibitory protein with regard to neurogenesis, consistent with the autosomal dominant genetics of the KE family (Fisher et al., 1998). How might it do this? We report here that the

KE mutant FoxP2, which is mutated at a single amino acid in the forkhead DNA binding domain, localizes to both the nucleus and cytoplasm of cortical precursors. A similar cytoplasmic localization was seen with this mutant in cell lines (Vernes et al., 2006; Mizutani et al., 2007a), and cytoplasmic aggregates of this protein were observed in neurons when it was knocked-in to the murine FoxP2 locus (Fujita et al., 2008). Since the KE mutant FoxP2 can modify the localization of wild-type FoxP2 (Mizutani et al., 2007a), then we propose that it acts as a dominant-inhibitory protein by binding to the endogenous protein and sequestering it in the cytoplasm. It may also mediate this dominant-inhibition function in part by inhibiting appropriate DNA binding and transcriptional activation within the nucleus.

In summary, our findings support a novel role for FoxP2 in developmental neurogenesis, and indicate that human FoxP2 acquired an enhanced ability to promote neurogenesis coincident with the evolution of a larger human cortex. While our findings do not preclude a later role for

FoxP2 in neuronal development, we propose that perturbations in the numbers or timing of cortical neuron genesis would ultimately cause aberrant neural circuit formation, thereby potentially providing a neural template for the deficits in speech and language seen in individuals with FoxP2 mutations.

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CHAPTER 5: CBP REGULATES INTERNEURON DIFFERENTIATION AND

MATURATION

Abstract

While the majority of cells in the cortex are generated from neural progenitors in the dorsal telencephalon, two major cell types that reside in the mature cortex are generated from the medial ganglionic eminence (MGE) in the ventral telencephalon – cortical interneurons and the earliest wave of oligodendrocytes. Over the past decade, epigenetics has gained increasing attention in cell fate determination. Recently, our lab has shown that CBP, a coactivator protein that also has histone acetylation activity and is associated with the developmental disorder

Rubinstein-Taybi syndrome, regulates differentiation of progenitors of the dorsal telencephalon by binding to the promoters for neural genes and promoting local histone acetylation. However, little is known about the role of CBP in the regulation of progenitors in the ventral telencephalon.

Here, we developed an in vitro culture system to examine the role of CBP in the development of

MGE precursors. siRNA knockdown of CBP in MGE precursors modestly reduces the number of newly-born neurons, and inhibits their maturation into GABAergic interneurons in culture. No effect on proliferation or cell death is observed when CBP is knocked down. The reduction in interneurons that is observed when CBP is knocked down is rescued by coincident expression of ectopic CBP. Moreover, this reduction is also rescued by the histone deacetylase inhibitor trichostatin-A. Taken together, these results suggest that CBP is required for the generation and/or maturation of MGE-derived interneurons through its histone acetyltransferase activity.

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Introduction

The mammalian cerebral cortex consists of two major classes of neurons: glutamatergic, excitatory projection neurons, and GABAergic, inhibitory interneurons. While projection neurons are generated by radial precursors within the developing cortex (Malatesta et al., 2000;

Noctor et al., 2001), interneurons are generated in the medial ganglionic eminence (MGE) before migrating along the superficial and deep tangential migratory streams to enter the cortex (Lavdas et al., 1999; Xu et al., 2008). A clonal analysis study has shown that the process by which interneurons are generated is very similar to the radial units in the cerebral cortex, with a radial precursor-like cell serving as a scaffold for migration of neuronal progeny (Brown et al., 2011), with each precursor able to give rise to several subtypes of interneurons. It is also thought that the MGE precursors are bipotent, capable of giving rise to interneurons and oligodendrocytes

(He et al., 2001; Yung et al., 2002; Petryniak et al., 2007).

Rubinstein-Taybi syndrome is a genetic disorder characterized by distinct facial features, broad thumb and great toe, short stature, and moderate to severe intellectual disability (Wiley et al., 2003). It is a heterogeneic disorder, with most of the cases caused by mutation of a single allele of the gene encoding for CREB-binding protein (CBP), which include microdeletions, translocations and point mutations, in the gene encoding CBP (Petrij et al., 1995). In addition, several studies have shown that approximately 3% of the cases result from a mutation in the gene encoding a close homologue of CBP, p300 (Roelfsema et al., 2005; Zimmermann et al., 2007;

Schorry et al., 2008; Foley et al., 2009). CBP is a transcriptional co-activator that binds to many different transcription factors, and is thought to activate gene transcription by acting as a physical bridge to the transcriptional apparatus, and via its histone acetyltransferase activity

(Bannister and Kouzarides, 1996). Insights into how haploinsufficiency for CBP might cause

108 intellectual disability comes from work showing that CBP promotes differentiation of embryonic cortical precursors by modifying chromatin at the promoters of neural genes and thereby enhancing transcription of those genes (Wang et al., 2010a). As a consequence, when CBP is haploinsufficient in mice, this causes decreased genesis of cortical neurons and glia and behavioral perturbations in early life (Wang et al., 2010a).

In addition to intellectual disability, a meta-analysis of 732 cases of RTS found that

57.6% of these individuals have EEG anomalies and 27% exhibit epilepsy (Cantani and Gagliesi,

1998). Intriguingly, a seizure phenotype was also seen in a small proportion of mice carrying a human CBP mutation that results in expression of a truncated protein (Oike et al., 1999). Since seizures in both mice (Kash et al., 1997; Cobos et al., 2005) and humans (Kitamura et al., 2002) are frequently due to dysfunction in the generation of GABAergic interneurons and/or the inhibitory neurotransmitter GABA, then this suggests that CBP might also be essential for the genesis of interneurons from embryonic ventral forebrain precursors. Here, we have tested this hypothesis and provide evidence that CBP is required for the maturation of MGE derived interneurons.

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Results

CBP is expressed in embryonic precursors of the ganglionic eminence

We first asked if CBP is expressed in embryonic precursors of the medial ganglionic eminence (MGE) by analyzing CBP mRNA expression. RT-PCR of RNA isolated from the murine cortex at embryonic day 12 (E12) demonstrated that CBP mRNA is expressed at this timepoint (Fig. 5.1A). To ask specifically whether CBP is expressed in neural precursors of the

MGE, we double-labelled sections through the E12 ventral forebrain with an antibody against

Nestin, a marker for neural precursors, and with a CBP antibody that we have previously characterized and used to identify expression of CBP in precursors of the dorsal forebrain (Wang et al., 2010a). This analysis demonstrated that CBP was expressed in almost all cells of the embryonic forebrain, including the precursors labelled by Nestin (Fig. 5.1B).

To confirm that CBP is expressed in ventral forebrain precursors, we developed a culture system for MGE precursors. Specifically, we dissected the murine MGE at embryonic day 12

(E12), dissociated the cells mechanically and plated them in neurobasal media supplemented with B27, FGF2 and L-glutamine, conditions we use for culturing embryonic cortical precursors.

Immunostaining demonstrated that, upon plating, the vast majority of cells expressed the precursor marker nestin and the proliferation marker Ki67, consistent with a precursor phenotype

(Fig. 5.1C). Even at two days in culture, approximately 85% of the cells were nestin-positive and approximately 66% were proliferating (Fig. 5.1D). At 3 days in culture, these proportions decreased so that by 5 days, only approximately 32% and 23% of the cells were positive for nestin and Ki67, respectively. To ask whether these precursors were differentiating into interneurons, we immunostained the same cultures for βIII-tubulin, an early neuronal marker, and Gad67, which is expressed in GABAergic interneurons. By 2 days in culture approximately

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28% of the cells already expressed βIII-tubulin, and this proportion increased to approximately

48% over the ensuing 3 days (Fig. 5.1D). However, at 2 days, only approximately 14% of the cells expressed Gad67, but this number increased so that approximately 48% expressed this interneuron marker by 5 days (Fig. 5.1D). At this later timepoint, some of these neurons also started to express detectable somatostatin (Fig. 5.1C) and calretinin, markers for interneuron subtypes.

These data suggested that βIII-tubulin was expressed prior to expression of Gad67 as these precursors generated neurons. We therefore analyzed the time course of expression of

Gad67 relative to βIII-tubulin by double-labeling cultures over this same timeframe.

Quantitation revealed that while only 23% of βIII-tubulin-positive neurons expressed Gad67 at 2 days in vitro, by 4 days approximately 88% of βIII-tubulin-positive neurons expressed Gad67

(Fig. 5.1E). Thus acquisition of an inhibitory neurotransmitter phenotype lags several days behind the first neuron-specific gene expression.

Having established the cell culture system, we asked when and in what cell type CBP is expressed in the MGE precursor cultures. Consistent with the ubiquitous pattern of expression by immunohistochemistry in tissue sections (Fig. 5.1B), CBP is expressed in both precursor cells and neurons in culture throughout the time course of our analysis (Fig. 5.1C), suggesting it may be important in both precursors and post-mitotic neurons.

CBP regulates differentiation but not proliferation of MGE precursors

To ask if CBP is required for interneuron differentiation, we used a CBP siRNA that has been previously characterized in cortical precursors (Wang et al., 2010a; 2012). First, we assessed the efficacy of siRNA-mediated CBP knockdown in cultured MGE precursors. MGE

111 precursors were cultured from E12 embryos and cotransfected with a plasmid encoding EGFP plus either control or CBP siRNA. Three days later, cells were immunostained (Fig. 5.2A) and the level of CBP immunofluorescence in EGFP-positive cells was categorized as high or low/undetectable expression. Quantification showed that while approximately 70% of control- siRNA transfected cells expressed high levels of CBP, only 30% of CBP-siRNA-transfected cells expressed similarly high levels (Fig. 5.2A, B).

Having established the efficacy of the CBP siRNA in MGE precursor cells, we asked whether CBP knockdown affected their survival or proliferation. We cultured E12 MGE precursor cells and transfected them with a plasmid encoding EGFP together with either control or CBP siRNA. Two days later, we assessed proliferation by quantifying the percentage of

EGFP-positive cells that stain for the proliferation marker Ki67. We also assessed cell death by quantifying the percentage of EGFP-positive cells that had condensed nuclei. No difference was observed for Ki67 (Fig. 5.2C, D) or condensed nuclei (Fig. 5.2E, F) in cells transfected with the scrambled siRNA versus the CBP siRNA. Next, we asked if CBP is required for neurogenesis by quantifying the percentage of EGFP-positive cells that were positive for the early neuronal marker βIII-tubulin 3 days after transfection. This analysis demonstrated that there was a modest but significant decrease in βIII-tubulin-positive neurons when CBP is knocked down in MGE precursors (Fig. 5.2G, H). These results suggest that, as was seen for cortical precursors (Wang et al., 2010a), CBP plays a role in MGE precursor differentiation.

CBP regulates MGE interneuron differentiation

To ask if there is a role for CBP in the genesis and/or maturation of interneurons, we knocked down CBP and assessed the effect on expression of markers associated with maturation

112 of interneurons. Initially, we characterized the expression of interneuron markers in MGE cultures at 7 days. In particular, we immunostained cultures for the GABA synthesis enzyme

Gad67, which is expressed in almost all interneurons, as well as for parvalbumin, somatostatin and calretinin, markers of the three major interneuron subtypes (Gonchar and Burkhalter, 1997).

In this regard, parvalbumin- and somatostatin-positive interneurons are thought to derive from the MGE, while calretinin-positive interneurons arise predominantly from the caudal ganglionic eminence (Xu et al., 2004). Double-labeling for Gad67 and these three different markers demonstrated somatostatin-positive, Gad67-positive interneurons were readily detectable in

MGE cultures at 7 days in vitro, when they comprised approximately 35% of the cells in these cultures (Fig. 5.3A). In contrast, none of the cells were positive for parvalbumin, which is a relatively late interneuron marker (Mukhopadhyay et al., 2009). Calretinin was also expressed in a very small percentage of cells in the culture but did not always colocalize with Gad67 (Fig.

5.3A).

On the basis of these findings, we knocked down CBP in MGE precursors and asked whether it affected interneuron differentiation by quantifying cells expressing Gad67 and somatostatin. Specifically, E12 MGE precursors were cultured and transfected with a plasmid encoding EGFP together with control or CBP siRNAs, and analyzed 3 days later for Gad67 and

7 days later for somatostatin. Immunostaining for Gad67 demonstrated that CBP knockdown decreased the proportion of EGFP-positive, Gad67-positive interneurons by almost 50% (Fig.

5.3B, C). Consistent with this, CBP knockdown also caused a significant reduction in the percentage of EGFP-positive, somatostatin-positive interneurons (Fig. 5.3D, E).

Since the reduction in interneurons is much larger than that seen for βIII-tubulin-positive neurons at 3 days (Fig. 5.2G, H), and since these interneuron markers turn on after βIII-tubulin in

113 these cultures (Fig. 5.1D), then this suggests that CBP might play a role in interneuron maturation. To address this possibility, we performed two additional experiments. First, we knocked down CBP and, 4-5 days post-transfection, immunostained for the more mature panneuronal marker NeuN (Fig. 5.4A). Quantification demonstrated that CBP knockdown decreased the proportion of EGFP-positive, NeuN-positive neurons by an amount similar to that seen for the interneuron markers (Fig. 5.4B), suggesting that CBP is important for MGE interneuron maturation. Second, we knocked down CBP and quantified cultures 3 days post- transfection by triple-labeling them for EGFP, Gad67 and βIII-tubulin (Fig. 5.4C). This analysis demonstrated that, in control siRNA-treated cultures, approximately 90% of the βIII-tubulin- positive cells also expressed Gad67 (Fig. 5.4D). In contrast, this percentage was only approximately 60% in cultures transfected with CBP siRNA (Fig. 5.4D). Thus, CBP knockdown inhibited the transition from βIII-tubulin-positive newborn neurons to more mature neurons expressing a Gad67-positive, somatostatin-positive interneuron phenotype.

One potential explanation for this finding is that CBP knockdown caused a switch to another neuronal phenotype, since MGE precursors also generate cholinergic neurons (Xu et al.,

2008; Fragkouli et al., 2009), and LGE precursors can generated immature Tbr1-positive glutamatergic neurons when transduced with Ngn2 or NeuronD1 (Roybon et al., 2010). We therefore knocked down CBP and immunostained these cultures 7 days later with antibodies for the cholinergic neuron marker choline acetyltransferase (ChAT) and the glutamatergic neuron marker vGlut1. Cells expressing these two markers were not found in either the control or CBP siRNA-transfected cultures suggesting that the decrease in Gad67-positive, somatostatin-positive neurons is due to a decrease in maturation, not a switch in neuronal fate.

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CBP overexpression promotes interneuron differentiation and rescues the interneuron differentiation deficit caused by CBP knockdown

These data indicate that endogenous CBP is important for newborn interneurons to mature. We therefore asked whether overexpressing CBP was sufficient to enhance interneuron maturation. To do this, we cotransfected cultured MGE precursors with plasmids encoding

EGFP and wild-type CBP. This CBP plasmid has been previously characterized (Wang et al.,

2010a). Immunostaining of these cultures 3 days later demonstrated that CBP overexpression caused a robust increase in the proportion of transfected cells that expressed Gad67 at this timepoint (Fig. 5.5A). We therefore asked whether overexpression of CBP is able to rescue the deficit in interneuron differentiation caused by CBP knockdown. To do this, we cotransfected

E12 MGE cultures with a plasmid encoding EGFP and either control or CBP siRNA, together with either an empty vector or the plasmid encoding for wild-type CBP. Quantification of these cultures by immunostaining 3 days later showed that overexpression of wild-type CBP significantly rescued the decrease in Gad67-positive interneurons that occurred following CBP knockdown (Fig. 5.5B). These results suggest that CBP is normally limiting in MGE precursors so that overexpression alone is sufficient to induce expression of the mature interneuron marker

Gad67. They also confirm the specificity of the phenotype caused by CBP siRNA,

CBP regulates interneuron differentiation in part by histone acetylation

Previous work on CBP indicated that it mediates its effects on cortical precursor differentiation via its histone acetyltransferase activity. We therefore asked whether we could rescue the deficit in interneuron differentiation caused by CBP knockdown by treating MGE precursors with the HDAC inhibitor trichostatin A (TSA) to increase histone acetylation. To do

115 this, we cotransfected E12 MGE precursors with a plasmid encoding EGFP together with either control siRNA or CBP siRNA, and then treated the cells the next day with either DMSO or 20 nM TSA, a concentration that was previously shown to increase H3K9/K14 acetylation in cortical precursors (Wang et al., 2010a).. We then analyzed these cultures 3 days later by immunostaining for Gad67. Quantification demonstrated that addition of TSA rescued the interneuron differentiation deficit caused by CBP knockdown (Fig. 5.5C). Thus, CBP likely promotes interneuron maturation by promoting histone acetylation.

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Figure 5.1

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Figure 5.1: Expression of CBP in medial ganglionic eminence (MGE) precursors. A, RT-

PCR analysis for FoxP2 mRNA in total RNA isolated from E12 murine MGE using two different primer sets. Molecular weight markers are shown to the left of the panel. B, Confocal micrographs of coronal sections through the E13 cortex immunostained for CBP (red) and Nestin

(green; the far right panel shows the merge); the right three panels show higher magnification images of the left-most panel. C, Fluorescence micrographs of E12 MGE precursors cultured for

3-7 days, immunostained for CBP (red) and Nestin (green; 3 days), Ki67 (green; 3 days), Gad67

(green; 3 days), or somatostatin (green; SST; 7 days), as indicated. D, E12 MGE precursors were cotransfected with EGFP and immunostained for EGFP and one of the four markers at timepoints indicated on the x axis. Note the percentage of Nestin-positive, Ki67-positive precursors decreased over time while the percentage of βIII-tubulin-positive, Gad67-positive interneurons increased. E, E12 MGE precursors were plated and immunostained for βIII-tubulin and Gad67 at timepoints indicated on the x axis. Note the percentage of βIII-tubulin-positive that were Gad67-positive increased over time.

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Figure 5.2

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Figure 5.2: CBP knockdown in MGE precursors reduces neurogenesis without affecting precursor survival or proliferation. E12 MGE precursors were cotransfected with EGFP and control siRNA or CBP siRNA and analyzed 2 days (C-F) or 3 days (A-B, G-H) later. A,C,E,G,

Fluorescence micrographs of transfected cells immunostained for EGFP (green) and CBP (A, red), Ki67 (C, red), or βIII-tubulin (E, G, red). In (A), arrows denote cells that express high level of CBP, while arrowheads denote cells that do not express CBP. In (C,E,G), arrows denote

EGFP-positive cells that also are positive for Ki67 (C) or βIII-tubulin (G), or cells that have condensed nuclei (E). B, Quantification of the percentage of transfected cells that express high levels of CBP or that have low or no CBP expression. D,F,H, Quantification of the percentage of transfected, Ki67-positive cells (D), cells with condensed nuclei (F), or βIII-tubulin-positive cells (H) in experiments similar to those shown in A,C,E,G. Graphs show pooled data from three

(B, D) or four (F, H) independent experiments. (*p < 0.05; ***p < 0.001; two-tailed paired

Student’s t-test). Error bars denote S.E.M.

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Figure 5.3

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Figure 5.3: CBP knockdown in MGE precursors reduces the percentage of neurons expressing mature interneuron markers. A, E12 MGE precursors were plated and immunostained 7 days later for Gad67 (green) and somatostatin (SST, top row) or calretinin

(bottom row). For the top row, arrows indicate cells that are both Gad67-positive and SST- positive. For the bottom row, arrows indicate cells that are Gad-positive while arrowheads indicate cells that are calretinin-positive. B-E, E12 MGE precursors were cotransfected with

EGFP and control siRNA or CBP siRNA and analyzed 3 days (B,C) or 7 days (D,E) later. B,D,

Fluorescence micrographs of transfected cells immunostained for EGFP (green) and Gad67 (B, red) or SST (D, red). Arrows denote EGFP-positive that is also positive for Gad67 (B) or SST

(D). C,E, Quantification of the percentage of transfected, Gad67-positive (C) or SST-positive

(E) cells in experiments similar to those shown in B,D. Graphs show pooled data from four independent experiments. (*p < 0.05; two-tailed paired Student’s t-test). Error bars denote

S.E.M.

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Figure 5.4

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Figure 5.4: CBP knockdown in MGE precursors reduces the percentage of neurons expressing mature interneuron markers. A-D, E12 MGE precursors were cotransfected with

EGFP and control siRNA or CBP siRNA and analyzed 4 days (A,B) or 3 days (C,D) later. A,

Fluorescence micrographs of transfected cells immunostained for EGFP (green) and NeuN (red).

Arrows denote an EGFP-positive cell that is also positive for NeuN. B, Quantification of the percentage of transfected, NeuN-positive cells in experiments similar to those shown in A. Graph shows pooled data from four independent experiments. (*p < 0.05; two-tailed paired Student’s t- test). C, Fluorescence micrographs of cells transfected with control siRNA (top row) or CBP siRNA (bottom row), and immunostained for EGFP (green), βIII-tubulin (white) and Gad67

(red). For the control siRNA condition (top row), arrows denote cells that are EGFP-positive,

βIII-tubulin-positive and Gad67-positive. For the CBP siRNA condition (bottom row), arrows indicate cells that are EGFP-positive, βIII-tubulin-positive and Gad67-negative. D,

Quantification of the percentage of EGFP-transfected and βIII-tubulin-positive cells that express the interneuron marker Gad67 in experiments similar to those shown in C. Graph shows pooled data from three independent experiments. (**p < 0.01; two-tailed paired Student’s t-test). Error bars denote S.E.M.

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Figure 5.5

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Figure 5.5: The interneuron development deficit caused by CBP knockdown is rescued by WT-

CBP and the HDAC inhibitor Trichostatin A (TSA). A, E12 MGE precursors were cotransfected with EGFP and empty vector (Control) or a plasmid encoding WT-CBP and quantified for the percentage of Gad67-positive cells after 3 days. B, E12 MGE precursors were cotransfected with EGFP, and control siRNA (control siRNA/EV), CBP siRNA plus empty vector (CBP siRNA/EV), or CBP siRNA plus a plasmid encoding wild-type CBP (CBP siRNA/WT-CBP), and quantified for the percentage of Gad67-positive cells after 3 days. C, E12

MGE precursors were cotransfected with EGFP, and control siRNA (control siRNA/DMSO) in the presence of DMSO, or CBP siRNA in the presence of DMSO (CBP siRNA/DMSO) or TSA

(CBP siRNA/TSA), and quantified for the percentage of Gad67-positive cells after 3 days.

Graphs show pooled data from three independent experiments, normalized to the control treatment. (*p < 0.05, **p < 0.01, **p < 0.001). Error bars denote S.E.M.

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Discussion

In this study, we provide evidence that CBP regulates the maturation of interneurons that are generated from MGE precursors, at least in part by promoting histone acetylation. These findings build upon a previous study from the Miller laboratory showing that CBP is required for differentiation of cortical precursors both in culture and in vivo (Wang et al., 2010a). However, in contrast to that study, where CBP was found to play an essential role in the transition between embryonic neural precursors and their differentiated progeny, here we identify an additional role for CBP in the maturation of newborn neurons after they have already adopted a neuronal fate.

Together, these two studies indicate that CBP is required for multiple aspects of neurogenesis within the forebrain, a role that might generalize throughout the developing CNS.

Cellular differentiation is a multistep process that depends on a cascade of genetic programs and CBP has been shown to be required for multiple steps in different tissue systems.

For example, muscle cell differentiation is composed of the initial myoblast determination, terminal differentiation and muscle fiber maturation. Each of these steps involves the expression of certain genes such as the early markers p21 and MyoD, and the late markers MHC and MCK.

Using a selective inhibitor of CBP/p300 HAT activity and a transdominant mutant of CBP, it was shown that CBP/p300 is required for myotube fusion and histone acetylation at the promoters of late but not early muscle marker genes (Polesskaya et al., 2001). While CBP is thus required for a late differentiation stage in myogenesis, it plays an earlier role in the nervous system, regulating both early neuronal specification and neuronal migration. For example, in the spinal cord, retinoic acid induces the recruitment of CBP to the retinoic acid receptor/Ngn2 (a bHLH transcription factor) complex, facilitating transcription of motor neuron genes and thereby promoting motor neuron specification (Lee et al., 2009). In the developing mouse cortex, CBP is

127 required for differentiation of precursors into both neurons and glia (Wang et al., 2010a). In addition, it has also been shown that Ngn2 promotes radial migration of cortical neurons, potentially by recruiting CBP to the doublecortin (Dcx) promoter and displacing CBP from the

RhoA promoter (Ge et al., 2006). Finally, in Drosophila, CBP also regulates functional synaptic development at the neuromuscular junction (Marek et al., 2000). Data presented here further support the idea that CBP is required for multiple stages of nervous system development. In particular, we show that CBP is required at various stages of interneuron differentiation including the initial genesis of these neurons from precursors, and their later maturation as assayed by expression of the late neuronal marker NeuN and expression of their neurotransmitter phenotype as marked by Gad67 and somatostatin.

One explanation for our results is that CBP regulates important upstream events in interneuron genesis so that when it is knocked down, this causes a deficit in interneuron maturation. A second explanation is that CBP does not work as a "master regulator" of interneuron maturation, but instead independently regulates expression of genes important for interneuron maturation. Indeed, one way it could do this is by association with CREB since various late interneuron markers are known to be regulated by CREB signaling. For example, the somatostatin gene is regulated by a cAMP-protein kinase A-CREB pathway (Gonzalez and

Montminy, 1989) and CREB has been shown to bind to the cAMP responsive element (CRE) in the promoter of somatostatin genes in cortical neurons (Cha-Molstad et al., 2004). Gad65 expression has also been shown to be regulated by BDNF/TrkB signaling via the Ras-ERK-

CREB pathway in cortical interneurons (Sanchez-Huertas and Rico, 2011). Using ChIP and luciferase assays, the authors found that CREB binds the CRE in the promoter of Gad65 gene and directly regulates its expression. Interestingly, they also found a reduction in Gad67

128 expression in the neocortex of two week old animals that had TrkB ablated specifically in interneurons. While a CRE has not been definitively identified in the Gad67 promoter, the Gad67 promoter region does contain a motif with high homology to CREB binding sites (Kobori and

Dash, 2006). Given that it is thought that CBP recruitment via phosphorylation of Ser-133 of the

CREB protein is required for cAMP-mediated transcription (Nakajima et al., 1997), it is plausible that CBP itself is crucial for the expression of these genes.

Our study also suggests that histone acetylation plays an important role in interneuron development. In particular, we demonstrate that an HDAC inhibitor, TSA, can rescue the deficit in interneuron maturation caused by CBP knockdown, suggesting that CBP regulates interneuron maturation either directly by its own HAT activity, or indirectly by its interaction with other

HATs. This finding is reminiscent of the previous study showing that CBP regulates cortical precursor differentiation via its histone acetyltransferase function (Wang et al., 2010a).

Intriguingly in this regard, mice that are homozygous for a mutation in the querkopf gene, which encodes a MYST family histone acetyltransferase, have reduced Gad67-positive interneurons in the cerebral cortex (Thomas et al., 2000). The hypothesis that histone acetylation plays important roles in interneuron development is further strengthened by the observation that the promoters of interneuron genes are epigenetically regulated by histone modification. For example, various

HDAC inhibitors have been shown to up-regulate Gad67 expression either directly or indirectly through histone hyperacetylation (Dong et al., 2007; Kundakovic et al., 2009). In NIH3T3 cells, recruitment of the CBP homologue p300 to the somatostatin promoter leads to acetylation of histone H4 and subsequent transcription of a reporter construct (Asahara et al., 2001).

p300 is another histone acetyltransferase that is highly homologous to CBP and shares several functional domains with CBP. For example, CBP and p300 share 90% amino acid

129 sequence homology within the KIX domain, which contains the binding surface for some of

CBP’s transcription factor partners including CREB and p53 (Karamouzis et al., 2007). Given that p300 is highly homologous to CBP, a key question is to what extent their function overlaps with regard to interneuron genesis and maturation. In the hematopoietic system, CBP is required for precursor self-renewal while p300 is required for differentiation (Rebel et al., 2002). Also, mutation in p300 resulted in more severe phenotypes in heart, lung and small intestine than the corresponding mutation in CBP (Kalkhoven, 2004). Moreover, CBP and p300 differ in the histone and non-histone substrates that they acetylate, the proteins that they interact with, and their target genes (Ramos et al., 2010). On the basis of these findings, it would be intriguing to ask whether p300 also plays a role in interneuron differentiation. Experiments addressing this question are discussed in the “Discussion and future directions” section, and conclusions drawn from the results of these experiments may also improve our understanding on the function of gene duplication and divergence in neural development.

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CHAPTER 6: DISCUSSION and FUTURE DIRECTIONS

FoxP2 regulates neurogenesis in the developing cortex

In chapter 4, I examined the role of FoxP2 in cortical development and my work led to three major conclusions. First, FoxP2 promotes neurogenesis in the developing murine cortex at least in part via the generation of intermediate progenitors. Second, ectopic overexpression of human FoxP2 promotes the generation of intermediate progenitors and neurons while overexpression of mouse FoxP2 does not, suggesting that the minor difference in the amino acid sequence between these two highly conserved homologues may be critical for neurogenesis.

Third, ectopic expression of the FoxP2 allele that carries the mutation identified in the KE family phenocopies FoxP2 knockdown in cortical precursors, suggesting that the KE mutant FoxP2 allele encodes a dominant negative form of the protein.

FoxP2 has previously been implicated in a number of key neurogenesis pathways. Foxp2 was found to be a putative target of Pax6 in a bioinformatics screen and this was validated by

ChIP using zebrafish embryos (Coutinho et al., 2011). Most importantly, in situ hybridization showed that foxp2 expression is abolished in the dorsolateral telencephalon of embryonic day

11.5 Pax6 knockout mouse embryos, suggesting that Pax6 may be upstream of FoxP2 in the developing cortex. Foxp2 is initially expressed throughout the neuroepithelium and overlaps with

Sox2 expression in the spinal cord (Rousso et al., 2012), suggesting that FoxP2 and Sox2 may share an upstream regulator or FoxP2 may be downstream of Sox2. The same study also found that FoxP2 expression is reduced in the developing cortex of embryonic day 12.5 FoxP4 mutant cortex, suggesting that FoxP2 is downstream of FoxP4 in the developing cortex. Furthermore, overexpression of Ngn2 failed to rescue the deficit in neurogenesis caused by knockdown of

FoxP2 and FoxP4, thus placing both FoxP proteins downstream of the Ngn2. Finally, in the

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FoxP2-S321X mutant, which does not express any FoxP2 protein, Shhrs, a non-coding RNA also known as Dlx6as or Evf1/2, is upregulated in the subventricular zone of the cortex at embryonic day 16, suggesting that FoxP2 downregulates the expression of Shhrs in the developing cortex

(Vernes et al., 2011). Interestingly, Shhrs is normally suppressed in the cortex but expressed in the subventricular zone of the ventral forebrain (Kohtz and Fishell, 2004), and is downstream of the ventral morphogen Sonic Hedgehog. This raises the possibility that FoxP2 may also play a role in repressing ventral forebrain identity, which is also a known function for Pax6 and Ngn2, consistent with the hypothesis that FoxP2 is in the same pathway as Pax6 and Ngn2. Figure 6.1 represents a summary of our proposed pathway for FoxP2 in cortical neurogenesis.

How does FoxP2 regulate neurogenesis?

FoxP2 is a transcription factor that forms homodimers or heterodimers with FoxP1 or

FoxP4 via their leucine zipper domain in order to bind DNA (Li et al., 2004a). Interestingly, an

X-ray crystallography study showed that FoxP2 forms domain-swapped dimers, and modeling suggests that domain-swapped FoxP dimers can bind cognate DNA sites that are far away from each other or on different DNA strands, thereby raising the possibility that the FoxP dimers promote higher order protein/DNA complexes (Stroud et al., 2006). While most studies showed that FoxP2 acts as a transcriptional repressor in both mouse and primates (Li et al., 2004a; Zhou et al., 2008; Konokpa et al,. 2009; Vernes et al., 2011), FoxP2 can also act as transcriptional activator (Konopka et al., 2009).

Rousso et al. proposed that in the mouse and chick spinal cord, FoxP2 and FoxP4 promote neurogenesis by downregulating N-cadherin thereby causing detachment of differentiating neurons from the neuroepithelium (Rousso et al., 2012). The initial step of

132 delamination appears to be important for fate determination, as precursor-precursor interaction is required for β-catenin-mediated effects on cell fate (Zhang et al., 2010). Specifically, in vivo knockdown of N-cadherin in cortical precursors resulted in premature neuronal differentiation and migration. In a different study, when N-cadherin was specifically ablated in the developing cortex, the organization of the cortex was disrupted, such that both neurons and mitotic figures were distributed throughout the entire cortex, and radial precursor morphology was severely disrupted so that there were no radial fibers in contact with the pia (Kadowaki et al., 2007).

However, the authors did not report any changes in cell fate in this study. This difference in results may be due to the mosaic nature of in utero electroporation, since use of this approach in the former study allowed for observation of relatively cell-autonomous effects of loss of N- cadherin on precursor biology in an otherwise normal developing cortex.

Does FoxP2 regulate cilia development?

In radial precursors, primary cilia arise on the apical plasma membrane and are thought to be in a strategic location to sense signals that are present in the ventricular fluid that may regulate cortical development (Cohen and Meininger, 1987; Breunig et al., 2008). Recently, it was discovered that the development of basolateral cilia may be the first biological feature of cells that will delaminate from the apical adherens junction belt and differentiate into intermediate progenitors (Wilsch-Brauninger et al., 2012). Interestingly, these basolateral cilia are not randomly distributed but are preferentially located in the vicinity of spot-like adherens junctions which include N-cadherin and β-catenin. It would be interesting to see if FoxP2 plays any role in regulating this first step of delamination, given that it is thought to play a role in delamination via regulation of N-cadherin, at least in the developing spinal cord (Rousso et al.,

133

2012). One way to test this hypothesis is to quantify the occurrence of apical and basolateral cilia in the GFP transfected population of ventricular zone precursors in cortices transfected with control versus FoxP2 shRNA using electron microscopy and immunogold labelling of ultrathin cryosections for GFP to identify transfected cells. If FoxP2 plays a role in regulating cilia development, one would expect to observe a shift in the proportion of apical versus basolateral cilia in the FoxP2 shRNA transfected cells compared to control shRNA transfected cells.

What brain region is required for FoxP2’s role in ultrasonic vocalization?

As mentioned above, several different lines of FoxP2 mutants exhibit abnormal ultrasonic vocalization. Many laboratory rodent pups emit ultrasonic vocalization when they are separated from their mothers (Portfors, 2007). The function of these calls, while still debatable, is thought to act as a distress call for their mothers. This is because pups quickly lose body temperature due to the lack of fur and subcutaneous fat, and these calls elicit retrieval behaviour from their mothers. This idea is supported by the finding that anti-anxiolytics such as benzodiazepines can reduce ultrasonic vocalization (Takahashi et al., 2009). However, the underlying biology behind this defect is still unclear. A previous study in our lab revealed a role for CBP in the differentiation of all neural lineages of cortical precursors, and showed that mice heterozygous for CBP exhibited alterations in ultrasonic vocalization, suggesting that the cortex is an important substrate for ultrasonic vocalization (Wang et al., 2010a). However, it is thought that the cerebellum is also important for ultrasonic vocalization because both the FoxP2-R553H knockin mutants and a mutant of the synaptic adhesion molecule CADM1 exhibit defective ultrasonic vocalization and have smaller cerebelli (Fujita et al., 2012). Finally, the striatum has

134 also been implicated in ultrasonic vocalization deficits because FoxP2 mutants also display deficit in striatal neurite outgrowth (Vernes et al., 2011).

Ablation studies in monkeys have shown that various brain regions are required for different aspects of vocalization. These regions include the anterior cingulate cortex, the subcallosal gyrus, the anterior supplementary motor area, the cortical facial area, the thalamomidbrain junction, and the ventral central gray (Sutton et al., 1974; Kirzinger and

Jurgens, 1982; MacLean and Newman, 1988). While several neural structures have been implicated as important substrates for ultrasonic vocalization, there has been no single molecular/anatomical pathway that has been correlated with ultrasonic vocalization. One potential approach to this question would take advantage of the conditional FoxP2 null mutant that has been generated (French et al., 2007). It would be very interesting to ask which part of the brain is required for FoxP2 to regulate ultrasonic vocalization, by crossing these animals with animals carrying Cre under promoters expressed in cortical precursors (Emx1-Cre), striatal precursors (Gsh2-Cre) and cerebellar precursors (Engrailed1/2-Cre). Answers to this question would also potentially shed light on the cause of language impairment in the KE family.

Neurogenesis in different niches may be governed by similar mechanisms, albeit with different molecular players

In chapter 5, I examined a role for CBP in neurogenesis of interneurons derived from the

MGE. I found that similar to the cortex (Wang et al., 2010a), CBP also mediates neurogenesis in

MGE derived precursors. My data also indicate that CBP plays a prominent role in expression of late neuronal markers such as NeuN, Gad67 and somatostatin and that CBP mediates this cellular

135 response either via its own histone acetyltransferase activity or that of its protein partners, some of which also contain histone acetyltransferase activity.

Neuronal differentiation is composed of many biological processes. Once the daughter cell of a precursor division is determined to become a neuron, it has to leave the cell cycle and express early neuronal markers. Then the cell has to adopt various characteristics of a mature neuron, including neurotransmitter phenotype, dendritic morphology, formation of synapses with other neurons, and the expression of neuronal subtype-specific proteins, such as ion channel subtypes. One can also consider learning and memory, or synaptic plasticity as a continuation of neuronal development in response to changes in the cell’s environment. Interestingly, in addition to its role in neurogenesis and neuronal migration as discussed in the CBP section of the introduction of this thesis, CBP has either been directly or indirectly implicated in each of these biological processes. In cerebellar granular neurons, both CBP/p300 and P/CAF mediate TSA- induced neurite outgrowth (Gaub et al., 2010). The canonical transient receptor potential channel

TRPC6 promotes neurite outgrowth through phosphorylation of cAMP-response element binding protein (CREB) at the Ser-133 residue, which is site required for CREB to associate with CBP

(Tai et al., 2008). In Drosophila, CBP activates transcription of sox14 via its histone acetyltransferase activity and mediates axonal pruning in sensory neurons during metamorphosis

(Kirilly et al., 2011). Finally, adult CBP+/- mutants show behavioural deficits in long-term fear memory and object recognition as well as deficits in the physiological counterpart – long-term potentiation (Alarcon et al., 2004). Interestingly, viral delivery of a transgene encoding CBP is sufficient to rescue the learning and memory deficits in a mouse model of Alzheimer’s disease

(Caccamo et al., 2010). Thus CBP is important for many different biological functions from the precursor stage to mature neuronal function. My work adds to this repertoire of CBP functions,

136 and show that CBP is also required for at least some components of the neuronal maturation process, including expression of neurotransmitter-synthesizing enzymes and neuropeptides such as somatostatin, in interneurons derived from MGE precursors.

Consistent with its ubiquitous expression, CBP is also required for neurogenesis in several different niches – both in the central and peripheral nervous system. First, CBP is required for differentiation of precursors in the developing cerebral cortex (Wang et al., 2010a).

Second, CBP mediates metformin-induced neurogenesis in the subgranular zone of the adult hippocampus (Wang et al., 2012). Third, CBP cooperates with Ngn2 and retinoic acid receptor to specify motor neuron fate in spinal cord precursors (Lee et al., 2009). Fourth, in the autonomic nervous system, the bHLH transcription factor Hand2 promotes generation of sympathetic neurons by regulating Mash1 expression, and this is dependent on formation of transcriptional complexes with CBP/p300 (Morikawa et al., 2005). In addition, Hand2 is also essential for maintaining the noradrenergic properties of differentiated sympathetic neurons, including expression of noradrenergic marker genes that encode dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) (Schmidt et al., 2009), which also requires interaction with CBP/p300 and a homeodomain factor Phox2a (Xu et al., 2003). Finally, my work shows that CBP is required for the generation and/or maturation of interneurons derived from the ventral forebrain.

Taken together, these studies all point to a role for CBP in neurogenesis for different populations of neural precursors. They also suggest that despite the involvement of different transcription factors in these different neurogenic niches, an underlying theme of neuronal differentiation is the formation of a higher order transcriptional complex which includes histone aceyltransferases such as CBP to drive the expression of neuron-specific genes.

137

Does CBP phosphorylation regulate interneuron differentiation in MGE precursor cultures?

Recruitment of CBP to at least some transcriptional complexes requires phosphorylation of CBP by growth factor signalling pathways at serine 436. As a consequence, a single amino acid change from serine to alanine at this site blocked growth-factor-dependent gene activation in vitro (Zanger et al., 2001). In this regard, our lab has tested whether phosphorylation of CBP at this serine 436 (S436) site by atypical PKC is required for differentiation of cortical precursors

(Wang et al., 2010a). Specifically, wild-type CBP was able to rescue the differentiation deficits in CBP+/- precursors but a phosphomutant CBP-S436A was unable to do so, indicating that phosphorylation of the S436 site is required for differentiation of cortical precursors. Mice that carry CBP-S436A substitution have been generated, and these mice have been shown to exhibit reduced CBP phosphorylation as well as increased gluconeogenesis (Zhou et al., 2004b). It will be interesting to ask whether S436 phosphorylation is important for interneuron development in vivo by quantifying interneurons by immunohistochemistry and Western analysis in the striatum and cortex in early postnatal animals with this genetic background.

Are CBP and p300 functionally interchangeable with respect to neural development?

p300 is another histone acetyltransferase that is highly homologous to CBP and shares several functional domains with CBP (Fig. 6.2). For example, CBP and p300 share 90% amino acid sequence homology for the KIX domain, which contains the binding surface for some of

CBP’s transcription factor partners including CREB and p53. A key question in evolutionary biology is whether proteins with highly similar structures and shared conserved functional domains play similar roles in biological processes such as development. Previous studies suggest that CBP and p300 are functionally similar but have unique biological roles. How could this

138 happen? There are at least three, not mutually-exclusive possible explanations. First, CBP and p300 may be functionally similar but may be expressed in different tissues or different compartments of a specific tissue. Second, CBP and p300 may have subtle functional differences despite their structural similarity. Third, CBP and p300 may share similar molecular properties, but make unique contributions to biological processes by being differentially regulated by upstream pathways. Therefore, it will be interesting to ask whether p300 plays a role in neural development by knocking down p300 in both cortical and MGE precursors and assessing neurogenesis in vitro and in vivo, using our cell culture systems and in utero electroporation.

Even if CBP and p300 knockdown showed different phenotypes, this could be due to differences in expression rather than differences in function. Therefore, to further ask if CBP and p300 are functionally interchangeable, one could overexpress p300 in MGE precursors when

CBP is knocked down in culture, or overexpress p300 in the developing CBP+/- cortex in vivo using in utero electroporation, to ask if neural differentiation in the cortex is restored compared to controls.

Finally, it is also possible that while CBP and p300 have similar functions, they may be regulated differentially and this may account for any differences in their function. Importantly for our purposes, the CBP serine 436 phosphorylation site, which is a substrate of aPKC and is critical for cortical neural differentiation, is not found in p300 (Zanger et al., 2001). A mouse has been generated with the equivalent residue (ie. corresponding to serine 436 of CBP) in p300 changed from glycine to serine to mimic the phosphorylation site in CBP. These mice are therefore potential gain-of-function mutants. With these transgenic mice, one can ask if introduction of this aPKC regulated phosphorylation site into p300 enhances differentiation of cortical projection neurons and interneurons. If the phosphomutant p300 mice rescue the neural

139 phenotypes in CBP+/- animals, then this will support the idea that while CBP and p300 share similar molecular properties, they make unique contributions to neural differentiation by being differentially regulated by upstream pathways.

140

Figure 6.1

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Figure 6.1. Schematic of the proposed pathway of FoxP2 in cortical neurogenesis. Solid lines represent known activation and inhibition steps in murine cortices or cultured cell lines. Dashed lines represent putative activation steps. The presence of PKC phosphorylation site in human

FoxP2 makes it a potential target for aPKC.

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Figure 6.2

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Figure 6.2: Schematic of the protein structure of CBP and p300. CBP and p300 are highly homologous proteins. Coloured boxes represent domains that are labelled in the figure legend below the protein structures. CH1: cysteine/histidine-rich 1 domain; BD: bromodomain; HAT: catalytic acetyltransferase domain; CH3: cysteine/histidine-rich 3 domain; SID: SRC/p160- interacting domain; polyQ: polyglutamine. S301 and S436 represent two phosphorylation sites that are not conserved between CBP and p300.

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