The Dual Role of Notch Signaling During Motor Differentiation

Glenn Christopher Tan

Submitted in partial fulfillment of the

requirements for the degree of

Doctorate of Philosophy

under the Executive Committee

of the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY

2012

© 2012

Glenn Christopher Tan

All Rights Reserved

Abstract

The Dual Role of Notch Signaling During Differentiation

Glenn Christopher Tan

Throughout the developing , Olig2+ progenitors in the motor neuron progenitor domain give rise to an impressive array of motor , and . Motor neurons are further diversified into motor columns and pools based on cell body settling position, general axonal trajectories, and the individual muscles they innervate.

Elegant studies have demonstrated that motor neuron columnar and pool diversity, along the rostral-caudal axis of spinal cord, is programmed by extrinsic signals that confer a combinatorial

Hox code at each rostral-caudal coordinate. However, we are only beginning to understand the signals that control motor neuron diversification and neuronal versus glial competency within a given rostral-caudal segment level of spinal cord.

As a key mediator of cell-to-cell communication, the has been implicated as a primary player in the generation of intra-domain cellular diversity throughout development. Despite this, the role of Notch signaling in contributing to neural diversity within the motor neuron progenitor domain has remained elusive. The major hurdle to studying the role of Notch in the motor neuron progenitor domain has been the inability to specifically manipulate

Notch signaling in motor neuron progenitors. In this dissertation, I use embryonic stem cell (ESC) to motor neuron differentiation

technology to demonstrate that Notch signaling has a dual role during motor neuron

differentiation. In Chapter 2, I demonstrate that Notch signaling is required for inhibiting motor

neuron differentiation and maintaining a subset of progenitors for genesis via

lateral inhibition. Activation or inactivation of Notch signaling during ESC to motor neuron

differentiation is capable of disrupting lateral inhibition and generating homogenous cultures of

either glial precursors or motor neurons. Interestingly, induction of Notch signaling during

differentiation is sufficient to upregulate glial markers Sox9 and Sox10, suggesting that Notch also plays an instructive role in specifying glial cell fate.

In Chapter 3, I show that Notch signaling regulates motor neuron columnar identity.

Specifically, I demonstrate that Notch signaling is required for selection of medial motor column

(MMC) identity and that inhibition of Notch signaling during motor neuron differentiation leads

to rostral-caudal appropriate conversion of MMC identity into hypaxial motor column (HMC) identity in cervical conditions or lateral motor column (LMC) identity in brachial conditions. I further identify the transition from progenitor to postmitotic motor neuron as the critical period where Notch activity is necessary to select motor neuron columnar identity.

Previous studies have proposed that an Olig2/Ngn2 competition model controls motor neuron differentiation. In Chapter 5, I show that contrary to this hypothesis, Olig2 does not

inhibit motor neuron differentiation and that Olig2 and Ngn2 largely bind and regulate different

sets of during motor neuron differentiation. Comparing -wide binding and expression data after Ngn2 induction, I identify the early gene expression program directly downstream of Ngn2 that drives motor neuron differentiation. Table of Contents

Table of Contents i List of Illustrations and Tables iv Acknowledgements vi Dedication viii

Chapter 1. Introduction 1 Mechanisms for generating neuronal diversity 2 Regional patterning by extrinsic signals 3 Specification of spinal identity 4 Dorsal-ventral patterning of the ventral spinal cord 5 Temporal patterning 8 Intrinsic mechanisms 8 Negative feedback inhibition 10 Intra-domain diversification of progenitors 11 Intra-domain diversification of postmitotic neurons 11 Neuronal diversity in the spinal cord 12 Motor neurons, oligodendrocytes, and astrocytes are derived from the pMN 13 domain Lineage relationships between motor neurons and oligodendrocytess 14 A bHLH competition model controls motor neuron 16 differentiation Motor neuron diversity 17 Subtype diversification of motor neurons 19 Hox genes control motor neuron columnar and pool identity 20 Regulation of motor neuron columnar identity 21 Generation of neuronal diversity by Notch signaling 22 Notch receptors, ligands, and effectors 23 Notch-mediated lateral inhibition 23 Lateral inhibition in vertebrate CNS 24 The role of Notch signaling in controlling the neuronal to glial cell fate switch 25 Notch selects alternative cell fates 27 Conclusion 29

Chapter 2. Notch controls the motor neuron to glial cell fate switch in the pMN 43 domain Introduction 43 Results 44 Cell-autonomous activation of Notch signaling prevents pMN differentiation 44 Cell-autonomous inhibition of Notch signaling is incompatible with pMN identity 45 In vitro ESC differentiation recapitulates the motor neuron to glial cell fate 45 switch Activation of Notch signaling inhibits motor neuron differentiation 47 Inhibition of Notch signaling promotes pMN differentiation into motor neurons 48

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DAPT treatment increases motor neuron yield 49 Inhibition of Notch signaling promotes pMN differentiation into motor neurons 49 by disrupting lateral inhibition Ngn2 is sufficient to drive motor neuron differentiation 50 Discussion 51 Notch controls motor neuron versus glial cell fate via lateral inhibition 51 Molecular effectors of Notch signaling in pMN domain 52 Motor neurons and are generated from a common progenitor 53 DAPT can be used as a general treatment for generating high-yield motor 54 neuron cultures

Chapter 3. Notch selects intra-segmental motor neuron column identity 76 Introduction 76 Results 77 Notch inhibition promotes HMC-like motor neuron identity during ESC to motor 77 neuron differentiation Notch inhibition promotes LMC identity in brachial spinal cord conditions 78 Changes in columnar markers are not due to acceleration of motor neuron 80 maturation Candidate HMC molecular profile 82 Sst is a marker of HMC motor neurons in vitro 84 Inhibition of Notch signaling converts Lhx3+ MMC motor neurons to Sst+ HMC 85 motor neurons Sst is a marker of cervical HMC motor neurons in vivo 86 Changes in columnar cell fate occur at early stages of motor neuron 87 differentiation Ngn2 does not alter motor neuron columnar identity 88 Discussion 89 Selection of cell fates is not controlled via disruption of lateral inhibition and 90 Ngn2 induction Selection of columnar identity by Notch signaling occurs during motor neuron 91 genesis Notch and non-canonical Wnt 92 Sst is a novel marker of cervical HMC motor neurons 93

Chapter 4. 115 Introduction 115 Results 117 Olig2 is not an in vivo repressor of Hb9 117 Ngn2 ChIP-Seq identifies genes directly regulated by Ngn2 119 Olig2 and Ngn2 do not have similar binding profiles 119 Olig2/Ngn2 do not constitute a cooperative motor neuron binding program 121 Genes directly regulated by Ngn2 define an early motor neuron differentiation 122 program Discussion 123 Olig2 is not an in vivo repressor of Hb9 124

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Ngn2 and Olig2 have disparate binding profiles 124 Genes directly regulated by Ngn2 represent the motor neuron differentiation 125 program downstream of Ngn2 induction Chapter 5. Discussion and Perspectives 136 Lineage relationship between motor neurons, oligodendrocytes, and astrocytes 136 Contextual Notch signaling 139 Temporal progression 141 Conclusion 143 Chapter 6. Materials and Methods 149 Chick electroporation 149 Generation of inducible lines 149 ESC to motor neuron differentiation 150 Induction of transgenes and DAPT treatment 151 Embedding of embryoid bodies for immunocytochemistry 151 Dissociation of embryoid bodies for motor neuron culture 151 Dissection of chick and mouse spinal cords 152 Immunocytochemistry/Immunohistochemistry 152 Antibodies 152 Imaging 153 Quantification of chick spinal cord electroporations 153 Quantification of embryoid bodies 153 Quantification of dissociated cells 154 Statistical analysis 154 RNA purification 154 qRTPCR 155 Microarray preparation and analysis 155 ChIP-Seq preparation 155 ChIP-Seq analysis 155

Bibliography 159

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List of Illustrations and Tables

Figure 1.1 SHh patterning of the ventral spinal cord into distinct domains 31 Figure 1.2 The pMN domain gives rise to motor neurons and glia 33 Figure 1.3 Motor neurons are organized into columns with distinct molecular 35 profiles Figure 1.4 Hox genes control motor neuron diversity along the rostral-caudal axis of 37 the developing spinal cord Figure 1.5 The Notch signaling pathways contributes to neuronal diversity via 39 lateral inhibition and selection alternative cell fates. Figure 1.6 Notch signaling is active in the ventricular zone of the spinal cord 41 Figure 2.1 Electroporation of cell intrinsic activators and inhibitors of Notch 56 signaling Figure 2.2 ESC to motor neuron differentiation protocol recapitulates the motor 58 neuron to glial progenitor cell fate switch Figure 2.3 Induction of Notch signaling inhibits motor neuron differentiation and 60 promotes glial cell fate Figure 2.4 NICD induced Olig2 cells are glial progenitors 62 Figure 2.5 DnMaml1-eGFP promotes motor neuron differentiation and depletes 64 progenitors Figure 2.6 DAPT treatments effectively inhibits Notch signaling and increases 66 motor neuron yield. Figure 2.7 Inhibition of Notch signaling depletes progenitors 68 Figure 2.8 Notch inhibition disrupts lateral inhibition and induces Ngn2 70 Figure 2.9 Ngn2 induction is sufficient to promote motor neuron differentiation and 72 deplete progenitors Figure 2.10 Proposed model based on results of Notch activation and inhibition 74 during motor neuron differentiation Figure 3.1 Inhibition of Notch signaling leads to loss of MMC motor neuron marker 95 Lhx3 Figure 3.2 Inhibition of Notch signaling in brachial conditions leads to conversion 97 of MMC motor neurons into LMC motor neurons Figure 3.3 Gene expression changes in DAPT treated motor neurons are not 99 correlated with motor neuron maturation Table 3.1 Microarray analysis identifies a candidate gene expression profile for 101 HMC motor neurons Figure 3.4 Validation of candidate genes identified in microarray study 103 Figure 3.5 Sst and Lhx3 are mutually excluded in ESC derived motor neurons 105 Figure 3.6 Sst is a marker of rostral-cervical HMC motor neurons in-vivo 107 Figure 3.7 Day 4 pMN stage is the critical period for Notch activity in controlling 109 motor neuron columnar identity Figure 3.8 Changes in motor neuron columnar identity after Notch inhibition are not 111 mediated by Ngn2 Figure 3.9 Model for Notch selection of columnar cell fate 113 Figure 4.1 Olig2 does not repress Hb9 126

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Figure 4.2 ChIP-Seq identifies Dll1 as a direct target of Ngn2 128 Figure 4.3 Comparative analysis of Ngn2 and Olig2 genome wide binding profiles 130 Figure 4.4 Genes directly regulated by Ngn2 constitute a motor neuron gene 132 expression program Table 4.1 Genes directly regulated by Ngn2 during motor neuron differentiation 134 Figure 5.1 The iterative role of Notch signaling during pMN development 145 Figure 5.2 Genetic network controlled by Notch signaling 147 Table 6.1 qRTPCR primers 157

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Acknowledgements

This thesis could not have been done without the support of many kind friends and colleagues. Chief among these people, I would like to thank Dr. Hynek Wichterle for his years of mentorship and for giving me the opportunity to perform this work in his laboratory. His enthusiasm for science is truly contagious and will inspire me throughout my future career. I am grateful to all the many past and present members of the Wichterle lab who have created a congenial work environment and contributed many insights and discussions to this work. I thank

Mirza Peljto for taking an extreme novice at experimental biology under his wing and teaching me how to survive the pressures of graduate school. Jun-An Chen has been a great friend throughout the years and his mentorship and empathy have been critical in my development as a scientist. I am grateful to Esteban Mazzoni and Stephane Nedelec, who can always offer a good joke or story, but are also brilliant scientists whose criticisms and opinions I hold in high regard.

Many thanks to Michael Closser, Annete Gaudino, Yu Chen, Yuan-Ping Huang, Rosie Bogan,

Carolyn Morrison, Susan Jun, Jane Kim, Elena Abiramov, Dosh Whye, and Chelsea Culbert for all the good years.

I am grateful to the members of my thesis committee Dr. James Goldman, Dr. Richard

Mann, and Dr. Boris Reizis for all the productive meetings we’ve had over the years. Their advice has been instrumental in guiding the direction of my thesis project. I thank Dr. Jan

Kitajewski and Thaned Kangsamakin for early discussions on the mechanics of Notch signaling.

I thank the following people for their help or for providing regents critical for my work: Kristie

Gordon, Sandra Tetteh, Susan Brenner-Morton, Molly Golden, Christopher Reeder, Dr. Nicholas

Gaiano, and Dr. Warren Pear.

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I thank Rosario and Johnny for being wonderful parents and for continuing to being in awe of everything I do, even at the age of 34. I thank my sister Amy and brother in-law Austin for being a constant source of familial support in New York, and for always offering their home as a haven away from the rigors of graduate school. To my nieces Naomi and Mikaela, thank you for constantly putting a smile on my face. I am grateful for my brother Fritz, who has always been my comrade in arms for all our endeavors.

Most importantly, I thank Qing Wang for her love, support and patience. She has been a constant source of calmness, and without her editing skills this thesis could not have been completed.

vii

DEDICATION

For my family.

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1

Chapter 1. Introduction

One of the amazing aspects of developmental biology is the ability of a single cell to give rise to all specialized postmitotic cells of a . The mammalian central (CNS) alone is comprised of thousands of different types of neurons and glia that all have to be integrated into functional circuits requiring developing neurons to migrate to the right position, project their along the proper path, with correct targets, and have their axons myelinated by oligodendrocytes (Pearson and Doe, 2004). All of this is predicated by the necessity for neurons and glia to be generated in the correct space and time. Diseases such as Spinal Muscular (SMA), Huntington’s Chorea, and Parkinson’s Disease provide best evidence that selective loss or disruption of a single or few classes of neurons can lead to severe neuronal disorders (Markowitz et al., 2012; Rochet et al., 2012; Zheng and Diamond,

2012) .

Clearly, precise mechanisms are necessary to tightly control the proliferation and specification of progenitor cells in order to generate the proper numbers of each cell type at the appropriate times. Over the last few decades, experiments in diverse organisms spanning from invertebrates with relatively simple nervous systems to mammals have started to elucidate the molecular players behind neuronal diversity (Lin and Lee, 2012; Livesey and Cepko, 2001;

Pearson and Doe, 2004). These studies have revealed that neuronal diversity is established by several mechanisms, including regional patterning of progenitors, temporal patterning of progenitors and intra-domain diversification of both progenitors and postmitotic neurons.

Importantly, many molecular mechanisms governing these processes are re-used in different 2 areas of the , during different developmental stages, and across different species.

In the mammalian CNS, these principles can be best illustrated in the developing ventral spinal cord, a region that was studied in particular detail over the past thirty years. Following closure, the ventral half of the spinal cord is exposed to a gradient of Sonic

Hedgehog (SHh) protein secreted from the and . The concentration gradient of SHh patterns the region into five distinct progenitor domains that each gives rise to a different cell class (Briscoe and Ericson, 2001; Price and Briscoe, 2004). During the early neurogenic phase, progenitors within each of the cardinal domains generate multiple subtypes of neurons.

Later in development, progenitors undergo a temporal patterning switch from neurogenesis to gliogenesis. For example, the Olig2-expressing motor neuron progenitor (pMN) domain first produces motor neurons of diverse columnar, divisional and motor pool identity, followed by production of oligodendrocytes and astrocytes (Briscoe and Novitch, 2008). While molecular mechanisms controlling regionalization of the developing spinal cord are relatively well understood, mechanisms that contribute to temporal changes in progenitors and intra-domain diversification of neurons are less clear. Thus, the goal of this dissertation will be to explore the mechanisms controlling cell fate decisions within the pMN domain, with a particular focus on the Notch signaling pathway that has been previously implicated in diversification of neural progenitors in simpler organisms and in other regions of the CNS.

Mechanisms for generating neuronal diversity

Despite the complexity of the CNS, nascent neuroepithelial tissue initially forms as morphologically homogenous sheets of undifferentiated neural progenitors. The vertebrate CNS 3 utilizes many mechanisms to specify naïve tissue into progressively more specialized cell types.

The first step in this process is the regionalization of the developing neural tube into distinct compartments by morphogens secreted from patterning centers along both the rostral-caudal

(RC) and dorsal-ventral (DV) axis of the developing embryo. Integration of RC and DV signals generates a Cartesian like map where cells in each progenitor domain have unique [RC, DV] coordinates (Edlund and Jessell, 1999). One consequence of RC patterning is the compartmentalization of the neural tube into four distinct regions that will eventually form the forebrain, , hindbrain, and spinal cord. In this section I will describe the general mechanisms utilized in the CNS to generate cellular diversity, starting with regionalization of the neural tube into distinct domains.

Regional patterning by extrinsic signals

During gastrulation, neural tissue is initially specified with a default anterior cell fate and formation of caudal CNS compartments requires caudalizing signals secreted by patterning centers or organizers (Nieuwkoop, 1985; Stern, 2005; Stern et al., 2006). Over the past few decades, many studies have started to elucidate the signals involved with compartmentalization of the vertebrate neural tube into forebrain, midbrain, hindbrain, and spinal cord.

The first indication of a caudalizing patterning center was provided by transplantation experiments in chicken and quail embryos, where tissue from the midbrain/hindbrain border, known as the isthmus, was grafted into forebrain or caudal hindbrain. In either case, the grafted tissue was able to induce the formation of midbrain or caudal hindbrain structures, indicating that the isthmus secretes a factor that is capable of inducing midbrain/hindbrain character (Bally-Cuif et al., 1992; Bally-Cuif and Wassef, 1994; Martínez et al., 1995; Martinez et al., 1991). The 4 ability of beads with soaked fibroblast growth factor 8 (FGF8) to induce ectopic midbrain and hindbrain tissue later revealed the patterning factor secreted by the isthmus to be FGF8 (Crossley et al., 1996; Martinez et al., 1999). Consistent with the role of FGF8 as a specifier of midbrain/hindbrain character, FGF8-/- mice completely fail to form midbrain and rostral hindbrain. Instead, FGF8-/- embryos develop neural tubes with the caudal hindbrain and spinal cord immediately adjacent to the forebrain, indicating that FGF8 signaling is required for specifying midbrain and rostral hindbrain (Sun et al., 1999).

Specification of spinal identity

Developmental studies have established that specification and patterning of the spinal cord relies on a complex set of caudalizing signals that include the Wnt, RA, FGF, and TGFβ families of factors (Dasen and Jessell, 2009). Gain and loss of function studies have shown that

Wnt signaling plays a critical role in primary axis formation and specification of caudal identity in neural tissues. Wnt3A-/- mice exhibit loss of caudal neural tissues and expansion of forebrain and hindbrain character throughout the RC axis of the neural tube (Liu et al., 1999), and increasing concentrations of Wnt3a induces chick neural explants to adopt progressively more caudal neural identities (Nordström et al., 2002). After the initial acquisition of caudal neural identity by Wnt activity, additional signals further compartmentalize the caudal neural tube into cervical, brachial, thoracic, and lumbar sacral spinal cord.

Significant evidence implicates retinoic acid (RA) as having a crucial role in patterning caudal neural tissue into caudal hindbrain and rostral spinal cord territories. Early studies treating whole mouse, rat, zebrafish and Xenonpus embryos to high levels of retinoic acid (RA) led to suppression of caudal neural territories (Maden, 2002). It was later found that somites of 5 the paraxial mesoderm express the retinaldehyde dehydrogenase 2 (Raldh2) enzyme that is necessary for synthesizing RA, and that Raldh2-/- mice have normal forebrain and rostral midbrain but form expanded rostral hindbrains at the expense of caudal hindbrain and spinal cord

(Niederreither et al., 2000). Further studies using a retinoic acid receptor (RAR) blocking agent were able to disrupt individual rhombomeres at different levels of retinoid inhibition, demonstrating that retinoid specification of posterior identity occurs in a concentration dependent manner(Dupé and Lumsden, 2001).

The RC patterning effects of RA in the hindbrain and spinal cord are primarily mediated through control of expression in the developing embryo. The Hox genes are a unique class of homeodomain transcription factors that are collinearly organized on chromosomes in the same order that they are expressed in the RC axis, with 3’ Hox genes expressed rostrally and 5’ genes expressed caudally (Durston et al., 2011; Kmita and Duboule, 2003). Early experiments in

Drospophila melanogaster highlighted the ability of Hox genes to confer regional identity to embryonic tissue. Most famously, gain-of-function (GOF) experiments driving the expression of the Hox gene Antennepedia from a heat shock inducible promoter converted antenna bearing segments of Drosphila into leg bearing segments (Schneuwly et al., 1987).

Regional specification by Hox genes is an evolutionarily conserved mechanism for specifying RC identity in bilateral development. In mammals, Hox genes are ordered into four distinct Hox clusters generated by gene duplication events (Swalla, 2006). Similar to

Drosophila, GOF and loss-of-function (LOF) genetic manipulations of Hox genes leads to homeotic transformations along the RC axis of developing murine embryos (Davis and

Capecchi, 1994; Horan et al., 1994; Jegalian and De Robertis, 1992; Kessel and Gruss, 1991; 6

Small and Potter, 1993; Zhang et al., 1994). The ability of Hox genes to define tissue structures along the RC axis suggests that they are the downstream effectors of extrinsic RC patterning signals.

Indeed, several studies have demonstrated links between regional patterning by extrinsic factors and Hox gene expression. For example, in Raldh2-/- mice, the loss off caudal hindbrain structures is reflected by the loss of expression of caudal hindbrain Hox genes Hoxa3 and

Hoxd4. The concomitant expansion of the caudal hindbrain was also accompanied by a caudal expansion of rostral Hoxb1 into the Hoxa3 and Hoxd4 domains (Niederreither et al., 2000;

Niederreither et al., 2002). The identification of retinoic acid response elements (RARE) in the regions of 3’ Hox genes, combined with recent data showing direct binding of RAR receptors to Hox enhancers, further suggests that RA-mediated changes in Hox gene expression are the result of direct transcriptional regulation by RA (Huang et al., 2002; Kashyap et al., 2011;

Langston and Gudas, 1992; Mahony et al., 2011; Moroni et al., 1993; Oosterveen et al., 2003;

Packer et al., 1998).

Though their mechanisms are likely not direct, Fgf, Wnt, and TGFβ signaling have all been identified as caudalizing factors responsible for inducing caudal Hox genes and specifying caudal spinal cord identity. At the regressing end of the developing neural tube, a patterning center known as Henson’s node in chick, secretes FGF caudalizing signal (Dubrulle and

Pourquié, 2004; Liu et al., 2001). Multiple studies have shown in vitro and in vivo that low levels of FGF8 induce expression of brachial Hox6-8, while higher levels induce expression of thoracic Hoxc9 (Bel-Vialar et al., 2002; Dasen et al., 2003; Liu et al., 2001). TGFβ family member GDF11 is expressed in caudal tissues and induces the expression of lumbar Hox10 7 genes (Liu, 2006). Integration of these extrinsic Hox regulators ultimately results in the patterning of the developing spinal cord into cervical, brachial, thoracic and lumbar sacral segments of spinal cord and proper generation of position-appropriate neuronal diversity (Dasen and Jessell, 2009).

Dorsal-ventral patterning of the ventral spinal cord

In parallel to the extrinsic signals that pattern the RC axis of the developing neural tube, cells in the notochord and floor plate secrete SHh protein that diffuses dorsally and creates a gradient that is high ventrally and low dorsally. The graded levels of SHh signaling pattern the ventral spinal cord into distinct progenitor domains that express different combinations of SHh- sensitive transcription factors (Fig. 1.1A) (Price and Briscoe, 2004). These transcription factors are divided into Class 1 and Class 2, depending on whether they are repressed or induced by SHh signaling, with each transcription factor repressed or induced at different levels of SHh signaling.

Cross-repressive interactions between the Class 1 and Class 2 transcription factors establishes progenitor domains with sharp boundaries (Fig. 1.1B). The pMN, for example, expresses the basic helix loop helix (bHLH) transcription factor Olig2 which directly represses the ventral p2 domain marker Irx3 and ventral p3 domain marker Nkx2.2, both of which in turn repress Olig2. Some cells at domain borders might transiently co-express markers for two domains, but this state is inherently unstable due to the cross-repressive activities of the transcription factors involved, and individual cells will stochastically resolve into expressing only one marker (Briscoe and Ericson, 2001). The resulting five domains have sharp boundaries and can be identified by distinct combinations of transcription factors. Each of these unique domains generates different classes of neurons with distinct properties and functional roles in 8 motor circuitry. The Olig2-expressing pMN domain that gives rise to motor neurons and oligodendrocytes is the primary focus of this dissertation and will be discussed in detail later in this chapter.

Thus, RC and DV signals pattern the developing neural tube into compartments with distinct molecular profiles, cellular composition, and functional roles. However, even within individual domains, many types of neural cells are generated and additional mechanisms must exist to generate the necessary diversity for proper CNS function. In the next few sections I will review additional methods for generating diversity within a given progenitor domain.

Temporal patterning

The temporal patterning of progenitors through both intrinsic and extrinsic cues is another method through which neuronal diversity can be specified. Temporal generation of neurons has best been documented in regions exhibiting complex layered organization such as the neocortex and retina (Desai and McConnell, 2000; Livesey and Cepko, 2001). However even simpler regions undergo temporal switches between neuronal cell fates, albeit with different mechanistic details.

Intrinsic mechanisms

Due to their precise and stereotypical generation of neurons, melanogaster neuroblast lineages have been the prototypical system for studying temporal patterning. The

Drosophila CNS is composed of a bi-lateral array of thirty neuroblasts running along the anterior-posterior axis of the developing embryo (Bossing et al., 1996; Schmid et al., 1999;

Schmidt et al., 1997). Neuroblasts undergo asymmetric divisions that maintain the neuroblast 9 and generate a mother cell (GMC) (Goodman and Doe, 1993). GMCs divide to generate two neurons, with younger GMCs pushing older GMCs and neurons deeper into the developing embryo. The result is a laminar organization of neurons, with older neurons located deep within the embryo and new born neurons located in superficial layers (Isshiki et al., 2001;

Schmid et al., 1999). While individual neuroblasts generate anywhere from three GMCs to over twenty GMCs, the order of GMC and neuron genesis in each lineage is invariant, indicating a precise control of cell identity at each cell division step (Pearson and Doe, 2004).

Genetic studies have since uncovered that Drosophila neuroblast lineages are controlled by a dynamic transcriptional network that cell-intrinsically confers temporal identity on each neuroblast and its progeny. Neuroblasts sequentially express the transcription factors that define their temporal competence: Hunchback (Hb), Krüppel (Kr), Pdm and Castor (Cas) (Isshiki et al.,

2001). While overexpression of Hb confers early temporal identity and forces later born GMCs to differentiate into early neurons, mis-expression of Kr, Pdm, or Cas can induce neuroblasts to skip early lineages and generate neurons associated with the respective transcription factor

(Isshiki et al., 2001; Novotny et al., 2002; Pearson and Doe, 2003).

Current evidence strongly suggests that progression through the transcription factor cascade occurs through cell intrinsic mechanisms. Neuroblasts isolated from embryos and grown in vitro in the absence of extrinsic factors still progress through the transcription factor cascade normally (Grosskortenhaus et al., 2005). Additionally, isolated neuroblasts undergo asymmetric division and generate the number of GMCs and neurons expected for the given neuroblast (Broadus and Doe, 1997; Pearson and Doe, 2004). 10

Neuroepithelial progenitors in both the rodent retina and neocortex also progress through a series of competence states in which they generate different classes of neurons. Newborn cortical neurons migrate radially and settle at a position immediately distal to the previous cohort of neurons, resulting in a laminar organization with deep layers containing early-born neurons and superficial layers containing late-born neurons (Livesey and Cepko, 2001; McConnell,

1988). Similar to Drosophila, the competence states of retinal progenitors seems to be an intrinsic property, as heterochronically transplanted retinal progenitors are insensitive to their transplanted environments and continue to generate neurons consistent with their own age

(Rapaport et al., 2001). Furthering the analogy with Drosophila neuroblast lineages, the HB ortholog Ikaros was recently found to confer competence for early lineages to retinal progenitors.

Overexpression of Ikaros at a time when photoreceptors are normally produced induces retinal progenitors to instead differentiate into early-born lineages (Elliott et al., 2008).

Negative feedback inhibition

Although competency states are intrinsically defined, there is evidence that extrinsic factors play a role in temporal control of cell fates in retinal progenitors. In heterochronic in vitro cultures, postmitotic retinal ganglion cells (RGC) and amacrine cells are capable of inhibiting retinal progenitors from differentiating into RGCs or amacrine cells. This result suggests that newly born RGCs and amacrine cells secrete factors that inhibit progenitors from differentiating into those cell types (Belliveau and Cepko, 1999; Waid and McLoon, 1998). It was later shown that in the case of RGCs, the secreted factor is SHh (Wang et al., 2005). This negative feedback provides a key mechanism through which the retina can regulate the number 11

RGCs and amacrine cells that are generated and may be a component in the signaling machinery that controls changes in retinal progenitor competency transitions.

Intra-domain diversification of progenitors

Extrinsic signals can also play a role in generating cell diversity within one progenitor domain at one developmental time-point. This is achieved by diversification of progenitors within one progenitor domain or by diversification of postmitotic progeny. Oligodendrocytes and inhibitory GABAergic neurons are generated by neural progenitors in both the ganglionic eminence (MGE) and the anterior entopeduncular area (AED) of the ventral telencephalon

(Kessaris et al., 2006). Recent studies have revealed that non-canonical Wnt signaling through the receptor Ryk is responsible for controlling neuronal versus glial generation in this context.

While the number of cycling progenitors is not changed in Ryk-/- mice, there is a significant increase in oligodendrocytes and loss of GABAergic neurons. Conversely, activation of non- canonical Wnt signaling through administration of Wnt3A or overexpression of Ryk intracellular domain promotes GABAergic neuron differentiation and suppressed oligodendrocyte differentiation (Zhong et al., 2011).

Intra-domain diversification of postmitotic neurons

Intra-domain diversification of neurons can also occur at postmitotic stages.

Photoreceptors in the mouse retina can be divided into three subtypes based on their morphology and the type of opsin they express. Rod cells are the most common photoreceptors in the retina and are named for their rod-shaped outer segment. Cone cells have coned-shaped outer segments and are further subdivided into M or S cones based on their use of M or S opsins to detect light. While rods are highly excitable photoreceptors capable of resolving low levels of 12 light, cones require many times more light for activation and are excitable at specific wavelengths of light due to their the S and M opsins, which allow for color discrimination.

Thus, the rodent photoreceptor family consists of morphologically, functionally, and molecularly distinct classes of neurons (Oh et al., 2007; Swaroop et al., 2010).

In contrast to previously described mechanisms, photoreceptors are born as generic photoreceptor precursor neurons and selection of subtype identity occurs postmitotically. The transcription factors Neural Retina Leucine Zipper Factor (NRL) and nuclear orphan receptor

Thyroid Hormone Receptor β2 (TRβ2) control selection of the three photoreceptor subtypes in a two-step process. Early-born photoreceptors express low amounts of S opsin transcript revealing a default S cone fate. In the first step, if levels of NRL reach a specific threshold, the precursor is specified as a rod cell. In the absence of NRL the photoreceptor will become a cone cell, and exposure to circulating thyroid hormone (TH) will induce TRβ2 activity and M cone cell fate. In the absence of both NRL and TRβ2 activity, photoreceptors will adopt the default S cone fate

(Ng et al., 2011). Additionally, NRL exerts transcriptional dominance over TRβ2 so that cells expressing both factors will still adopt rod photoreceptor identity (Oh et al., 2007). In the mature rodent retina, rod photoreceptors outnumber cone photoreceptors thirty to one. This serves as a built-in mechanism for ensuring that rod photoreceptors are generated in a preferential fashion.

Thus, intra-domain neural diversity can be generated by mechanisms that specify cell fate in both progenitors and postmitotic cells.

Neuronal diversity in the spinal cord

Together these general mechanisms comprise a set of evolutionarily conserved tools for progressive differentiation of initially homogenous and undifferentiated tissue into individual 13 neurons and glia. The same principles for generating neuronal diversity in other parts of the

CNS apply to the vertebrate spinal cord. In many cases, the ventral spinal cord has served as the prototypical model system for studying regional patterning of mammalian CNS and for intra- domain specification of neuronal subtypes. In the next section, I will review diversity within the spinal cord as well as the mechanisms that generate this diversity.

Motor neurons, oligodendrocyte, and astrocytes are derived from the pMN domain

Earlier in this chapter, we discussed the confluence of RC and DV signals that result in specification of the Olig2+ pMN domain. Detailed genetic and immunohistochemical studies have revealed that progenitors from the pMN domain give rise to motor neurons during early stages of development and to oligodendrocytes and astrocytes later in development (Fig. 1.2A)

(Lu et al., 2002; Zhou and Anderson, 2002). However, the lineage relationship between motor neurons, oligodendrocytes and astrocytes is unclear, as it is currently not known whether a common Olig2 progenitor is temporally patterned to give rise first to motor neurons and then to glial cells (Fig. 1.2B), or separate Olig2+ progenitors give rise to motor neurons, oligodendrocytes, or astrocytes (Fig. 1.2C).

Olig2 expression first appears and marks the pMN domain as early as E8.5 (Wu et al.,

2006). During these early stages of development, Olig2+ pMNs in the ventricular zone of the spinal cord differentiate into motor neurons and migrate laterally to the ventral horn of the spinal cord. While motor neuron differentiation is completed by E12.5, Olig2+ progenitors are not depleted after this period (Novitch et al., 2001). As development proceeds, the remaining Olig2+ cells acquire expression of the p3 domain marker Nkx2.2, as well oligodendrocyte precursor

(OPC) markers Sox9, Sox10, and Platelet Derived Growth Factor Alpha (PDGRα) (Fu et al., 14

2002; Zhou et al., 2001). These Olig2+/Nkx2.2+ cells continue to proliferate and eventually migrate throughout the spinal cord where they differentiate into mature oligodendrocytes (Zhou et al., 2001).

Chick neural tube electroporation experiments indicate that the acquisition of Nkx2.2 reflects a switch from neuronal to glial competence of pMN progenitors. Dorsal mis-expression of Olig2 alone leads to a dorsal expansion of Olig2 into the p2 domain and generation of ectopic motor neurons at the expense of v2a (Novitch et al., 2001). In contrast, co- electroporation of Olig2 and Nkx2.2 in chick neural tube results in the generation of ectopic oligodendrocytes, even at early stages of development prior to normal OPC development (Zhou et al., 2001).

Lineage relationship between motor neurons and oligodendrocytes

The sequential generation of motor neurons and oligodendrocytes from the same progenitor domain raises the question of whether these two lineages share a common progenitor and what pathways control the switch from neuronal to glial cell fate. Olig2 mutant mice lose all motor neurons and oligodendrocytes confirming that these cell types are generated by Olig2 expressing progenitors in the pMN and suggesting that motor neurons and oligodendrocytes share a common progenitor (Novitch et al., 2001; Takebayashi et al., 2002; Zhou and Anderson,

2002). Alternatively it is possible that two distinct Olig2+ progenitor populations separately give rise to motor neurons and oligodendrocytes.

Major evidence supporting the two progenitor hypothesis came from a study using a genetic system to ablate Olig1-expressing cells. In the pMN, Olig2 expression is closely followed by Olig1 expression. Wu et al. crossed the Olig1-Cre knock-in mouse with a 15 conditional Rosa26-DTA mouse to generate progeny that would produce Olig2+ pMNs but immediately kill them by Olig1-driven expression of diphtheria toxin. In these , newly born Olig2+ cells are continually ablated, leading to a dramatic loss of both motor neurons and oligodendrocytes, reminiscent of Olig2-/- mice (Novitch et al., 2001; Wu et al., 2006; Zhou et al.,

2001). Despite the ablation of early-born Olig2+ cells, these mice continue to generate Olig2+ cells, even after motor neuron differentiation has ceased, leading the authors to conclude that these later-born Olig2+ cells signify a separate OPC lineage (Wu et al., 2006). However, these results occur in an injury background where Olig2+ cells are constantly depleted; thus the possibility that the late-born Olig2+ cells are the result of homeostatic mechanisms that generate new Olig2+ cells to compensate for their loss cannot be ruled out.

In contrast, significant evidence points to a common progenitor for motor neurons and oligodendrocytes. The experiment most directly addressing this question used lineage tracing through Olig2-CreER-driven recombination to label progeny of Olig2-expressing cells at different time points. A single dose of tamoxifen at the onset of motor neuron differentiation at

E9.5 leads to labeling of motor neurons, oligodendrocytes, and astrocytes (Masahira et al., 2006).

Additionally, transplantation studies demonstrated that FACS-sorted Olig2-GFP cells from E9.5 murine spinal cord transplanted into chick spinal cord gave rise to both motor neurons and oligodendrocytes (Mukouyama et al., 2006). Together these studies demonstrate that early-born

Olig2+ progenitors contribute to both motor neuron and oligodendrocyte lineages. However, they do not rule out the possibility that Olig2+ cells found in E9.5 embryos are a heterogeneous population that harbors subsets of progenitors committed to generating only motor neurons or only oligodendrocytes. 16

A bHLH transcription factor competition model controls motor neuron differentiation

Given the strong evidence that both motor neurons and oligodendrocytes are generated from early Olig2+ cells in the pMN domain, precise mechanisms must exist to control differentiation of pMNs into either motor neurons or OPCs. Compelling evidence suggests that the pro-neural bHLH transcription factor Neurog2 (Ngn2) is a key determinant of motor neuron differentiation. Overexpression of Olig2 in chick spinal cord is capable of inducing Ngn2 expression and Olig2/Ngn2 co-electroporation is capable of inducing ectopic motor neurons in chick midbrain where motor neurons are not normally generated (Mizuguchi et al., 2001;

Novitch et al., 2001). Subsequently it was demonstrated that Ngn2 forms a hexamer complex with Islet1 (Isl1) and LIM 3 (Lhx3) and can bind to the promoter of critical motor neuron determinant Homeobox 9 (Hb9). Thus, Ngn2 drives Hb9 expression and the differentiation of pMNs into postmitotic motor neurons (Lee and Pfaff, 2003).

Immunohistochemical analysis of Olig2 and Ngn2 in chick spinal cord discovered that

Ngn2 is expressed in Olig2+ cells in a salt and pepper fashion during motor neuron differentiation and is extinguished when motor neuron genesis is completed. Olig2 and Ngn2 are both bHLH transcription factors with similar binding affinities (Murre et al., 1989), but Olig2 is known to be a transcriptional repressor while Ngn2 is a transcriptional activator (Sommer et al.,

1996). In vitro studies demonstrating that Olig2 can bind and repress an Hb9 enhancer element, led to the proposal of a bHLH competition model for controlling motor neuron differentiation

(Lee et al., 2005). In this model, Olig2 promotes progenitor identity by binding to the promoters of motor neuron genes and inhibiting motor neuron differentiation. Factors downstream of Olig2 then stochastically induce the expression of Ngn2, which, at high levels, can out-compete Olig2 17 at these promoter regions to induce motor neuron differentiation. This model indicates that

Ngn2 is a crucial determinant of whether a cell differentiates into motor neurons or remains as a progenitor for later OPC differentiation. While the factors regulating Ngn2 expression in the pMN context are not known, Ngn2 is a member of the Atonal family of pro-neural bHLH transcription factors that are frequently regulated by Notch signaling during lateral inhibition of neurogenesis (Kageyama et al., 2008). The putative role of Notch signaling in controlling the pMN neuronal to glial cell fate switch will be covered in detail, later in this chapter.

Motor neuron diversity

Motor neurons themselves are not a uniform class of neurons but are a diverse group of cells that can be classified into dozens of subtypes (Kanning et al., 2010). At the highest level of organization, classic studies using retrograde labeling techniques in chick embryos revealed that motor neurons are organized into three primary motor columns defined by their cell body settling position and general axonal trajectories (Hollyday and Jacobson, 1990; Landmesser, 2001). The

Medial Motor Column (MMC) spans the entire spinal cord and is located medially at a position closest to the neural tube and its axons project dorsally towards axial muscles of the back (Fig

1.3A-B). The Lateral Motor Column (LMC) is positioned in the lateral aspect of brachial and lumbar spinal cord and innervates muscles in the limb (Fig 1.3B). The spatial organization of the

Hypaxial Motor Column (HMC) is less well-defined but is generally thought to reside in a position intermediate to the MMC and LMC columns and is predominantly present at trunk levels of spinal cord. HMC motor neurons project their axons to hypaxial muscles including the diaphragm and intercostal muscles (Fig 1.3A). The pre-ganglionic column (PGC) is a fourth 18 column present only in thoracic spinal cord and contains autonomic motor neurons that innervate the sympathetic ganglia (Dasen and Jessell, 2009).

In addition to their physical characteristics, motor columns also have distinct molecular profiles (Fig. 1.3 A-B). With a few exceptions, all motor neurons can be labeled by the expression of transcription factors Hb9 or Islet 1/2 (Isl1/2) (Arber et al., 1999; Pfaff et al., 1996).

While Lhx3 is initially expressed in all nascent motor neurons, it is downregulated in HMC,

LMC, and PGC motor neurons and serves as a marker of MMC identity (Fig. 1.3A) (Arber et al.,

1999; Tsuchida et al., 1994). Genetic studies in mice have further demonstrated that Lhx3 plays an instructive role in specifying MMC cell fate. Driving Lhx3 expression using a transgenic Hb9 promoter, Sharma et al. showed that constitutive expression of Lhx3 leads to a downregulation of genes associated with HMC or LMC identity. Additionally, Lhx3 overexpression redirects axons away from limb and ventral muscles to project dorsally towards axial muscles that are normally innervated by MMC motor neurons (Sharma et al., 1998).

Similar to Lhx3 for MMC motor neurons, Forkhead Box Protein 1 (Foxp1) is both a marker and instructive cue for LMC and PGC identity. Foxp1 overexpression studies in chick neural tube showed that high levels of Foxp1 expression induces an increase in LMC motor neurons at the expense of other columnar types, and low levels of Foxp1 expression leads to a

PGC cell fate. Furthermore, Foxp1 mutant mice show complete loss of both LMC and PGC motor neurons with a dramatic corresponding increase in the number of HMC motor neurons generated. Together these data show that Foxp1 is both sufficient and necessary for specifying

LMC and PGC columnar cell fate (Dasen et al., 2008; Rousso et al., 2008). 19

Unlike MMC and LMC motor neurons, there is no universal marker for HMC motor neurons. Instead, HMC motor neurons are identified by their lack of expression of both Foxp1 and Lhx3.

Subtype diversification of motor neurons

Motor columns are further subdivided into small clusters of motor neurons that innervate individual muscles. In many cases, these motor pools have also been found to have their own unique transcriptional profiles. The flexor carpi ulnaris, for example, is innervated by motor neurons expressing the Pou-domain transcription factor Scip, while the neighboring cutaneous maximums motor pool expresses the ETS transcription factor Pea3 (Dasen et al., 2005; Lin et al.,

1998; Livet et al., 2002). The human body contains more than 300 pairs of muscles that must each be innervated by their own motor neuron pool, with each pool potentially having its own distinct molecular profile (Kanning et al., 2010).

Individual motor neurons in each motor pool can be further functionally classified into either gamma or alpha motor neurons. Alpha motor neurons (α-MN) are large neurons that innervate extrafusal muscle fibers and control contraction of skeletal muscles. In contrast, gamma motor neurons (γ-MN) are much smaller, innervate intrafusal muscle fibers, and play a proprioceptive role in detecting muscle tension (Kanning et al., 2010). Recent studies have started to identify molecular markers that distinguish these two classes of neurons, with γ-MNs expressing Estrogen-related Receptor Gamma (Esrrg), and both NeuN and Osteopontin (OPN) marking α-MNs (Friese et al., 2009; Misawa et al., 2012). However, markers thus far identified only distinguish between alpha and gamma motor neurons postnatally, and it is not yet known whether these neuronal subtypes are specified embryonically, or if postnatal interaction with 20 muscle targets is required for their specification. Finally, α-MNs are sub-divided into motor neurons that innervate either fast- or slow-twitch muscle, and a comprehensive gene expression analysis recently identified Chondrolectin (Chodl) and Paired-like Homeodomain Transcription

Factor 2 (Pitx2) as novel markers for fast- twitch motor neurons (Enjin et al., 2010).

Hox genes control motor neuron columnar and pool identity

As mentioned earlier in the chapter, extrinsic signals pattern the neural tube along the

RC axis, resulting in collinear expression of Hox genes along the rostral caudal axis. The Hox genes, in turn specify the motor neuron columns and pools that are generated at each level of spinal cord. While the MMC column is insensitive to Hox gene expression and spans the entire

RC axis of the spinal cord, the HMC and LMC columns are only present at precise levels, and their specification is controlled by Hox gene expression. At brachial and lumbar levels of spinal cord, Hoxc6 and Hoxd10 induce the expression of Foxp1 and specify LMC cell fate. At thoracic levels of spinal cord, Hoxc9 represses Foxp1, resulting in motor neurons adopting HMC cell fate

(Fig 1.4) (Dasen et al., 2008).

Hox proteins also control the diversification of LMC motor neurons into individual motor pools that innervate specific limb muscles. Within the Hoxc6 domain of brachial spinal cord, nascent motor neurons initially express a broad range of additional Hox paralogs. Similar to the

Class I and Class II transcription factors during DV patterning, Hox paralogs exhibit cross- repressive interactions between each other, and resolution of these cross-repressive interactions results in a specific motor neuron pool identity (Dasen et al., 2005).

Together, these results have led to a model of motor neuron subtype acquisition centered around Hox genes. In this model, extrinsic factors first specify RC identity and confer an 21 initially unstable Hox combinatorial profile. Sequential rounds of Hox gene resolution progressively define column and pool identity (Dasen and Jessell, 2009). This model offers an elegant explaination for how postmitotic resolution of Hox genes can diversify motor neuron cell fate. However, several questions remain. First, expression of the ETS transcription factor Pea3, which marks the several motor pools, requires Glial Derived Neurotropic Factor (GDNF) from limb tissue (Haase et al., 2002; Lin et al., 1998), suggesting that some aspects of motor pool identity might require additional extrinsic signals. More importantly, how motor neurons select

MMC versus HMC, LMC, or PGC identity within a given segment of spinal cord cannot be explained by this Hox-based model.

Regulation of motor neuron columnar identity

Two recent studies have provided some insight into how motor neuron column identity may be selected within a given segment. In the first study, Agalliu et al. noticed that non- canonical Wnts were highly expressed in the ventral-most region of the spinal cord. In chick electroporation experiments, overexpression of non-canonical Wnt5a and Wnt4 promotes MMC over HMC fate in thoracic spinal cord. These observations support the idea that motor neurons generated in a more ventral position are exposed to higher levels of non-canonical Wnt signaling, which in turn promotes acquisition of MMC over HMC fate. However, while mouse mutants retaining only one copy of Wnt5a, Wn5b, or Wnt4 generate significantly reduced numbers of

MMC motor neurons, MMC motor neurons are not completely ablated, indicating that one copy of Wnt is sufficient to promote MMC identity, or that other currently unknown pathways may be involved (Agalliu et al., 2009). 22

In the second study, Sabharwal et al. showed that motor neurons express the six transmembrane protein GDE2 and that GDE2-/- mice are deficient in neurogenesis and generate fewer motor neurons. As motor neuron survival is not affected, they proposed that GDE2 mediates a novel positive feedback mechanism through which motor neurons induce additional pMNs to differentiate. Interestingly, while HMC and LMC motor neuron numbers in GDE2-/- mice are decreased, MMC motor neurons are spared. Autoradiography birthdating experiments have shown that motor neurons are born in an inside-out fashion with MMC motor neurons being born first (Fujita, 1964; Langman and Haden, 1970; Price and Briscoe, 2004). This led

Sabharwal et al. to conclude that in the GDE2-/-, MMC motor neurons are born normally, but disruption of GDE2 feedback signaling impairs later rounds of differentiation that generate HMC and LMC motor neurons (Sabharwal et al., 2011). This study identified a novel feedback mechanism through which temporal generation of motor neurons is regulated, however, the actual inductive signals that specify MMC, HMC and LMC cell fates remain to be determined.

Generation of neuronal diversity by Notch signaling

Two critical questions remain in regards to neural diversity in the pMN domain. First, what controls the temporal switch of motor neuron to OPC potential of Olig2+ progenitors, and second, what pathways control segregation of motor neurons into distinct columns within a given spinal cord segment? The Notch signaling pathway was original discovered almost 100 years ago as a gene necessary for proper wing development in Drosphila melanogaster (Moohr, 1919) and has been implicated as a critical player in many biological processes (Greenwald, 2012). As one of the primary methods for relaying information between neighboring cells, Notch plays a critical role in cell fate decisions throughout development. Thus, Notch signaling serves as a 23 potential candidate for regulating cell fate decisions in the pMN domain of the ventral spinal cord.

Notch receptors, ligands, and effectors

The Notch receptor and its ligands are an evolutionarily conserved class of large single- pass transmembrane receptors. While Drosophila has only one type of Notch receptor and two ligands, Delta and Jagged, mammals have four Notch receptor paralogs (Notch 1-4), three Delta ligands (Delta 1,3,4) and two Jagged ligands (Jag 1,2) (Gazave et al., 2009). Upon interaction between the extracellular domain of Notch with ligand from an adjacent cell, a series of cleavage events ultimately leads to the release of the Notch intracellular domain (NICD) from the plasma membrane (Kopan and Ilagan, 2009). The newly freed NICD translocates to the where it forms a complex with DNA binding protein CSL, and the co-activator Mastermind1 (Maml1)

(Nam et al., 2006; Wilson and Kovall, 2006). Together, this complex binds to DNA and recruits transcriptional activators to induce the expression of target genes (Fig. 1.5A) (Petcherski and

Kimble, 2000; Wu et al., 2000). LOF in Maml1, CSL, or the Notch targets Hes1 and

Hes5 leads to common caused by loss of Notch signaling (Yoon and Gaiano, 2005).

Two key processes that generate intra-domain neural diversity that are directly controlled by

Notch signaling are the acquisition of alternative cell fates during asymmetric division (Fig.

1.5C) and lateral inhibition (Fig.1.5B).

Notch-mediated lateral inhibition

One of the primary ways Notch signaling contributes to neuronal diversity is through its ability to diversify a field of uniform progenitors through the process of lateral inhibition. The first evidence for specification of neuronal precursors via Notch-mediated lateral inhibition was 24 in the Drosophila peripheral nervous system (PNS) (Simpson, 1990). Sensory precursor

(SOP) cells that are destined to become sensory bristles develop from a cluster of uniform neuroectodermal progenitors. Each of these progenitors has the ability to differentiate into an

SOP cell, but Notch signaling actively inhibits their differentiation. During development, a single progenitor will stochastically escape Notch signaling and differentiate into an SOP cell.

The stochastic process is stabilized by lateral inhibition, as a differentiating SOP cell upregulates the expression of Delta ligand, thereby inducing Notch signaling in neighboring cells and inhibiting them from differentiating into additional SOP cells (Fig. 1.5B) (Heitzler and Simpson,

1991).

The primary downstream effectors of Notch-mediated lateral inhibition are two classes of bHLH transcription factors (Greenwald, 1998). The Achaete-scute and Atonal families of bHLH transcription factors are well known for their ability to specify neuronal identity and differentiation. During lateral inhibition, Notch activates another bHLH transcription factor, enhancer of split (Espl), which inhibits neuronal differentiation by repressing transcription of

Achaete-scute and Atonal transcription factors (Bailey and Posakony, 1995; Lecourtois and

Schweisguth, 1995; Schweisguth, 1995; Schweisguth and Posakony, 1994). However, as a cell escapes Notch signaling, it upregulates pro-neural bHLH transcription factors, which promote neuronal differentiation and activate transcription of Delta, thereby exerting negative feedback

Notch signaling on surrounding cells (Heitzler et al., 1996).

Lateral inhibition in vertebrates CNS

There is significant evidence that Notch plays a similar role in controlling neurogenesis via lateral inhibition in the vertebrate CNS. The Notch receptors and vertebrate homologues of 25

Espl, Hes1 and Hes5 are expressed in neurogenic regions throughout the CNS (Lindsell et al.,

1996). Notch1-/- mutants lose Hes5 expression, exhibit increased expression of pro-neurogenic bHLH transcription factors, and undergo precocious neurogenesis throughout the CNS (Yoon and Gaiano, 2005). Precocious neurogenesis results in an overall decrease in neurons generated as a result of progenitor depletion at early stages (Yoon et al., 2004). Together, these results are consistent with the idea that Notch signaling inhibits differentiation and promotes maintenance of progenitor identity.

In addition to directly controlling neuron versus progenitor cell fate, Notch control of progenitor maintenance indirectly leads to control of cell fates generated during temporally patterned neurogenesis. Multiples studies have demonstrated that ectopic Notch expression maintains cells as retinal progenitors and prevents neuronal differentiation; conversely, inhibition of Notch signaling leads to an increase in neurogenesis (Henrique et al., 1997; Riesenberg et al.,

2009). More specifically, inhibiting Notch signaling at various developmental times points during retinal development leads to increased generation of neurons normally born at those time points (Nelson et al., 2007). Interestingly, Notch inhibition in chick retinas leads to an increase exclusively in retinal ganglion cell (RGC) numbers (Austin et al., 1995). Thus, Notch controls cell identity by forcing progenitors to differentiate into temporally relevant neuronal cell types but may also have a role for specifically inhibiting RGC cell identity.

The role of Notch signaling in controlling the neuronal to glial cell fate switch

In situ hybridiziation and immunohistochemical analysis have demonstrated that the

Notch1 (Fig. 1.6A), Notch2, and Notch3 receptors and the Notch ligand Dll1 (Fig. 1.6C) are all expressed in the pMN domain, suggesting that Notch plays a role in the control of motor neuron 26 diversity or motor neuron to OPC cell fate switch (Genethliou et al., 2009; Lindsell et al., 1996;

Marklund et al., 2010; Yang et al., 2006). However, while several studies have attempted to assess the role of Notch signaling in the ventral spinal cord, their results are unclear and often contradictory, and hence a clear role for Notch has not been defined. Unfortunately, conditional knockouts of Notch1 exhibit neural tube fusion and delayed generation of the pMN domain, rendering analysis of the specific role of Notch in the pMN domain difficult in these mutants

(Yang et al., 2006). Instead, several groups have turned to studies in zebrafish and chicken embryos to assess the role of Notch signaling in controlling the temporal switch in motor neuron and OPC differentiation.

Zebrafish GOF and LOF studies were the first to demonstrate that Notch signaling may control neuronal versus glial differentiation in the spinal cord. Zebrafish Delta mutants exhibit increased neurogenesis and a complete loss of oligodendrocytes, while heat shock-inducible expression of intracellular Notch blocks neurogenesis and leads to dramatic increases in the number of OPCs generated during OPC differentiation (Appel et al., 2001; Park and Appel,

2003). These results are consistent with a role for Notch in inhibiting neuronal differentiation and preserving progenitors for later OPC differentiation, but the lack of good antibodies marking pMN cell types in zebrafish prevented a more detailed analysis of pMN cell fates in this context.

Overexpression of Jag2 by electroporation in chick neural tube or its knockdown using siRNAs further supports the role of Notch signaling in the pMN domain. Jag2 knockdown leads to precocious generation of motor neurons and depletion of pMNs, while Jag2 overexpression inhibits motor neuron differentiation and increases the number of undifferentiated Olig2+ cells present in the pMN domain (Rabadán et al., 2012). These results are consistent with a role for 27

Notch in lateral inhibition of neurogenic differentiation, but manipulation of Notch ligand instead of intrinsic members of the Notch signaling pathway makes it difficult to determine if these results are specifically due to disruption of Notch signaling in pMNs. This consideration is especially relevant considering previous reports that Jag2 is only expressed in postmitotic motor neurons that have migrated to the ventral horn and that pMNs express fringe proteins that insulate them from the Serrate family (Jag1 and Jag2) of Notch ligands (Marklund et al., 2010;

Ramos et al., 2010; Valsecchi et al., 1997). These points, combined with earlier studies showing dramatic effects of Notch signaling in spinal cord development, have raised the question of whether Jag2 manipulation phenotypes may be the result of off-target effects that disrupt proper formation of the pMN domain.

Further complicating the issue is a recent study on the role of Notch signaling during adult zebrafish spinal cord regeneration. Zebrafish undergo limited motor neuron regeneration after spinal cord transection. However, inhibition of Notch signaling by treatment with the gamma secretase inhibitor DAPT leads to a dramatic increase in the number of regenerated motor neurons. Conversely, heat shock-inducible NICD expression reduced the numbers of motor neurons generated after injury. These findings are seemingly consistent with previous results but were later found to occur through an entirely different mechanism. In this system,

Notch inhibition leads to an increase in the number of cycling pMNs, while Notch activation reduces the number of cycling pMNs. Thus, in an adult regeneration context, Notch signaling inhibits proliferation of pMNs, and increases in motor neuron numbers after Notch inhibition are due to the increased rate of pMN proliferation (Dias et al., 2012).

Notch selects alternative cell fates 28

Reminiscent of SOP development, Drosophila neuroblasts in the CNS are selected from clusters of neuroepithelial progenitors by lateral inhibition (Campos-Ortega, 1993; Doe et al.,

1985). However, while the asymmetric division generating a GMC cell occurs through Notch- independent mechanism, the asymmetric division of GMC cells into two distinct daughter cells depends on cell-to-cell communication via Notch signaling. Detailed analyses of cell fates produced by various neuroblast lineages in Notch mutants have revealed that disruption of Notch signaling results in symmetric division into only one of the daughter cell fates (Buescher et al.,

1998; Guo et al., 1996; Lundell et al., 2003; Skeath and Doe, 1998; Spana and Doe, 1996). In isolated GMCs cultured in vitro, both GMC daughters differentiate into the Notch-independent cell fate, indicating that Delta/Notch interactions between the two daughter cells are not sufficient to activate Notch, and that Notch signaling must be activated by the embryonic environment (Spana and Doe, 1996). Asymmetry in Notch signaling is generated by unequal distribution of the Notch inhibitor Numb during GMC division (Frise et al., 1996). Numb mutants also display lack of asymmetry in GMC divisions, but GMC daughter cells of these mutants differentiate into the opposite neuronal fate from that of Notch mutants.

Recent studies in the p2 domain of the ventral spinal cord suggest that Notch control of binary cell fates are an evolutionarily conserved mechanism that also operates in vertebrates.

Reducing Notch signaling by mutating the gamma secretase complex member Presenelin1

(Psen1) or by conditionally knocking out Dll1 have demonstrated that Notch signaling promotes v2a over v2b cell fates in the p2 progenitor domain of the spinal cord (Del Barrio et al., 2007;

Peng et al., 2007; Rocha et al., 2009). However, conditional Notch mutants die before establishment of motor neuron subtypes; thus, it has not been possible to determine whether or not Notch control of neuronal subtype identity also extends to motor neurons (Marklund et al., 29

2010). It was suggested that the change in motor neuron columnar subtypes in GDE2-/- mice was the result of increased Notch signaling, but a careful analysis of motor neuron subtype markers after specifically manipulating the Notch pathway was not performed (Sabharwal et al., 2011).

Thus, whether or not Notch plays a role in specifying motor neuron columnar identity within a particular segment of spinal cord remains to be determined.

Conclusion

Multiple mechanisms spanning from large scale regional patterning to binary fate decisions between pairs of postmitotic neurons were co-opted during evolution to ensure generation of sufficient neuronal diversity necessary for the formation of the functional mammalian nervous system. In the ventral spinal cord, DV patterning specifies the pMN domain, which sequentially gives rise to motor neurons and oligodendrocytes. Extrinsic signals along the RC axis of the spinal cord confer a unique Hox code on motor neurons that is resolved into columnar and pool cell fates appropriate for the given level of spinal cord. However, several questions still remain. First, what are the lineage relationships between motor neurons and glia within the pMN domain, and what are the signals that control temporal generation of these cell types? Secondly, within a given RC segment of spinal cord, what signals control motor neuron columnar identity?

Significant evidence points to Notch as a mediator of motor neuron versus OPC differentiation in the ventral spinal cord. However, the use of overexpression studies in broad domains of spinal cord combined with usage of Notch ligand manipulators has made it difficult to assess whether these results are specifically due to manipulation of Notch signaling in pMNs.

Furthermore, the varying results in different model systems raises the question of whether Notch 30 is exerting its control on motor neuron versus OPC cell fate through lateral inhibition, regulation of progenitor proliferation, or perhaps direct specification of OPC identity.

Despite the clear role for Notch in controlling alternative cell fates in Drosophila lineages, a role for Notch signaling in specifying motor neuron subtype identity has not been established. Emerging evidence demonstrating Notch control of subtype fate in the p2 progenitor domain suggests that Notch signaling may also control motor neuron subtype identity. A role for Notch signaling in controlling intra-segmental acquisition of motor column identity would fill a gap in our current knowledge of how motor column identity is established.

Thus, the focus of this dissertation will be to address these questions and to determine the role of

Notch signaling in both general motor neuron differentiation and in the specification of motor neuron columnar cell fate. 31

A B

Class 1

Class 1

Figure 1.1

32

Figure 1.1. SHh patterning of the ventral spinal cord into distinct domains

Schematic of dorsal-ventral patterning of the ventral spinal cord, adapted from Ribes and

Briscoe, 2009. A. SHh secreted from cells in the notochord and floor plate of the ventral spinal cord diffuse dorsally, generating a SHh gradient that is high ventrally and low dorsally. The SHh gradient patterns the ventral spinal cord in to 5 distinct progenitor domains that give rise to distinct types of neurons. B. Each progenitor domain is characterized by the expression of different combinations of SHh sensitive transcription factors. Class II transcription factors are induced at varying levels of SHh signaling and repress the expression of Class II transcription factors. Class I transcription factors are either repressed or insensitive to SHh signaling, and in turn repress Class II transcription factors. Cross-repressive interactions result in resolution of the spinal cord into distinct domains with sharp boundaries. 33

A E9.5 Neurogenesis E14.5 Gliogenesis

Olig2 Olig2/Nkx2.2

B Temporal progression MN Oligodendrocyte

Olig2 Olig2 Olig2 Nkx2.2 ?

Time C Mixed Progenitors

MN Olig2

Olig2 Oligodendrocyte Nkx2.2 Olig2 ? Astrocyte

Time Figure 1.2

34

Figure 1.2. The pMN domain gives rise to motor neurons and glia

A. During early stages of murine development, from E9.5-E12.5, Olig2 progenitors in the pMN domain differentiate into spinal motor neurons. Later in development, Olig2 progenitors adopt a gliogenic identity and differentiate into astrocytes and oligodendrocytes. While all three cell types are generated by Olig2+ cells, the lineage relationships between them are not clear. B.

Temporal progression model of pMN development. In this model, a single Olig2 progenitor progresses through competency states and sequentially gives rise to motor neurons and glial cells. C. In the mixed progenitor model, the early pMN domain harbors a heterogeneous population of Olig2+ progenitors that separately give to motor neurons, oligodendroctyes and astrocytes at different developmental times. 35

A B Cervical/Thoracic Brachial/Lumbar

Axial Axial

LMC HMC

MMC MMC

MMC Lhx3+ MMC Lhx3+/Foxp1-

HMC Lhx3- Hypaxial LMC Lhx3-/Foxp1+ Limb

Figure 1.3

36

Figure 1.3. Motor neurons are organized into columns with distinct molecular profiles

Spinal motor neurons are organized into three distinct columns with distinct cell body settling positions, axonal trajectories, and molecular profiles. A. Cervical and thoracic levels of spinal cord are populated by MMC and HMC motor neurons. The MMC motor neuron column is located medially, expresses Lhx3, and projects its axons dorsally towards the axial muscles of the back. HMC motor neurons are thought to reside lateral to the MMC motor neuron column, do not express Lhx3, and project their axons ventrally towards hypaxial muscle. B. At limb levels of spinal cord, the HMC motor neuron column is largely reduced and is replaced by

Foxp1+ LMC motor neurons that project their axons towards the limbs. 37

A GDF11 FGF RA B Hox6 Hox9 Hox10 C

Figure 1.4

38

Figure 1.4. Hox genes control motor neuron diversity along the rostral-caudal axis of the developing spinal cord

Schematic of rostral-caudal patterning of the spinal cord taken from Dasen and Jessell, 2009. A.

Extrinsic signals (RA, FGF, and GDF11) confer a positional Hox identity to developing motor neurons. B. Brachial spinal cord expresses Hoxc6, thoracic spinal cord expresses Hoxc9, and lumbar spinal cord expresses Hoxd10. In a similar manner to SHh transcription sensitive transcription factors during DV patterning, cross-repressive interactions between the Hox genes resolve the spinal cord into distinct rostral-caudal domains with sharp boundaries. C. The MMC is insensitive to Hox gene expression, and spans the entire spinal cord. Hoxc6 and Hoxd10 promote the expression of Foxp1 and specify LMC motor neurons at brachial and lumbar levels of spinal cord. In thoracic spinal cord, Hoxc9 represses Foxp1 and specifies HMC motor neurons at thoracic levels of spinal cord. 39

A B Lateral Inhibition

Delta Notch

NICD Hes5 Ngn2 Dll1 C Selection of Alternative Cell Fate

CSL Notch

Figure 1.5

40

Figure 1.5. The Notch signaling pathways contributes to neuronal diversity via lateral inhibition and selection of alternative cell fates.

A. Schematic of Notch signaling taken from Ersvaer and Bruserud, 2011. 1.) Interaction of

Notch receptor with ligand from a neighboring, cell initiates a cascade of events that lead to transcription of Notch targets. 2.) ADAM-family protease cleaves the Notch receptor at the S2 extracellular juxtamembrane position, releasing the Notch extra-cellular domain. 3.)

Subsequently, γ-secretase complex cleaves the Notch receptor at the S3 intracellular juxtamembrane position, releasing the Notch intracellular domain (NICD) from the plasma membrane. 4.) The free NICD translocates to the nucleus where it forms a complex with DNA binding protein CSL, Mastermind1, and Co-activator to activate transcription of Notch targets.

B. Notch diversifies a uniform field of progenitors via lateral inhibition. As a cell differentiates into a neuron it upregulates expression of proneural bHLH transcription factor Ngn2. Ngn2 promotes neuronal differentiation, but also induces expression of Notch ligand Dll1, resulting in increased Notch signaling in surrounding cells, and inhibiting their differentiation. C. Notch signaling promotes asymmetric differentiation of progenitors into Notch dependent and Notch independent daughter cell fates.

41

E11.5 Spinal Cord Notch 1 Hes5 A B

pMN pMN

Stg 17 Spinal Cord Dll1

pMN

C Figure 1.6

42

Figure 1.6. Notch signaling is active in the ventricular zone of the spinal cord

In situs probing the expression of Notch effectors in developing spinal cord. Red brackets indicate the approximate location of the pMN domain. A-B. In situ hybridization of Notch1 and

Hes5 in E10.5 mouse spinal cord, taken from Yang and Shen, 2006. Notch1 and Hes5 are expressed in the ventricular zone throughout the spinal cord, indicating that Notch signaling is active in spinal progenitor domains . C. In situ for Dll1 in ~Stg 17 chick spinal cord taken from

Marklund and Ericson, 2010. Dll1 is expressed in alternating regions along the dorsal-ventral axis of the spinal cord. The pMN domain expresses high levels of Dll1.

43

Chapter 2: Notch controls the motor neuron to glial cell fate switch in the pMN domain

Introduction

Several studies have attempted to define the role of Notch signaling in controlling the temporal cell fate switch from motor neurons to oligodendrocytes in the pMN domain (Appel et al., 2001; Dias et al., 2012; Park and Appel, 2003; Rabadán et al., 2012). Studies using electroporation to overexpress or knockdown Jag2 in chick spinal cord most directly addressed this question. These experiments showed that constitutive Notch activation inhibits motor neuron differentiation and increases progenitor numbers during spinal cord development.

Conversely, siRNA knockdown of Jag2 leads to precocious motor neuron generation and reduces the number of Olig2 progenitors in the ventricular zone of the spinal cord. These findings suggest that Notch signaling laterally inhibits motor neuron differentiation and preserves progenitors for later OPC differentiation. Interestingly, Jag2 knockdown also leads to precocious oligodendrocyte differentiation, suggesting that Notch plays an additional role in maintaining progenitors during oligodenrdrocyte genesis (Rabadán et al., 2012).

A major caveat of this study is that manipulation of Jag2 expression targets Notch ligands, thereby alterating Notch signaling in neighboring cells, which are not targeted when electroporation efficiency is low. The results observed in this study are difficult to interpret, as the greatest changes occur not cell-autonomously but rather in these non-electroporated cells.

Thus, there is a need for manipulations that distinguish between cells with activated or inactivated Notch signaling.

In this chapter, I performed a series of experiments to clarify the role of Notch signaling in controlling the motor neuron to glial cell fate switch in the ventral spinal cord. In order to 44 analyze the effects of Notch manipulation in targeted pMN progenitors only, I utilized in ovo electroporation to deliver cell intrinsic inhibitors or activators of the Notch signaling pathway to chick spinal cord. These experiments demonstrate that Notch controls motor neuron differentiation versus progenitor maintenance via lateral inhibition of Olig2+ pMN progenitors. I then show that an in vitro embryonic stem cell (ESC) to motor neuron differentiation protocol recapitulates the motor neuron to glial progenitor cell fate switch. Using this protocol in conjunction with inducible stem cell technology, I show that Notch inhibition or activation in a mostly homogenous population of Olig2+ pMNs is capable of biasing the differentiation to either motor neuron or glial cell fate. Finally, we use the ESC system to study the mechanistic details of this cell fate change and confirm that Notch indeed regulates motor neuron differentiation via lateral inhibition.

Results

Cell-autonomous activation of Notch signaling prevents pMN differentiation

To examine the effect of Notch signaling on motor neuron differentiation, I electroporated a plasmid encoding the intracellular domain of Notch receptor (NICD) into

Hamburger-Hamilton (HH) stage 11-14 chick spinal cord, a stage prior to the onset of motor neuron genesis (Fig. 2.1A). After two days, the majority of the NICD electroporated cells are located medially within the progenitor domain (Fig 2.1B). In contrast, control cells electroporated with a plasmid expressing histone H1B-eGFP (H1-eGFP) populated both the medial progenitor zone and the lateral zone containing postmitotic neurons (Fig 2.1B). Spinal cord sections immunostained with antibodies against pMN marker Olig2 (Novitch et al., 2001;

Zhou et al., 2001) and postmitotic motor neuron marker Hb9 (Arber et al., 1999; Tanabe et al., 45

1998; William et al., 2003) revealed that NICD expressing cells fail to differentiate into Hb9+ motor neurons, remain within the pMN domain, and retain expression of Olig2 (Fig 2.1C and D).

This leads to an expansion of the Olig2+ progenitor domain on the electroporated side of neural tube. Together, these findings indicate that activation of Notch signaling prevents terminal differentiation of motor neuron progenitors.

Cell-autonomous inhibition of Notch signaling is incompatible with pMN identity

Since activation of Notch signaling prevents motor neuron differentiation, I next examined whether inactivation would have the opposite effect. To block Notch signaling in a cell-autonomous manner, I electroporated a plasmid driving the expression of a dominant- negative form of Maml1 fused to eGFP (DnMaml1-eGFP) (Maillard et al., 2004; Wu et al.,

2000) and an H1B-eGFP control plasmid into stage 11-14 chick spinal cord (Fig 2.1A). Cells expressing DnMaml1-eGFP show a striking difference in position and molecular identity, compared to control or NICD-electroporated cells at 48 hours post electroporation. The

DnMaml1-eGFP-expressing cells are predominantly located in the lateral aspect of the spinal cord, indicating that they exited the progenitor zone and populated the postmitotic mantel region of the developing neural tube (Fig 2.1B). Accordingly, within the ventral horn of the spinal cord, most DnMaml1-GFP+ cells lacked expression of pMN marker Olig2 and expressed postmitotic motor neuron marker Hb9 (Fig. 2.1C and D). These results reveal that inhibition of Notch signaling is incompatible with pMN identity and directs them to differentiate into postmitotic

Hb9+ motor neurons.

In vitro ESC differentiation recapitulates the motor neuron to glial cell fate switch 46

The observation that Notch signaling plays a key role in regulating the transition from pMNs to postmitotic motor neurons prompted me to examine whether the same process can be modeled in vitro during directed differentiation of embryonic stem cells (ESCs) to spinal motor neurons. ESCs grown as embryoid bodies under differentiation conditions can be efficiently patterned on day 2 of differentiation with retinoic acid (RA) and agonist (SHh) to become pMNs (Fig. 2.2G) (Wichterle et al., 2002). The in vitro motor neuron differentiation protocol follows a synchronous progression through stages that closely parallel in vivo development. At day 4 of the protocol ~80% of total cells will express pMN marker Olig2 (Fig.

2.2A). While OPC marker Nkx2.2 is completely excluded from Olig2+ cells at this stage, a subset of Olig2+ cells co-express Ngn2, indicating that they are indeed pMNs with the potential to differentiate into motor neurons (Fig. 2.2B-C) (Lee et al., 2005).

Although ~80% of cells express Olig2 by day 4, only ~40% of cells progress to become postmitotic motor neurons expressing Hb9 by day 6 of differentiation (Fig. 2.2D).

Immunostaining revealed that numerous Olig2+ cells are present as small clusters in the embryoid bodies, indicating that a subset of pMNs failed to differentiate into postmitotic motor neurons (Fig. 2.2D). Cells positive for Olig2 on day 6 express proliferative marker Ki67 but, in contrast to pMNs, do not express Ngn2. Instead, most Olig2+ cells co-expressed glial progenitor markers Sox9 and Sox10, and a subset express oligodendrocyte progenitor marker Nkx2.2 (Fig.

2.2E-F, 2.3B) (Liu et al., 2007; Stolt et al., 2003; Zhou et al., 2001). Together these results indicate that, similar to the developing spinal cord, a subset of Olig2+ cells remains in embryoid bodies after motor neuron genesis and acquires OPC or glial progenitor identity (Zhou et al.,

2001). 47

Activation of Notch signaling inhibits motor neuron differentiation

Given the similarity between the behavior of Olig2+ cells in vitro and in vivo, I was keen to examine whether manipulation of Notch signaling in pMNs on day 4 of differentiation may promote more homogenous differentiation of pMNs into either motor neurons or glial progenitors. To test this hypothesis, I generated an inducible ESC line carrying a V5-tagged

NICD construct under the control of a doxycycline-inducible promoter (Fig. 2.3A). Addition of doxycycline on day 3 of differentiation results in robust expression of NICD-V5 in the majority of cells in day 4 embryoid bodies and leads to a greater than twofold increase in the expression of the direct Notch target Hes5 (Fig 2.3B-C).

Activation of Notch signaling has profound effects on motor neuron differentiation.

Motor neuron yield at the end of differentiation decreases from 35% of cells to less than 3%, and the percentage of Olig2+ cells dramatically increases from 17% of cells to over 86% (Fig. 2.3E).

While NICD expression marked by V5 immunostaining closely correlates with Olig2 expression, the few Hb9 motor neurons that did differentiate are V5 negative, indicating that they have escaped NICD-V5 induction (Fig 2.3D). Interestingly, induced Olig2+ cells take on radial glial- like morphology and have elongated nuclei with the elongated axis perpendicular to the exterior perimeter of the embryoid body (Fig. 2.4A). Co-staining of Olig2 and nuclear marker Topro revealed that Olig2 cells are arranged as neuroepithelial sheets around the perimeter of the embryoid body and that the center of embryoid bodies is largely devoid of cells (Fig. 2.4A).

Motor neurons that manage to escape NICD induction are positioned immediately adjacent to the sheets of Olig2 cells on the inside of the embryoid bodies (Fig 2.3D). 48

I next asked whether NICD-induced Olig2 cells are able to adopt glial identity or if they remain as pMNs that can still give rise to motor neurons after release of ectopic Notch activation.

To define the cell fate status of these NICD-induced Olig2 cells, I immunostained for glial markers Sox9, Sox10, and Nkx2.2. Both control and Notch-induced Olig2+ cells co-express

Sox9 and Sox10, but only Notch-induced Olig2+ cells are completely devoid of Nkx2.2 (Fig.

2.4B). The expression of Sox9 and Sox10 marked these Olig2+ cells as likely glial progenitors incapable of differentiating into motor neurons. Previous reports have demonstrated that Nkx2.2 is downstream of Sox10 during OPC specification, suggesting that Notch inhibits OPC differentiation and maturation by repressing Nkx2.2 (Liu et al., 2007). Alternatively, the loss of

Nkx2.2 may indicate that NICD induction directs Olig2+ progenitors into the astrocyte lineage.

Inhibition of Notch signaling promotes motor neuron differentiation

Given the ability of Notch signaling to abolish motor neuron differentiation, I was interested in testing whether inhibition of Notch signaling in day 4 pMNs promotes more homogeneous and efficient differentiation of postmitotic motor neurons. As we did with NICD, we generated an inducible ESC line carrying the DnMaml1-eGFP construct under the control of a doxycycline-inducible promoter (Fig. 2.5A). Addition of doxycycline on day 3 of differentiation results in robust expression of the transgene in the majority of cells in day 4 embryoid bodies (Fig. 2.5B). Quantitative real-time PCR (qRT-PCR) revealed that expression of

Notch target gene Hes5 is reduced by 72% after DnMaml1-eGFP induction, confirming that

DnMaml1-eGFP efficiently inhibits Notch signaling (Fig. 2.5C). Expression of DnMaml1-eGFP in Olig2+ pMNs strongly enhances motor neuron differentiation. Induced embryoid bodies exhibit a striking reduction in Olig2+ cells from 17% to less than 3% at the end of differentiation 49 on day 6 (Fig. 2.5 D-E), with a concomitant increase in the number of motor neurons produced.

Approximately 36% of cells differentiate into Hb9+ motor neurons under control conditions, while DnMaml1-eGFP inhibition of Notch signaling results in greater than 60% of cells becoming Hb9+ motor neurons (Fig. 2.5D-E).

DAPT treatment increases motor neuron yield

To determine whether inhibition of Notch signaling can be used to increase efficiency of motor neuron differentiation in other cell lines, we examined the effects of treating differentiating cells with DAPT, a small molecule that inhibits Notch signaling by interfering with the formation of the gamma-secretase complex that is necessary for cleaving the Notch receptor (Geling et al., 2002). To test the ability of DAPT to inhibit Notch signaling and increase motor neuron yield, we treated pMNs generated from an Hb9-GFP transgenic ESC line with DAPT. Twelve hours of 5µM DAPT treatment of pMNs leads to a 77% decrease in Hes5 expression, similar to DnMaml1-eGFP induction (Fig. 2.6A). DAPT-mediated Notch inhibition leads to a complete depletion of Olig2+ cells, which constitute less than 1% of all cells at the final stage of differentiation (Fig. 2.6B-C). Quantification of motor neurons after DAPT treatment using flow cytometry to measure the number of HB9-GFP positive cells revealed that 68% of cells are HB9-GFP positive motor neurons, compared to 49% in the untreated control cells (Fig.

2.6D). Thus, inclusion of DAPT treatment during in vitro ESC to motor neuron differentiation significantly increases the efficiency of motor neuron production.

Inhibition of Notch signaling promotes pMN differentiation into motor neurons by disrupting lateral inhibition 50

The ability to inhibit Notch signaling in a homogenous population of pMN progenitors provided me a method to address the mechanisms through which Notch inhibition increases motor neuron production. To test the hypothesis that Notch inhibition positively promotes progenitors to exit cell cycle, I dissociated DAPT-treated and untreated embryoid bodies at the end of differentiation and counted the total number of cells. DAPT-treated cultures produced

41% fewer cells than untreated cultures (Fig. 2.7C). Consistent with this result, DAPT treatment leads to a reduction in Ki67+ cycling cells from 25.7% to 0.7% of all cells (Fig 2.7 A-B). This loss of progenitors indicates that Notch signaling is necessary for maintaining progenitor identity.

One of the primary mechanisms through which lateral inhibition is executed is Hes- mediated inhibition of proneural genes, including Ngn2 (Shimojo et al., 2011). I analyzed Ngn2 expression after 12 hours of DAPT treatment and found that Ngn2 expression is increased greater than threefold and that the percent of Olig2+ cells that co-express Ngn2 increases from

40.5% to 57.5% (Fig. 2.7D-F). These findings are consistent with the function of Notch signaling in promoting progenitor maintenance via lateral inhibition and suggest that increased motor neuron differentiation after Notch inhibition is due to disruption of lateral inhibition and subsequent de-repression of Ngn2.

Ngn2 is sufficient to drive motor neuron differentiation

I posited that, if increased motor neuron yield and progenitor depletion is the result of

Ngn2 induction after disruption of lateral inhibition, Ngn2 expression should be sufficient to drive motor neuron differentiation. To test the sufficiency of Ngn2 in driving motor neuron differentiation, I generated an ESC line carrying a doxycycline-inducible Ngn2 transgene. I 51 treated day 3 embryoid bodies with doxycycline to achieve high levels of Ngn2 expression at the day 4 pMN stage (Fig. 2.9A). Similar to both DAPT treatment and DnMaml1-eGFP induction,

Ngn2 induction increases the percentage of motor neurons generated on day 6, from 49% to 65% of cells as quantified by FACS (Fig. 2.9B, C). While not as efficient as DAPT or DnMaml1- eGFP, Ngn2 induction also leads to a significant decrease in the number of Olig2 progenitors remaining at the end of differentiation from 15.8% to 5.5% of total cells (Fig. 2.9B, D).

Discussion

In this chapter, I have presented both in vitro and in vivo evidence demonstrating that

Notch signaling controls the balance of neurogenesis versus gliogenesis in the pMN domain of the ventral spinal cord. The ability to promote or inhibit motor neuron differentiation by inhibiting or activating Notch signaling is reminiscent of overexpression studies performed in zebrafish and chick spinal cord (Appel et al., 2001; Park and Appel, 2003; Rabadán et al., 2012).

However, use of intrinsic manipulators of the Notch pathway in chick spinal cord affords me the additional advantage of being able to follow cell-autonomous effects of Notch signaling. By doing so, I was able to confirm that changes in motor neuron and glial progenitor numbers after

Notch activation and inhibition are due to changes in Notch signaling in pMNs. This concept is further strengthened by the ability to force ESC-derived pMNs to differentiate into a homogenous population of glial progenitors by Notch activation or postmitotic motor neurons by

Notch inhibition (Fig. 2.9B).

Notch controls motor neuron versus glial cell fate via lateral inhibition

The depletion of Ki67+ progenitors and overall reduction of neurogenesis during ESC to motor neuron differentiation suggests that reductions in motor neuron numbers are not due to 52

Notch inhibition of progenitor proliferation, as suggested in a recent zebrafish regeneration study

(Dias et al., 2012). Instead, the loss of cycling progenitors and decreased total cell number suggest that Notch signaling is necessary to maintain progenitor character. Consistent with this idea, Notch induction induces a radial glial-like morphology and organization to Olig2+ progenitors.

Molecular effectors of Notch signaling in pMN domain

Notch-mediated lateral inhibition of neurogenesis is primarily achieved by Hes-mediated repression of proneural bHLH transcription factors. As neurons differentiate, they upregulate

Ngn2, which in turn induces expression of Notch ligand Dll1. Increased Dll1 expression in differentiating neurons promotes Notch signaling in surrounding cells and inhibits them from differentiating. The finding that Ngn2 is upregulated after Notch inhibition and is sufficient to promote pMN differentiation into postmitotic motor neurons suggests that Notch promotes progenitor maintenance via lateral inhibition in the pMN domain and that increases in motor neuron number after DnMaml1-eGFP or DAPT treatment are the consequence of disruption of lateral inhibition.

Interestingly, my results also suggest that Notch signaling plays an instructive role in promoting glial identity. I found that, in addition to promoting progenitor identity, Notch activation is sufficient to upregulate the expression of glial markers Sox9 and Sox10 in pMNs.

This finding suggests that Notch signaling is sufficient for inducing pMNs to progress to a glial identity. However, despite the upregulation of Sox9 and Sox10, prolonged Notch activity completely extinguishes Nkx2.2 expression in prospective glial progenitors. The loss of Nkx2.2 may reflect the inability of pMNs to progress to glial cell fate, but this is unlikely as NICD- 53 induced Olig2+ cells express Sox9 and Sox10. Studies have shown that Nkx2.2 is downstream of Sox10 and is required for proper differentiation and maturation of oligodendrocytes. Thus, repression of Nkx2.2 may be a novel mechanism through which Notch signaling inhibits OPC maturation and differentiation (Fu et al., 2002; Liu et al., 2007; Qi et al., 2001). Alternatively, loss of Nkx2.2 expression may reflect that prolonged Notch signaling has induced glial progenitors to adopt an astrocyte cell fate. Whether loss of Nkx2.2 is mediated by direct repression of Nkx2.2 by Hes genes or attenuation of SHh signaling, which is required for Nkx2.2 expression, is not clear (Dessaud et al., 2007). Future work will need to resolve which of these mechanisms are in play and what the ultimate cell fate adopted by Olig2+/Nkx2.2- cells is.

These findings lead me to propose the following model for Notch-mediated control of the temporal cell fate switch from neurogenesis to gliogenesis (Fig. 2.9B). During motor neuron stages of development, Notch signaling laterally inhibits a subset of progenitors from differentiation into motor neurons, thereby preserving them for later oligodendrocyte genesis.

Concomitantly, Notch signaling promotes glial progenitor identity by inducing the expression of both Sox9 and Sox10. Finally, Notch also inhibits OPC differentiation by repressing Nkx2.2.

Motor neurons and glia are generated from a common progenitor

The lineage relationship between oligodendrocyte and motor neurons has not been satisfactorily resolved. Existence of independent progenitors co-existing side-by-side in a single progenitor domain has recently been demonstrated in the developing neocortex (Franco et al.,

2012). While still indirect, our study provides strong support of the hypothesis that motor neurons and oligodendrocytes are generated from a common Olig2+ progenitor within the pMN domain. First, the ability to convert Olig2+ pMNs into uniform embryoid bodies containing only 54 motor neurons or glial progenitors, simply by manipulating Notch signaling, demonstrates that

Olig2+ pMNs are plastic in their cell fate decisions and can be artificially pushed towards either motor neuron or glial cell fate by inactivation or activation of Notch signaling. This finding is consistent with previous studies demonstrating pMN plasticity by altering neurogenic versus gliogenic potential by manipulating Cyclin D profiles of pMN progenitors (Lukaszewicz and

Anderson, 2011). Second, increased motor neuron differentiation after Notch inhibition depletes

Olig2+ progenitors from the culture resulting in a nearly complete loss of glial progenitors at the end of differentiation, indicating that Notch signaling is required to set aside a subset of pMNs for later gliogenesis. Alternatively, inhibition of Notch signaling could lead to selective death of glial progenitors, but this is unlikely as I did not observe any changes in after DAPT treatment or DnMaml1-eGFP expression. Together, these results strongly suggest that motor neurons and oligodendrocytes share a common pMN lineage. However, definitive proof will require clonal analysis of individual pMNs over time to determine if a single progenitor can give rise to both motor neurons and OPCs in vivo.

DAPT can be used as a general treatment for generating high-yield motor neuron cultures

ESC to motor neuron differentiation serves as the basis for many studies in motor neuron development and disease modeling. Cell lines from different genetic backgrounds can be used to study specific developmental questions or to generate disease-specific motor neurons that can model disease progression in vitro. Indeed, the advent of induced pluripotent stem cell (IPSC) technology has brought forth the ability to generate patient-specific IPSC lines, even when the underlying genetic mutations causing the disease are unknown. 55

One of the major concerns for disease modeling using IPSCs is the relatively low yield of motor neurons in differentiated cultures (Boulting et al., 2011) that might prevent identification of potential disease-relevant phenotypes. The presence of progenitors in cultures also poses a problem, as ongoing proliferation and neurogenesis complicate motor neuron survival analysis and require frequent passaging of cultures. The current strategy for eliminating progenitors and other cells is to use FACS to isolate cells expressing a fluorescent marker under the control of a motor neuron specific promoter. However, such a strategy requires the generation of transgenic lines that necessitates genetic manipulation and long periods of selection. In the case of large- scale high-throughput studies using many different patient-derived stem cell lines, such an approach is costly, time-consuming, and therefore impractical.

The finding that DAPT treatment increases motor neuron yield by 39% and almost completely depletes progenitors from the cultures leads to the attractive possibility of using

DAPT, or other pharmacological methods for inhibiting Notch signaling, to generate more homogenous populations of human motor neurons for the study of normal motor neuron biology and diseases, such as spinal muscular atrophy (SMA) and amyotrophic lateral schlerosis (ALS).

56

A HH Stg 14 D 110% pMN 90% %HB9

Cells Cells in 70% %Olig2/HB9 - + 50% %Olig2 - + 30% - + 10% Electroporated -10% H1-GFP DnMaml1 NICD % of%

B H1-GFP DnMaml1-eGFP NICD

C H1-GFP DnMaml1-eGFP NICD Olig2 Hb9

Figure 2.1 57

Figure 2.1. Electroporation of cell intrinsic activators and inhibitors of Notch signaling

H1B-eGFP, NICD and DnMaml1-eGFP plasmids were electroporated into HH stage 14 chick embryos and analyzed 48 hours later at approximately HH stage 21. A. Schematic of chick electroporation. B. Cross sections of stage 21 chick spinal cords at thoracic levels. H1B-eGFP electroporation has no effect on differentiation and H1B-eGFP expression can be detected broadly across the medial lateral axis of the electroporated side of spinal cord. DnMaml1-eGFP expression is restricted to the marginal zone of the spinal cord, suggesting that DnMaml1-eGFP promotes differentiation into neurons. NICD is restricted to the progenitor domains of the medial aspect of the spinal cord. C. Co-staining of H1B-eGFP, NICD, and DnMaml1-eGFP with Olig2 and Hb9 in the pMN domain of the ventral spinal cord. H1B-eGFP is expressed in

Olig2 progenitors, Hb9 positive postmitotic motor neurons, and Olig2+/Hb9+ differentiating motor neurons. In contrast, DnMaml1-eGFP expression is excluded from Olig2 progenitors and predominantly expressed in Hb9+ postmitotic motor neurons and Olig2+/Hb9+ differentiating motor neurons. NICD expression is completely incompatible with differentiation and is exclusively expressed in Olig2 single positive progenitors. D. Quantification of Olig2, Hb9,

Olig2/HB9 co-expression with electroporated cells. H1-GFP: 18.7% ± 8.45% Olig2+, 16.78 ±

1% Olig2+/Hb9+, 64.5 ± 8.3% Hb9+. DnMaml1-eGFP: 0% ± 0% Olig2 (P=.048), 11.7% ±

3.48% Olig2+/Hb9+ (P=.090 N.S.), 81.2% ± 4.0% Hb9+ (P=.0180). NICD: 98.4% ± 3.2%

(P<.001), 1.6% ± 0% Olig2+/Hb9+ (P=.006), 0.0% ± 0.0% Hb9+ (P=.004).

58

Day 4 Day 6 A D Hb9 Olig2

B E Ngn2 Olig2

C F Nkx2.2 Olig2

G Day 0 Day 2 Day 4 Day 6 ESC NP pMN GP/MN

+RA/Hh

Figure 2.2 59

Figure 2.2. ESC to motor neuron differentiation protocol recapitulates the motor neuron to glial progenitor cell fate switch

In vitro differentiation of ESCs to motor neurons using RA and SHh. A. The majority of cells at day 4 are Olig2 positive, and few motor neurons have been born. B-C. A subset of day 4 Olig2 progenitors co-express Ngn2 (B), but Nkx2.2 is largely absent from Olig2 cells (C). E. By day

6 of differentiation many Olig2+ cells have differentiated into motor neurons, and only a few small clusters of Olig2 cells remain. F-G. Remaining Olig2 cells do not express Ngn2 (E), and many co-express Nkx2.2. G. Summary of in vitro motor neuron differentiation. RA and SHh driven ESC to motor neuron differentiation protocol proceeds through synchronous stages that align with in vivo developmental stages, and recapitulates the motor neuron to glial progenitor cell fate switch.

60

A

teOP Lox NICD V5 PGK Lox Neo

B C Day 4 Control iNICD 2.5 2

1.5 V5 5 Expression 1 Hes Olig2 0.5

0 Relative Relative Control iNICD D E Day 6 Control iNICD 100 *** Control 90

V5 80 iNICD 70 Olig2 60 50 *** 40 30

V5 20 10 Hb9

Olig2, HB9 as % of Total Cells Total of %Olig2, HB9 as 0 %Olig2 %Hb9 Figure 2.3 61

Figure 2.3. Induction of Notch signaling inhibits motor neuron differentiation and promotes glial cell fate

Analysis of day 4 and day 6 embryoid bodies after induction of NICD on day 3 of differentiation.

A. Schematic of NICD-V5 inducible locus. B. Addition of doxycycline on day 3 of differentiation leads to robust induction of NICD-V5 in day 4 embryoid bodies. C. NICD induction results in a 2.14 ± .06 fold induction in Hes5 expression (n=3). D. At the end of differentiation, NICD induced embryoid bodies generate dramatically fewer motor neurons, and significantly more Olig2+ cells. NICD expression detected by V5 immunostaining is completely absent in the few motor neurons that are born. E. Motor neurons are reduced from 35.4 ± 1.6 to

2.7 ± 2.52 percent of total cells (n=3, P<.001). Olig2+ cells increase from 17.2 ± .6 to 86.2 ± 1.6 percent of total cells (n=3, P<.001).

62

A Day 6 NICD + Dox Olig2 Olig2 Topro

B Day 6 Control iNICD Sox10 Olig2 Sox9 Olig2 Nkx2.2 Olig2

Figure 2.4

63

Figure 2.4. NICD induced Olig2 cells are glial progenitors

Characterization of day 6 Olig2+ cells after NICD induction. A. Notch induced Olig2+ cells take on radial glial like morphology and line the perimeter of the embryoid body. Topro staining reveals that the interior of the embryoid body is devoid of cells. B. Both control and NICD induced day 6 Olig2+ cells co-express Sox9 and Sox10, indicating they have adopted a glial cell fate. In addition to Sox9 and Sox10, control Olig2+ cells express Nkx2.2. Nkx2.2 is almost completely absent in NICD induced embryoid bodies. 64

A

teOP Lox DnMaml1 eGFP PGK Lox Neo

B C Day 4

Control iDnMaml1-eGFP 1 0.9 0.8 0.7 GFP 0.6 0.5

Olig2 0.4 0.3 Day 6 0.2 D 0.1 Control iDnMaml1-eGFP Relative Hes5 expression 0 Control iDnMaml1-eGFP

E *** Olig2 70

60 Control

50 iDnMaml1-eGFP

40 Hb9

30 **

20

10 Hb9 Olig2, Hb9 as % of Total Cells Total ofOlig2,% Hb9 as

0

Olig2 %Olig2 %Hb9

Figure 2.5

65

Figure 2.5. DnMaml1-eGFP induction promotes motor neuron differentiation and depletes progenitors

Analysis of day 4 and day 6 embryoid bodies after induction of DnMaml1-eGFP expression on day 3 of differentiation. A. Schematic of DnMaml1-eGFP inducible locus. B. Administration of doxycycline on day 3 of differentiation leads to robust expression of DnMaml1-eGFP expression in day 4 embryoid bodies as detected by GFP immunohistochemistry. C. DnMaml1- eGFP expression potently inhibits Notch signaling and reduces Hes5 expression by 72.2 ± .05 percent (n=3) D. DnMaml1-eGFP leads to a dramatic depletion of Olig2 cells. Concomitantly, motor numbers are significantly increased and are uniformly distributed throughout the embryoid body in place of Olig2 progenitor clusters. E. DnMaml1-eGFP induction reduces the percentage of Olig2 cells from 16.8 ± 2.7 to 2.7 ± .4 percent of total cells (n=3, P=.006). Motor neuron numbers are increased from 36.0 ± 2.5 to 60.0 ± 2.5 percent of total cells (n=3, P<.001).

66

A 1.2

1

0.8

0.6

0.4

0.2 Relative Relative expression Hes5 0 Control DAPT B C Day 4.5 *** Control DAPT 20

15

10

5 Olig2

0 Control DAPT Olig2 as % of Total Cells Total % Olig2 of as D -5 ** 80 70 Hb9 60 50 40 GFP Cells GFP - 30

Hb9 20 %Hb9 10

Olig2 Olig2 0 Control DAPT Figure 2.6

67

Figure 2.6. DAPT treatment effectively inhibits Notch signaling and increases motor neuron yield

Analysis at day 4 and day 6 of differentiation after treating cultures with gamma secretase inhibitor DAPT. A. 12 hours of DAPT treatment early on day 4 of differentiation is sufficient to inhibit Hes5 expression by 77.00 ± .03 percent (n=7). B. Similar to DnMaml1-eGFP induction,

DAPT treatment leads to depletion of Olig2 progenitors, and promotes more homogenous differentiation of progenitors into Hb9 positive motor neurons. D. DAPT treatment reduces the number of Olig2 cells from 15.8 ± 1.4 to just .3 ± .4 percent of total cells (n=3, P<.001). E.

Quantification of Hb9-GFP expressing cells by FACS shows an increase from 49.0% ± 4.8 to

68.0 ± .5 % of total cells (n=3, P<.001). 68

A Day 6 Control DAPT Ki67

B ** C 3500 * 35

30 3000

25 2500

20 2000

15 1500 10 1000 5 Total Cells/Plated Cells/Plated Cell Total 500 Ki67 as % of Total Cells Total of as %Ki67 0 Control DAPT 0 -5 Control DAPT Figure 2.7 69

Figure 2.7. Inhibition of Notch signaling depletes progenitors

A. Disruption of Notch signaling leads to nearly complete loss of proliferating cells as measured by Ki67 staining. B. Ki67 positive cells decrease from 25.6 ± 6.8 to just 2.0 ± 2.3 percent of total cells. C. Consistent with the loss of Ki67 positive cells, DAPT treated cultures generated far fewer cells. At day 6 of the differentiation protocol, untreated cultures yielded 2896 ± 248 cells per cell plated on day 0. DAPT treated cultures only yielded 1709 ± 388 cells per originally plated cell, representing a 41% reduction in the total number of cells generated during differentiation (n=3, P=.01). 70

A Day 4.5 Control 12 Hrs DAPT Ngn2 Olig2

B C 6 * 70 5 60 Cellls 4 50 40 3 30 2 20 1 10 Relative Relative expression Ngn2 Ngn2 as % of Olig2 as %of Ngn2 0 0 Control DAPT Control 12 Hrs DAPT Figure 2.8

71

Figure 2.8. Notch inhibition disrupts lateral inhibition and induces Ngn2

Analysis of the status of Ngn2 12 hours after DAPT treatment in day 4 pMNs. A. 12 hours of

DAPT treatment increases the number of Olig2 cells that are co-immunoreactive for Ngn2 on day 4 of differentiation. B. Ngn2 expression increases by 3.3 ± 1.6 fold after treatment with

DAPT for 12 hours (n=3). C. Consistent with the increase in Ngn2 mRNA levels, the percentage of Olig2 cells that are Ngn2+ increases from 40.5% ± 5.7% to 57.5% ± 4.3% (n=2,

P=.047). 72

A Day 4 Control iNgn2 V5 Olig2

B Day 6 Control iNgn2 Hb9 Olig2

C ** D *** 80 18 70 16 60 14 50 12 10

GFP Cells GFP 40 - 8 30 6 20 4 % Hb9% 10 Cells Total Olig2 asof % 2 0 0 Control iNgn2 Control iNgn2 Figure 2.9

73

Figure 2.9. Ngn2 induction is sufficient to promote motor neuron differentiation and deplete progenitors

Analysis of motor neuron differentiation after induction of Ngn2 on day 3 of differentiation. A.

Addition of doxycycline on day 3 of differentiation results in robust expression of Ngn2 in day 4 embryoid bodies. B. Similar to inhibition of Notch signaling, Ngn2 induction results in depletion of Olig2+ progenitors and more uniform differentiation into motor neurons. C.

Quantification of motor neurons by FACS quantification of Hb9-GFP+ cells shows an increase in motor neurons from 49 ± 4.8 to 65 ± 5.5 percent of total cells (n=3, P=.006). D. Ngn2 induction results in a decrease in Olig2 cells from 15.8% ± 1.4% to 5.5% ± 1.0% of total cells

(n=2, P<.001). 74

A Day 0 Day 2 Day 4 Day 6 ES NP pMN GP

GP/MN +RA/Hh

MN

B

Olig2

Notch

Olig2 Sox9 Hb9 Sox10 Motor Neuron Notch

Olig2 OPC Sox9 Sox9 Sox10 Sox10 Nkx2.2

OPC? Oligodendrocyte Astrocyte?

Figure 2.10

75

Figure 2.10. Proposed model based on results of Notch activation and inhibition during motor neuron differentiation

A. Summary of results. Normal ESC to motor neuron differentiation protocol recapitulates normal in vivo development and generates both motor neurons and glial progenitors. Activation of Notch signaling is sufficient to inhibit motor neuron differentiation and drive the majority of cells into the glial lineage, as indicated by co-expression of Sox9 and Sox10. However, NICD induced Olig2+ cells do not express Nkx2.2 and the subsequent fates adopted by Olig2+/Nkx2.2- are not known. Notch inhibition leads to increased Ngn2 expression, which in turn, induces motor neuron differentiation and yields relatively homogenous motor neuron cultures. Enhanced motor neuron differentiation depletes the culture of Olig2 positive progenitors originally fated to become glial progenitors. B. Proposed model. Notch signaling inhibits motor neuron differentiation and promotes progenitor maintenance via lateral inhibition. At the same time,

Notch induces the expression of Sox9/Sox10 and specifies glial cell fate. Notch also regulates glial differentiation by repressing Nkx2.2.

76

Chapter 3. Notch selects intra-segmental motor neuron column identity.

Introduction

Specification of motor neuron subtype identity can be viewed as a three-step process.

First, generic motor neurons are subdivided into axial-innervating medial MMC motor neurons and other non-MMC motor neurons along the entire length of the developing spinal cord.

Second, expression of Hoxc9 transcription factor in thoracic non-MMC motor neurons represses the specification of limb innervating motor neurons, resulting in specification of hypaxial HMC and preganglionic PGC motor neurons. Finally, young LMC motor neurons at brachial and lumbar levels of spinal cord initially co-express a cohort of Hox genes and Hox co-factors that engage in complex cross-repressive interactions, resulting in further subdivision of LMC motor neurons into dozens of distinct motor pools committed to innervating discrete muscle groups within the developing limbs (Dasen and Jessell, 2009; Dasen et al., 2005). While motor neuron subtype diversification depends primarily on rostral-caudal signals that control the expression of

Hox genes (Wnt, FGF, RA, TGFbeta) and on non-canonical Wnt specification of MMC identity

(Agalliu et al., 2009), the role of Notch signaling in motor neuron diversity has not been systematically examined.

The Notch signaling pathway is one of the primary methods for generating intra-domain cellular diversity in the central nervous system. In Drosophia, Notch signaling controls the generation of alternative cell fates during asymmetric division of neuroblasts in every lineage studied to date (Udolph, 2012). Emerging evidence suggests that Notch also plays a role in the specification of alternative cell fates in the vertebrate CNS. In the spinal cord, using different approaches, several groups have shown that Notch signaling is responsible for controlling v2a 77 versus v2b interneuron cell fate (Marklund et al., 2010; Peng et al., 2007; Rocha et al., 2009).

This leads to the intriguing possibility that Notch signaling may also play a key role in controlling intra-segmental columnar identity.

The embryonic lethality of conditional Notch mutants at a time prior to motor column establishment has rendered studies on the role of Notch signaling in the regulation of motor neuron columnar identity difficult (Marklund et al., 2010; Yang et al., 2006). In Chapter 2, I generated a number of tools for manipulating the Notch pathway during in vitro ESC to motor neuron differentiation to study the role of Notch signaling in controlling the temporal neuron to glial cell fate switch. In this chapter, I use these same tools to determine whether Notch also plays a role in the selection of motor neuron columnar identity. Using DAPT treatment and the inducible DnMaml1-eGFP ESC line, I demonstrate that inhibition of Notch signaling converts

MMC motor neurons into non-MMC subtypes. I then assessed the gene expression profiles of sorted motor neurons from both DAPT-treated and control motor neurons and identified

Somatostatin (Sst) as a novel marker of cervical motor neurons in vivo. Finally, taking advantage of the relatively synchronous ESC to motor neuron differentiation protocol, I identified the critical period for which Notch inhibition is necessary to elicit these effects. Our results point towards a role for Notch signaling in selecting MMC over HMC or LMC cell fate, thereby filling a major gap in our understanding of how motor neuron columnar diversity is achieved within the developing spinal cord.

Results

Notch inhibition promotes HMC-like motor neuron identity during ESC to motor neuron differentiation 78

As RA specifies hindbrain and spinal territories through its ability to directly activate the transcription of 3’ Hox genes (Hox1-5) (Kashyap et al., 2011; Mahony et al., 2011), the use of

RA in ESC to motor neuron differentiation protocols results in motor neurons with a rostral cervical identity (Peljto et al., 2010). The cervical identity of ESC-derived motor neurons is confirmed by immunohistochemical analysis to demonstrate that day 6 embryoid bodies express cervical spinal cord marker Hoxa5 and not brachial spinal cord marker Hoxc8 (Fig. 3.2B). At cervical and thoracic levels of spinal cord, motor neurons are organized into the Hb9+/Lhx3+

MMC and Hb9+/Lhx3- HMC columns (Fig. 3.1A). To determine whether Notch plays a role in specifying intra-segmental motor neuron columnar identity, I inhibited Notch signaling using

DAPT treatment from days 4 through 6 of motor neuron differentiation and analyzed the expression of Hb9 and Lhx3. Under control conditions, 72% ± 6% of Hb9+ motor neurons expressed the MMC marker Lhx3. In contrast, only 15% ± 1% of motor neurons expressed Lhx3 in DAPT-treated conditions, suggesting a shift from MMC to HMC motor neuron columnar cell fate (Fig. 3.1 B-C).

Notch inhibition promotes LMC identity in brachial spinal cord conditions

Due to the dramatic change in motor neuron composition during ESC to motor neuron differentiation after Notch inhibition, I was eager to determine whether similar changes would be observed under caudal conditions that generate LMC motor neurons in addition to MMC and

HMC motor neurons (Fig. 3.2A). Unfortunately, current methods for generating caudal motor neurons result in low motor neuron yield and a heterogeneous Hox profile, consisting of both brachial and thoracic motor neurons (Peljto et al., 2010). To bypass these hurdles, I combined inducible ES technology with DAPT treatment. As induction of Hox genes in vivo is sufficient 79 to reprogram the RC identity of spinal motor neurons (Dasen et al., 2003; Dasen et al., 2005), I used an inducible Hoxc8 ESC line (generated by Esteban Mazzoni in the lab) to produce a homogenous population of brachial motor neurons. Addition of doxycycline on day 3 of motor neuron differentiation results in robust induction of Hoxc8 in day 6 motor neurons with a concomitant loss in cervical Hoxa5 expression (Fig. 3.2B). Since some LMC motor neurons downregulate Hb9 expression shortly after birth, I immunostained with a pan-Islet antibody that recognizes both Isl1 and Isl2 to identify all motor neurons (Alaynick et al., 2011). I found that

34.0% of motor neurons were Lhx3+/Foxp1- MMC motor neurons, 14.2% were Lhx3-/Foxp1-

HMC motor neurons, and 51.9% were Lhx-/Foxp+ LMC motor neurons (Fig. 3.2 C-D).

To test the role of Notch signaling in controlling motor neuron columnar identity at brachial levels of spinal cord, I induced Hoxc8 expression on day 3 of differentiation and treated embryoid bodies with DAPT inhibitor on day 4. Similar to cervical conditions, DAPT treatment of induced Hoxc8 motor neurons decreases the percentage of Lhx3+ MMC motor neurons from

34.0% to 3.4% (Fig. 3.2C-D). However, instead of increasing the percentage of HMC motor neurons in the culture, DAPT treatment results in an increase from 51.9% to 80.0% of Foxp1+

LMC motor neurons, while the number of HMC motor neurons remains constant (Fig. 3.2 C-D).

Thus, regardless of spinal level, Notch inhibition leads to loss of MMC motor neurons. The loss of MMC motor neurons is accompanied by an increase in either HMC or LMC motor neurons in accordance with the column normally found within the rostro-caudal segment.

LMC motor neurons can be further subdivided into medial (LMCm) and lateral LMC

(LMCl) divisions. While LMCm motor neurons express high levels of both Isl1 and Isl2, LMCl motor neurons express only Isl2. The control Hoxc8-induced motor neuron culture produces 80

Foxp1+ cells that are heterogeneous with respect to Isl1/2 expression (Fig. 3.2 D), revealing that both LMCm and LMCl motor neurons are generated. In addition to increasing the overall number of Foxp1+ cells, DAPT treatment results in uniformly high levels of Isl1/2 expression in

Foxp1+ cells (Fig. 3.2D). This finding indicates that inhibition of Notch signaling promotes conversion of MMC and LMCl motor neurons to LMCm motor neurons and suggests that Notch signaling also plays a role in establishing subtype divisions within the LMC.

Changes in columnar markers are not due to acceleration of motor neuron maturation

All nascent motor neurons transiently express Lhx3 (Arber et al., 1999; Sharma et al.,

1998; Tsuchida et al., 1994), but its expression is rapidly downgregulated within non-MMC motor neurons of the developing spinal cord. The surprisingly large number of Lhx3+ motor neurons generated during ESC to motor neuron differentiation raises the possibility that the normal process of Lhx3 clearance is delayed in vitro. Thus, changes in Lhx3 expression after inhibition of Notch signaling may not reflect a true MMC to non-MMC subtype conversion but rather a correction of in vitro motor neuron neoteny, leading to rapid clearance of Lhx3 expression and manifestation of their true non-MMC subtype identity.

To distinguish between these two possibilities, I first examined the timing of motor neuron birth following Notch inhibition. After treatment of motor neuron cultures with DAPT on day 4 of differentiation, I quantified the number of Hb9-GFP expressing motor neurons in 6 hour intervals until day 6 of differentiation by FACS. My results indicate that DAPT treatment does indeed lead to precocious motor neuron birth, with maximal motor neuron numbers achieved ~18 hours earlier than in untreated cells (Fig 3.3A). 81

To further probe the timing of motor neuron differentiation, I compared the global gene expression profiles of control motor neurons on day 5 (young) and day 7 (mature) of differentiation with that of DAPT-treated and untreated motor neurons on day 6 of differentiation. As brachial conditions generate a more heterogeneous populations of motor neurons and do not induce as dramatic a change in Lhx3 expression, I focused this study on cervical motor neurons, which exhibit an almost 80% loss in Lhx3+ motor neurons. If DAPT treatment simply accelerates the maturation of motor neurons, I would expect that gene expression changes between DAPT-treated and control motor neurons strongly correlate with changes in mature versus young motor neurons. However, comparison of genes greater than fourfold enriched in either DAPT versus control or mature versus young motor neurons revealed that there is very little correlation (R2=.048) between the two sets of data (Fig. 3.3B). Of 147 total genes that were differentially regulated more than fourfold between DAPT and control cells, only 28 (19%) of them were differentially regulated in the same direction in mature versus young motor neurons.

The expression array data confirmed that Lhx3 expression is reduced more than 7.7-fold in DAPT treated cells but is mildly enriched 1.8-fold in mature versus young motor neurons

(Table 3.1). Choline acetyltransferase (Chat), the enzyme that synthesizes the motor neuron , is a key marker of motor neuron maturation. While Chat was enriched greater than twofold in mature versus young motor neurons, it was not enriched in

DAPT treated motor neurons. These findings indicate that while DAPT treated motor neurons are born precociously, their molecular profile does not reflect significantly advanced aging, suggesting that the changes in motor neuron columnar markers after Notch inhibition reflect a bona fide switch from MMC to HMC motor neuron identity. 82

Candidate HMC molecular profile

A major challenge in studying HMC motor neurons has been the lack of a specific marker for this neuronal subtype. This is particularly true in cervical regions of the spinal cord, where distinctions between dorsal and ventral musculature are not as clear as in thoracic levels, and the corresponding motor columnar organization has not been as well characterized. The finding that Notch inhibition leads to molecular changes consistent with HMC motor neuron production leads to the enticing possibility that the previously generated expression data for

DAPT-treated motor neurons contains candidate markers for HMC motor neuron identity. The mature versus young motor neuron expression data also affords us the ability to exclude analysis of genes that are likely due to increased maturation of motor neurons after DAPT treatment.

Indeed, genes specifically regulated in DAPT-treated motor neurons include many that have been previously implicated in motor neuron or neuronal subtype identity (Table 3.1).

Two interesting genes that are down-regulated in DAPT-treated motor neurons are estrogen-related receptor gamma (Esrrg) and transcription factor Sp8. Sp8 shows the second strongest change in gene expression, with a downregulation of over 28-fold in DAPT-treated motor neurons relative to control motor neurons. While Sp8 has not been previously reported to be expressed in motor neurons, it has been implicated in several areas of the CNS as a gene responsible for specifying interneuron subtype identity (Li et al., 2011; Waclaw et al., 2010); thus, Sp8 may also be a marker and specifier of motor neuron subtype identity. The gamma motor neuron marker, Esrrg (Friese et al., 2009), decreases greater than 5-fold in DAPT-treated motor neurons, suggesting that Notch signaling may also regulate gamma versus alpha motor neuron identity. However, interpretation of Esrrg expression is complicated by the fact that, like 83

Lhx3, it is transiently expressed by the majority of young motor neurons and only postnatally marks gamma motor neuron identity. Immunostaining for Sp8, Esrrg, and Hb9 confirms that

Sp8 and Esrrg are both expressed in a subset of motor neurons during normal differentiation, but their expression is extinguished in DAPT treated motor neurons (Fig. 3.4A-B).

I focused my further analysis on genes that are exclusively enriched in DAPT-treated motor neurons as these may represent novel markers of cervical HMC motor neurons. Most interesting among the induced genes are the Onecut family transcription factors, Hoxa1 transcription factor, and neuropeptide Somatostatin. The transcription factors Onecut1 (OC1) and Onecut2 (OC2) are two of the most enriched genes in DAPT-treated motor neurons. Both genes are downregulated in day 7 relative to day 5 control motor neurons indicating that the enrichment is specific to DAPT treatment and not the result of general motor neuron maturation

(Table 3.1). Immunostaining and quantification confirms that there is a dramatic increase in the number of Hb9 motor neurons that co-express OC1 and OC2 after DAPT treatment, implicating them as putative markers of HMC motor neuron identity (Fig. 3.4C-D).

Onecut transcription factors are broadly expressed in young motor neurons but become restricted to MMC and HMC motor neurons later in development, with the highest levels of expression in HMC motor neurons (Francius and Clotman, 2010). A recent follow-up study established that, while compound loss of OC1 and OC2 results in partial conversion of both

MMC and HMC motor neurons into PGC motor neurons in thoracic spinal cord (Roy et al.,

2012), the ratio of HMC to MMC neurons remains the same. This result seemingly conflicts with our finding that the Onecuts are more strongly associated with HMC cell fate; however the 84 study did not report expression of Onecut transcription factors in cervical spinal cord, leaving open the possibility that Onecut is a selective marker of cervical motor neuron HMC subtypes.

Another transcription factor enriched 4.6-fold in DAPT treated motor neurons is Hoxa1

(Table 3.1). Hoxa1 is transiently expressed from rhombomere 4 caudally to spinal cord. Its loss of function results in defects in specification of abducens (6N) and facial (7N) cranial motor nuclei in the caudal hindbrain; however, similar to the case of Onecut transcription factors, the detailed pattern of Hoxa1 expression in cervical spinal cord motor neurons remains to be examined. The observed induction of Hoxa1 expression following DAPT treatment raises an interesting possibility that Notch signaling plays a currently unappreciated role in the regulation of Hox gene expression.

Sst is a marker of HMC motor neurons in vitro

Of the differentially expressed genes, I further focused my analysis on Somatostatin (Sst), which is induced over 5-fold in DAPT treated motor neurons (Table 3.1). In contrast to the nuclear localization of transcription factors, the cytoplasmic localization of Sst may facilitate tracing of Sst-positive motor axons towards their muscle targets. Sst is expressed in many different tissues where it generally acts as a hormonal inhibitor through interactions with one of its six receptors (Barnett, 2003). The function of Sst is perhaps best studied in the pancreas, where Sst secreted from the intestine and pancreatic delta cells acts as a hormone to inhibit secretion of insulin and glucagon from beta and alpha cells, respectively (Strowski and Blake,

2008). Although its function in the CNS is not well understood, Sst serves as a key subtype marker of GABAergic inhibitory interneurons of the neocortex (Kawaguchi and Kondo, 2002).

The greater than 5-fold enrichment in DAPT-treated motor neurons raises the possibility that Sst 85 is a candidate marker for a subset of cervical HMC motor neurons in the spinal cord, where its expression has not been previously described.

I first confirmed that protein levels of Sst are enriched after DAPT treatment.

Immunostaining of day 6 embryoid bodies with antibodies against Hb9 and Sst revealed that Sst is indeed enriched in DAPT-treated motor neurons. However, since Sst is a cytoplasmic protein,

I was unable to associate Sst staining with individual motor neurons in densely clustered embryoid bodies. To determine whether individual motor neurons expresses Sst, I dissociated day 6 embryoid bodies and plated motor neurons on laminin-coated cover slips. The cultured motor neurons were then fixed and stained for Sst, Hb9 and Lhx3 on day 9 of differentiation.

Strikingly, Sst expression is completely excluded from Lhx3+ MMC motor neurons and is expressed in approximately 58.5% of Lhx3- HMC motor neurons, suggesting that Sst is indeed a marker of a substantial fraction of HMC motor neurons in vitro (Fig. 3.5 A,B,E).

Inhibition of Notch signaling converts Lhx3+ MMC motor neurons to Sst+ HMC motor neurons

Together these data suggest that DAPT treatment converts Lhx3+ MMC motor neurons in to HMC motor neurons that predominantly express Sst. As discussed in Chapter 2, the gamma- secretase inhibitor DAPT strongly inhibits not only Notch signaling, but also other pathways, prompting us to more directly examine whether changes in subtype-specific patterns of gene expression are caused by inhibition of Notch signaling (Kopan and Ilagan, 2004). To address this concern, I induced DnMaml1-eGFP on days 3-6 of differentiation and plated dissociated motor neurons on coverslips for immunostaining. Analysis of Lhx3, Hb9, and Sst staining on day 9 revealed that DnMaml1 efficiently downregulates Lhx3 expression from 49.1% to only

2.0% of motor neurons and increases Sst expression from 29.7% to 66.0% of motor neurons, 86 supporting the conclusion that the conversion of MMC to HMC motor neurons results from

Notch inhibition (Fig. 3.5 C,D,F).

Sst is a marker of cervical HMC motor neurons in vivo

Given the striking segregation of Lhx3 and Sst in motor neurons produced in vitro, I wondered whether Sst is an in vivo marker of HMC motor neuron identity. I compared Hb9,

Lhx3, and Sst antibody staining in E13.5 mouse hindbrain and cervical spinal cord sections taken at 270 µM intervals and found Sst immunoreactivity in the dorsal region of all levels of hindbrain and cervical spinal cord, likely marking commissural interneurons residing in the dorsal horn. Sst immunoreactivity is also present in an area lateral to motor neurons in the hindbrain and dorsal to cervical spinal motor neurons.

Interestingly, Sst is expressed in a subset of medially positioned Hb9+ motor neurons in rostral-cervical spinal cord. This result seemingly conflicts with our hypothesis that Sst is an

HMC motor neuron marker and should be excluded from medially positioned Lhx3+ MMC motor neurons. However, closer inspection of Sst, Hb9, and Lhx3 expression reveals that, at this level of spinal cord, Lhx3+ MMC motor neurons are located lateral to the Lhx3- HMC motor neurons. While the lateral location of MMC motor neurons relative to HMC motor neurons in cervical spinal cord is incongruous with spatial organization of motor neurons at more caudal levels, it is consistent with previous retrograde labeling studies in chicken and rat showing that ventral muscles in cervical spinal cord are innervated by medially positioned motor neurons

(Gutman et al., 1993; Watson C., 2008). At more caudal levels of spinal cord, Lhx3+ and Lhx3- cells intermingle, and the spatial distinction between HMC and MMC motor neurons becomes less clear. At these spinal cord levels, Sst is largely absent from all motor neurons. These 87 findings indicate that while spatial organization of motor columns is different in cervical spinal cord, Sst is a marker of a defined subset of Lhx3- HMC motor neurons.

Changes in columnar cell fate occur at early stages of motor neuron differentiation

After confirming that the changes in expression of columnar markers after Notch inhibition likely reflect a bona fide change in motor neuron columnar identity, I took advantage of our ES to motor neuron differentiation protocol to determine the critical period for Notch signaling in motor neuron columnar identity conversion. Current models of motor neuron subtype acquisition propose that motor neuron identity is primarily specified by postmitotic resolution of Hox gene expression. This implies that young motor neurons are plastic in regards to their subtype identity and led me to hypothesize that Notch inhibition in young postmitotic motor neurons may be sufficient to drive conversion of motor neuron columnar identity. I tested this hypothesis by treating embryoid bodies with DAPT on day 5, when many motor neurons have just become postmitotic, and analyzed the expression of Lhx3 on day 6 of differentiation.

Remarkably, DAPT treatment of young motor neurons failed to repress Lhx3 expression and convert MMC motor neurons into HMC motor neurons (Fig. 3.7B).

Next, I tested whether shorter periods of DAPT treatment during the pMN stage would be sufficient to suppress Lhx3 expression in postmitotic motor neurons. Treating day 4 embryoid bodies with DAPT and washing the DAPT away on day 5 revealed that a short 24-hour treatment is sufficient to convert MMC into HMC motor neurons (Fig. 3.7B). These findings lead me to conclude that Notch activity selects motor neuron columnar identity early during motor neuron differentiation at either the progenitor stage or immediately after motor neuron birth. 88

In the zebrafish retina, where retinal progenitors are temporally patterned and give rise to different classes of neurons over time, DAPT treatment is found to increase the generation of neurons normally associated with the time of treatment (Nelson et al., 2007). Autoradiography experiments in chick spinal cord have demonstrated that MMC motor neurons are born prior to

HMC and LMC motor neurons (Fujita, 1964; Langman and Haden, 1970; Price and Briscoe,

2004). Is it possible that pMNs undergo a temporal switch from production of MMC motor neurons early in development to non-MMC cells later in differentiation? To test this possibility,

I reasoned that treatment of embryoid bodies earlier in differentiation should increase, rather than decrease, the yield of Lhx3+ MMC motor neurons. However, treatment of day 3 embryoid bodies with DAPT led to a downregulation of Lhx3, similar to day 4 treatment (Fig. 3.7B), suggesting that the changes in motor neuron columnar identity are not the result of a simple temporal progression of one motor neuron subtype to another and that Notch signaling serves as an instructive signal for MMC motor neuron subtype specification.

Ngn2 does not alter motor neuron columnar identity

Finally, I considered the molecular mechanisms that link Notch signaling to motor neuron subtype specification. It is unlikely that Lhx3 expression is simply induced by Notch activation. Even under conditions where Notch signaling is inhibited by DAPT or DnMaml1- eGFP expression from day 4 onwards, nascent HMC motor neurons express transiently high levels of Lhx3. I therefore reasoned that regulation of Lhx3 expression in day 6 motor neurons must be a downstream effect of a transcriptional cascade initiated by inhibition of Notch signaling on day 4. In Chapter 2, I demonstrated that Notch inhibition leads to suppression of

Hes gene expression and subsequent induction of the bHLH transcription factor Ngn2. I also 89 demonstrated that induction of Ngn2 alone is sufficient to recapitulate effects of Notch inhibition on motor neuron genesis and glial progenitor maintenance. I therefore asked whether Ngn2 upregulation also mediates the changes in motor neuron columnar identity exhibited after inhibition of Notch signaling. To test this possibility, I used the inducible Ngn2 ESC line to induce Ngn2 expression on day 3 of differentiation and analyzed Hb9 and Lhx3 expression on day 6 of differentiation. In contrast to Notch inhibition, Ngn2 induction does not lead to downregulation of MMC marker Lhx3 (Fig. 3.8 AB), indicating that Notch effects on motor neuron subtype identity are mediated by other molecular mechanisms.

Discussion

Through its ability to regulate differentiation via lateral inhibition and selection of alternative cell fates, the Notch signaling pathway is one of the primary players in the generation of intra-domain cellular diversity in the CNS. Using an ESC to motor neuron approach, I demonstrate that Notch signaling contributes to specification of motor neuron subtype diversity arising from the spinal pMN domain. Inhibition of Notch signaling results in the repression of

Lhx3 and conversion of MMC motor neurons into HMC or LMC motor neurons in accordance with the rostro-caudal positional identity of differentiated cells.

Three lines of evidence indicate that Notch does not control motor neuron subtype identity by directly regulating the expression of subtype specific markers in postmitotic motor neurons. First, I demonstrate that Notch activity is required relatively early in pMNs. Second, although Lhx3 repression is the key manifestation of Notch inhibition in the motor neuron lineage, this effect is likely indirect since even cells in which Notch signaling is inhibited exhibit a transient wave of Lhx3 expression during their transition to a postmitotic stage. Third, while 90 inhibition of Notch signaling leads to Lhx3 clearance from postmitotic motor neurons, activation of Notch signaling results in the clearance of Lhx3 expression in the adjacent p2 progenitor domain and promotes Lhx3- v2b interneuron cell fate over Lhx3+ v2a interneuron cell fate (Peng et al., 2007). Together, these results establish that Notch signaling does not directly specify

MMC cell fate (Sharma et al., 1998). Instead, my findings are consistent with the function of

Notch signaling in Drosophila, where Notch selects different cell fates within the context of each neuroblast lineage (Udolph, 2012).

Selection of cell fates is not controlled via disruption of lateral inhibition and Ngn2 induction

In Chapter 2, we confirmed that Notch signaling is necessary for maintaining progenitor identity via lateral inhibition, and disruption of Notch signaling using DAPT or DnMaml1-eGFP leads to depletion of progenitors and precocious motor neuron differentiation. In the zebrafish retina, where neural progenitors are temporally patterned and give rise to different neurons over time (Nelson et al., 2007), DAPT treatment at various stages is capable of inducing increased genesis of neurons appropriate for the stage of treatment. As motor neurons also have a temporal order to their birth, this raises the possibility that conversion of MMC to HMC or LMC motor neurons is the result of increased neurogenesis at times when HMC and LMC motor neurons are being generated.

In support of this hypothesis, Sabharwal et al. show that early-born MMC motor neurons express a novel inhibitor of Notch signaling, GDE2, whose function is required for the production of a full complement of HMC and LMC motor neurons. While GDE2 loss of function has little effect on specification of earlier born MMC motor neurons, HMC and LMC motor neurons are significantly reduced (Sabharwal et al., 2011). They proposed a model in 91 which GDE2 functions as a feedback inhibitor of Notch signaling, necessary for pMN differentiation in LMC and HMC motor neurons. Thus, in their model, loss of GDE2 signaling leads to increased Notch signaling and prevents neurogenesis during the HMC and LMC phases of motor neuron differentiation (Sabharwal et al., 2011). In this context, one would predict that disruption of lateral inhibition at earlier stages promotes specification of earlier born MMC motor neurons, leading to the depletion of pMN progenitors and a consequent decrease in HMC and LMC motor neuron specification.

My data provides two pieces of evidence suggesting that changes in motor neuron composition are not due to simple regulation of lateral inhibition. First, in Chapter 2, I show that

Ngn2 overexpression alone is sufficient to promote precocious motor neuron genesis, leading to the depletion of Olig2+ progenitors. However, Ngn2 induction in pMN progenitors does not affect motor neuron subtype identity and specification of Lhx3+ MMC motor neurons. Second, forcing early motor neuron genesis by DAPT treatment on day 3, prior to the birth of motor neurons, does not increase production of MMC motor neurons and instead results in Lhx3 downregulation and HMC motor neuron generation, similar to DAPT treatment on day 4.

Together these results indicate that Notch is not simply controlling subtype identity by regulating the timing of motor neuron differentiation. Instead, it regulates targets other than Nng2 to achieve a change in motor neuron subtype properties.

Selection of columnar identity by Notch signaling occurs during motor neuron genesis

Genetic analyses have demonstrated that young motor neurons are born with generic identity in regards to motor pool identity and that pool identity is only specified in postmitotic motor neurons by consecutive rounds of Hox gene refinement (Dasen et al., 2005). In contrast, 92 my data suggests that Notch activity regulates motor neuron columnar identity during earlier phases of motor neuron genesis. Inhibition of Notch signaling in nascent motor neurons on day

5 of differentiation is incapable of altering motor neuron columnar identity, indicating that columnar identity has already been stabilized in young postmitotic motor neurons. Instead, I found that 24 hours of Notch inhibition on days 4-5 of differentiation, when pMNs exit the cell cycle, is sufficient to convert motor neuron columnar identity. The requirement of Notch activity during a temporal window encompassing the terminal differentiation of pMNs also supports the idea that regulation of columnar identity depends on a cascade of molecular events initiated in late pMNs and transmitted to mature motor neurons two days later when expression of columnar markers is fully crystalized.

Notch and non-canonical Wnt

The finding that both Notch and non-canonical Wnt signaling are involved in specifying

MMC motor neuron identity (Agalliu et al., 2009) adds to the already long list of potential interactions between Notch and Wnt signaling (Hayward et al., 2008). At the molecular level, numerous studies have shown interactions between downstream effectors of Notch and Wnt signaling. Several studies have demonstrated that NICD is capable of physically interacting with key effectors of canonical Wnt signaling, including Dsh, Gsk3β, and β-catenin (Axelrod et al.,

1996; Espinosa et al., 2003; Hayward et al., 2005; Ramain et al., 2001).

While Notch and canonical Wnt signaling generally have antagonistic roles in regards to regulation of cell fate specification, Notch and non-canonical Wnt signaling are often found to work in concert to solidify cell fate. For example, both Notch signaling and non-canonical Wnts have been shown to induce differentiation of adult human circulating endothelial progenitor cells 93

(EPS) into cardiomyocytes (Koyanagi et al., 2007; Koyanagi et al., 2005). In this case, Notch signaling was found to induce the expression of non-canonical Wnt5a, suggesting that Notch is upstream of Wnt5a signaling (Koyanagi et al., 2007). Similarly, studies have shown that hair follicle keratinocytes fail to differentiate when Notch effector CSL is deleted from underlying dermal papilla cells. Again, Wnt5a was found to be a downstream mediator of Notch activity as

Wnt5a mutants exhibit similar defects in hair follicle differentiation, and exogenous Wnt5a is capable of rescuing hair follicle differentiation in CSL mutants (Hu et al., 2010).

These genetic studies place Wnt5a downstream of Notch signaling, raising the possibility that Wnt5a is also the downstream effector of Notch signaling in differentiating motor neurons.

Interestingly, non-canonical Wnt signaling has also been reported to act upstream of Notch signaling. In hematopoietic stem cells (HSC), exogenous Wnt5a expression promotes HSC quiescence and prevents HSC differentiation by inhibiting canonical Wnt signaling and promoting Notch signaling (Nemeth et al., 2007). Thus, non-canonical Wnts may engage in positive feedback interactions with Notch signaling and play a cooperative role in specifying

MMC cell fate.

The finding that Notch is required for the selection of MMC motor neuron identity bridges an important gap in our understanding of how intra-segmental motor neuron columnar diversity is achieved. At any given level of spinal cord, Notch activity acts in cooperation with non-canonical Wnt signaling to select MMC cell fate as opposed to HMC or LMC cell fate.

Disruption of either Notch or non-canonical Wnt signaling in turn leads to selective loss of

MMC columnar identity and conversion into HMC or LMC motor neurons.

Sst is a novel marker of cervical HMC motor neurons 94

The ability to manipulate motor neuron columnar composition of in vitro-derived motor neurons leads to the exciting possibility of being able to generate relatively pure populations of motor neurons with defined subtype identity for developmental and disease studies.

Highlighting this possibility, expression analysis of DAPT-treated versus untreated motor neurons identified Sst as putative marker for HMC motor neurons. Subsequent in vitro and in vivo immunocytochemical and immunohistochemical analysis revealed that Sst is indeed a highly specific marker of cervical HMC identity. Furthermore, Sst expression is found only in rostral cervical HMC motor neurons, reflecting the RC specificity of in vitro-derived motor neurons.

This expression analysis also demonstrated changes in expression of a number of genes that have been implicated in other aspects of motor neuron diversity. Esrrg, a marker of gamma motor neurons that innervate intrafusal muscles is significantly enriched in DAPT-treated motor neurons, implying that Notch plays an additional role in controlling alpha versus gamma motor neuron cell fate. Hoxa1 transcription factor is also upregulated, indicating that Notch may play a previously unknown role in regulating Hox gene regulation. This is a particularly interesting finding, as previous studies have argued for a largely cell autonomous Hox-based transcriptional network in the control of final motor pool identity at limb levels of spinal cord. Demonstration that Notch mediated cell-cell signaling modulates Hox gene expression raises an important question of the extent to which Notch signaling controls the intrinsic Hox transcriptional networks that direct motor pool diversification. Implication of Notch signaling in motor pool specification would be a significant departure from the current models based on cell-autonomous diversification of cell identity. 95

A B Cervical/Thoracic

*** 100

80

HMC 60 MMC 40

MMC: Hb9, Lhx3+ 20 HMC: Hb9, Lhx3- Lhx3 as % of%Cells Hb9as Lhx3 0 Control DAPT C Day 6 Control DAPT Lhx3 Hb9

Figure 3.1

96

Figure 3.1. Inhibition of Notch signaling leads to loss of MMC motor neuron marker Lhx3

Embryoid bodies were treated with DAPT from days 4-6 of differentiation and Hb9/Lhx3 expression was analyzed on day 6 to determine the role of Notch signaling in acquisition of motor neuron column identity. A. Schematic of motor columns present in cervical and thoracic spinal cord. MMC motor neurons express Lhx3 and are positioned medially closest to the neural tube. HMC motor neurons are located lateral to the MMC column and are marked by the absence of Lhx3. B. Quantification of Lhx3 in treated and untreated day 6 embryoid bodies.

Normal ESC to motor neuron differentiation protocol yields motor neurons that are 71.8% ±

6.5% Lhx3+. DAPT treatment leads to dramatic down regulation of Lhx3 to only 17.8% ± 3.9%

(n=4, P<0.001) of Hb9+ motor neurons. C. Immunostaining for Hb9/Lhx3 demonstrating loss of Lhx3 in DAPT treated motor neurons. 97

A B Brachial/Lumbar Day 6 Control iHoxc8

LMC Hoxc8 HMC MMC

MMC: Isl1/2+, Lhx3+ Hoxa5 HMC: Isl1/2+, Lhx3-, Foxp1- LMC: Isl1/2+, Lhx3-, Foxp1+ D Day 6 iHoxc8 + Dox C Control DAPT

** 100 Control

90 Foxp1 DAPT 80 70 Isl1/2 60 *** 50 40 30 N.S.

20 Lhx3 % of Motor Neurons Motor of% 10 0 Isl1/2 MMC HMC LMC

Figure 3.2

98

Figure 3.2. Inhibition of Notch signaling in brachial conditions leads to conversion of

MMC motor neurons into LMC motor neurons

To determine if Notch signaling plays a universal role in regulating motor neuron columnar fate, an inducible Hoxc8 ESC line was treated with DAPT to determine the role of Notch signaling in brachial spinal cord. A. Schematic of motor neuron columns in brachial spinal cord. Lhx3-

HMC column is reduced in size, and the Lhx3-/Foxp1+ LMC column occupies the lateral aspect of the spinal cord. B. Under normal ESC to motor neuron differentiation, high levels of RA induce expression of Hoxa5, conferring a rostral-cervical identity. Addition of doxycycline to inducible Hoxc8 ESC line robustly induces the expression of Hoxc8 in the vast majority of cells, and leads to repression of Hoxa5. C. Quantification of MMC (Isl+/Lhx3+/Foxp1-), HMC

(Isl+/Lhx3-/Foxp1-) and LMC (Isl+/Lhx3-/Foxp1+) motor neurons. Hoxc8 induced cultures yield

34.0% ± 2.5% MMC, 14.2% ± 3.2% HMC, and 51.9% ± 5.4% LMC motor neurons. DAPT treated cultures yielded 3.4% ± 1.3% MMC (P<0.001), 16.8% ± 5.1% HMC (P=0.45), 80.0% ±

6.3 % LMC motor neurons (n=3, P=0.002). D. Immunostaining for Isl1/2, Lhx3, and Foxp1 in

DAPT treated and untreated Hoxc8 induced motor neurons. DAPT treatment leads to conversion of Lhx3+/Foxp1- MMC motor neurons into Lhx3-/Foxp1+ LMC motor neurons. DAPT treatment also results in increased Isl1/2 immunoreactivity, suggesting that Notch signaling might control

LMCm versus LMCl identity. 99

A 1.2 18 hrs 1

0.8

0.6

0.4 No Treatment Dapt

GFP Normalized to Day 6 to Day Normalized GFP 0.2 -

0 4.5 5 5.5 6 % Hb9 % Days of differentiation

B Log2 of expression ratios 35

25

15 + DAPT +

5

-60 -40 -20 0 20 40 60 -5

-15

-25 Control

-35 Day 5 Day 7 Figure 3.3

100

Figure 3.3. Gene expression changes in DAPT treated motor neurons are not correlated with motor neuron maturation

Time series analysis of motor neuron birth and comparative gene expression arrays were used to determine if changes in columnar identity are the result of motor neuron maturation. A. Time series FACS quantification of Hb9-GFP to determine the rate of motor neuron generation during control and DAPT treated ESC to motor neuron differentiation. DAPT treatment resulted in increased rate of motor neuron birth, reaching maximum motor neuron differentiation 18 hours earlier than control motor neurons. B. Comparison of gene expression changes between

DAPT/Control and mature/young motor neurons. All genes greater than fourfold changed in either sets of the data were plotted. The Y-axis measures log2 fold change of DAPT/control log2 fold change in expression, while the X-axis measure day 7/day 5 log2 fold change in expression

(R2=.048). 101

Probe Gene DAPT/Control Day7/Day5 P-Value (DAPT/Control) Genes Downregulated in DAPT Treated MNs 1421753_a_at Lhx3 -7.7 1.8 0.017 1421483_at Lhx4 -4.6 -1.2 0.003 1440519_at Sp8 -28.9 1.0 0.000 1455267_at Esrrg -5.1 2.2 0.006 Genes Upregulated in DAPT Treated MNs 1456974_at Onecut1 9.7 -7.5 0.002 1460044_at Onecut2 7.1 -1.9 0.005 1417954_at Sst 5.5 1.2 0.003 1420565_at Hoxa1 4.6 -2.3 0.007 Table 3.1

102

Table 3.1. Microarray analysis identifies a candidate gene expression profile for HMC motor neurons

Table listing probeset ID, gene name, DAPT/control fold change, day 7/day 5 fold change and P- value for DAPT/control. Top half of the table lists genes down regulated in DAPT versus control and unchanged or increased in day 7 versus day 5 motor neurons. Bottom half of the table lists genes that are specifically enriched in DAPT versus control motor neurons and serve as potential markers for HMC motor neuron identity. 103

Control DAPT A Esrrg Hb9

B Sp8 Hb9

** C 100

50 Onecut1

% Onecut1+ % 0 Hb9 Control DAPT D *** 100

50 Onecut2 % Onecut2+ %

Hb9 0 Control DAPT E Sst Hb9

Figure 3.4

104

Figure 3.4. Validation of candidate genes identified in microarray study

Immunostaining for differentially regulated genes validates specificity of DAPT versus control microarray study. A-B. Esrrg and Sp8 were both identified as genes that are downregulated after DAPT treatment. Immunostaining confirms that both genes are downregulated in day 6

DAPT treated Embryoid bodies (Not quantified). C-D. Immunostaining and quantification of

Onecut1 and Onecut2 in motor neurons. DAPT treatment increases the percentage of Onecut1 positive motor neurons from 41.5% ± 4.1% to 89.0% ± 1.1% (n=3, P=0.001). Onecut2 increases from 36.1% ± 8.0% to 86.7% ± 4.3% of motor neurons (n=3, P<0.001). E. Sst is enriched in

DAPT treated embryoid bodies. 105

Day 9 Dissociated iDnMaml1-eGFP Control iDnMaml1-eGFP A D Sst Hb9

B E Sst Lhx3

C * F 80 Control ** 70 DAPT 80 *** 70 60 60 50 50 + 40 40 Sst 30

% 30 20 20 HB9 of Cells % 10 10 0 0 MMC HMC Lhx3 Sst -10 -10 Figure 3.5

106

Figure 3.5. Sst and Lhx3 expression are mutually excluded in ESC derived motor neurons

Day 9 dissociated motor neurons were stained with Lhx3, Hb9, and Sst to address the status of

Sst in individual MMC and HMC motor neurons. A. Immunostaining of Hb9 and Sst dissociated day 9 motor neurons showing Sst expression in a subset of motor neurons. B. Expression of

Lhx3 and Sst is mutually exclusive in motor neurons. White arrows marker cell bodies of Lhx3-

/Sst+ motor neurons. C. Sst is expressed in 0.61% ± 1.2% of Lhx3+ MMC motor neurons, and in

58.5% ± 15.2% of Lhx3- HMC motor neurons (n=3, P=0.016) D. To determine if conversion of

MMC to HMC motor neurons was specific to inhibition of Notch signaling, day 6 embryoid bodies were dissociated and immunostained on day 9 of differentiation for Hb9, Lhx3, and Sst.

DnMaml1-eGFP increases the number of Hb9+ motor neurons that express Sst, E. and down regulates Lhx3 in day 9 motor neurons. F. DnMaml1-eGFP induction decreases the percentage of Lhx3+ motor neurons from 49.1% ± 1.0% to 2.0% ± 2.3% (n=3, P<.001). Sst is increased from 29.7% ± 7.4% to 66.0% ± 1.4% of motor neurons (n=3, P=.009). 107

E13.5 Spinal Cord Hb9 Sst Hb9 Sst Lhx3 A A’ Hind Hind

B B’ Rostral Cervical Rostral

C C’ Caudal Cervical Caudal

Figure 3.6

108

Figure 3.6. Sst is a marker of rostral-cervical HMC motor neurons in vivo

Serial sections of hindbrain and cervical spinal cord were stained with Sst, Lhx3, and Hb9 at 270

µM intervals. Sst immunoreactivity is observed in the dorsal horn and in subsets of interneurons at all levels of spinal cord (A-C). A’. 5X magnification of dashed rectangle in A. Sst immunoreactivity is detected lateral to (dashed arrow), but not in Hb9+ expressing motor neurons. B’. High magnification reveals that medial motor neurons at rostral-cervical levels of spinal cord express Sst (solid arrow). Lhx3 immunostaining identifies Sst+ motor neurons as medially positioned Lhx3- HMC motor neurons. C’. Lhx3 staining reveals MMC and HMC motor neurons overlap the same general position at caudal-cervical spinal cord, with HMC motor neurons having a slightly broader distribution. Sst is largely absent in caudal cervical spinal- cord and is only expressed in a few Lhx3- HMC motor neurons. 109

A

Day 3 Day 4 Day 5 Day 6 pMN MN

DAPT 3-6 DAPT 4-6 DAPT 4-5 DAPT 5-6

B 120

N.S 100

80

60

40 *

Lhx3 as % of%Cells Hb9as Lhx3 *** *** 20

0 Control DAPT 3-6 DAPT 4-6 DAPT 4-5 DAPT 5-6

Figure 3.7

110

Figure 3.7. Day 4 pMN stage is the critical period for Notch activity in controlling motor neuron columnar identity

To identity the critical period necessary for down regulating Lhx3, DAPT was administered at several time points for different duration. A. Schematic depicting DAPT treatments used to determine the critical period for Notch signaling. DAPT was administered from days 3-6, days

4-6, day 4-5, and day 5-6. B. Quantification of Lhx3 as percentage of Hb9 after various DAPT treatments. Control: 71.8% ± 6.4%, DAPT 3-6: 18.4% ± 11.7% (n=2, P=.02), DAPT 4-6:

17.8% ± 3.9% (n=4, P<.001), DAPT 4-5: 13.2% ± 3.1% (n=3, P<.001), DAPT 5-6: 75.1% ±

23.1% (n=3, P=.80). 24 hour treatment of DAPT from day 4-5 was sufficient to down regulate

Lhx3 expression, identifying the transition from pMN to motor neuron as the critical period for converting motor neuron columnar identity. 111

A Day 6 Control iNgn2 Lhx3 Hb9

B 100 N.S. 80

60

40

20

0 Lhx3 as % of%Cells Hb9as Lhx3 Control iNgn2 Figure 3.8

112

Figure 3.8. Changes in motor neuron columnar identity after Notch inhibition are not mediated by Ngn2

Analysis of Lhx3/Hb9 expression after Ngn2 induction to determine if Ngn2 mediates changes in motor neuron columnar identity. A. Hb9 and Lhx3 staining in day 6 embryoid bodies after induction of Ngn2 on day 3 of differentiation. In contrast to Notch inhibition, Ngn2 overexpression did not lead to down regulation of Lhx3. B. Quantification of Lhx3 as a percentage of Hb9+ cells. Control motor neurons were 73.0% ± 6.4% Lhx3+, while Ngn2 induced motor neurons were 73.7% ± 2.8% Lhx3+ (n=3, P=0.84). 113

Cervical Brachial Hoxa5 Hoxc8

pMN pMN

MMC HMC MMC LMC Lhx3 Sst Lhx3 Foxp1

Figure 3.9

114

Figure 3.9 Model for Notch selection of columnar cell fate

Notch signaling promotes selection of MMC motor neuron cell fate over non-MMC cell fates.

At Hoxa5 cervical levels of spinal cord, inhibition of Notch signaling results in conversion of

Lhx3+ MMC motor neurons into Lhx3-/Sst+ HMC motor neurons. Inhibition of Notch signaling at Hoxc8 brachial levels of spinal cord converts MMC motor neurons in to Foxp1+ LMC motor neurons.

115

Chapter 4: Characterization of the bHLH transcription factor network

Introduction

Accumulated studies, including the data we presented in Chapters 2 and 3, have squarely placed the bHLH transcription factors Olig2 and Ngn2 as critical players in motor neuron differentiation. Most prominently, knockout studies for both Olig2 and Ngn2 have identified severe defects in motor neuron differentiation after loss of either gene. While Olig2 mutants exhibit a complete loss of motor neurons, few motor neurons are born in Ngn2 mutants, and those that are born fail to upregulate Hb9 (Novitch et al., 2001; Scardigli et al., 2001).

Moreover, electroporation experiments in chick spinal cord and hindbrain imply that Olig2 and

Ngn2 cooperate to specify motor neuron cell fate. Individually, Olig2 and Ngn2 have a limited ability to promote motor neuron differentiation outside of the pMN domain (Mizuguchi et al.,

2001; Novitch et al., 2001). However, co-electroporation of Olig2 and Ngn2 induces motor neuron differentiation more broadly in the spinal cord and even in midbrain, where motor neurons are not normally generated (Mizuguchi et al., 2001).

In the classic model for cooperative Olig2/Ngn2 regulation of motor neuron differentiation, Olig2 first carves out the pMN domain by repressing alternative progenitor identities. Within the Olig2+ pMN domain, Ngn2 is upregulated by a subset of progenitors and, in conjunction with Isl1 and Lhx3, drives differentiation of progenitors into postmitotic motor neurons (Briscoe and Novitch, 2008). My results in Chapter 2, as well as the work of many others, suggest that the selection of Olig2+ pMNs for upregulation of Ngn2 and differentiation is controlled via Notch-mediated lateral inhibition. 116

Interestingly, the cooperative nature of Olig2 and Ngn2 in specifying motor neuron cell fate seemingly contradicts our current understanding of their molecular roles in the regulation of transcription. Ngn2 is a member of the Atonal family of pro-neural transcription factors, known for their ability to activate transcription of pro-neural gene expression programs (Sommer et al.,

1996). In contrast, Olig2 has been demonstrated to act primarily as a transcriptional repressor during motor neuron differentiation. In the previously discussed chick electroporation experiments, expression of the Olig2 bHLH domain fused to the repressor domain of the

Drosophila Engrailed (Enr) protein conveys the same patterning effects and promotion of motor neuron differentiation as full-length Olig2. In contrast, Olig2 bHLH domain fused to the Herpes simplex virus protein 16 transactivation domain (VP16) acts as a dominant negative form of

Olig2 and inhibits normal motor neuron differentiation. All bHLH transcription factors bind to

E-Box consensus sequences (CANNTG) (Murre et al., 1989), leading to the prediction that Olig2 and Ngn2 would have extremely similar binding profiles and regulate a similar cohort of genes.

This raises the interesting paradox of how two genes with similar binding profiles but opposing transcriptional regulatory activities can coordinate motor neuron-specific gene expression programs.

To address this paradox, Lee et al. characterized the function of Olig2 and Ngn2 in the transcriptional regulation of the motor neuron determinant Hb9 and found that, in agreement with their respective roles as transcriptional activator or repressor, Ngn2 activates a reporter driven by an Hb9 enhancer, while Olig2 represses it. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) experiments identified E-Box sequences in the Hb9 promoter that are bound by Ngn2 and Olig2, indicating that the effects were likely due to direct transcriptional regulation. Lee et al. then proposed a competition model for Olig2/Ngn2 117 regulation of motor neuron differentiation, where Olig2 maintains progenitor identity by repressing postmitotic motor neuron genes and differentiation, and subsequent high levels of

Olig2-induced Ngn2 would out-compete Olig2 at the Hb9 promoter to switch Hb9 transcription and initiate differentiation (Lee et al., 2005).

This model leads to the possibility that Olig2/Ngn2 competition represents a universal mechanism for controlling a cohort of motor neuron differentiation genes in addition to Hb9.

We therefore reasoned that Olig2/Nng2 co-binding might identify a cooperative gene expression program that directly promotes motor neuron differentiation. However, this hypothesis is based solely on the characterization of Olig2/Ngn2 transcriptional activity of a single gene, and whether other genes are regulated in a similar manner has yet to be established. To define the genetic motor neuron network downstream of Olig2 and Ngn2, we sought to compare genome- wide binding profiles for Olig2 and Ngn2 in pMNs.

In this chapter, I use a previously characterized inducible Olig2 ESC line to test if Olig2 induction will repress Hb9 in motor neurons (Mazzoni et al., 2011). I compared genome-wide binding profiles for Olig2 and Ngn2 to assess the level of correlation between their binding sites.

Finally, I generated gene expression profiles for pMNs after a short induction of Ngn2 and compared the consequent gene expression changes to the Ngn2, Olig2, and Ngn2/Olig2 binding data to determine the overall level of cooperation between Ngn2 and Olig2 in regulating the gene expression program driving motor neuron differentiation downstream of Ngn2.

Results

Olig2 is not an in vivo repressor of Hb9 118

The bHLH competition model predicts that changing the balance between Ngn2 and

Olig2 expression in pMNs will alter the balance of motor neuron differentiation versus progenitor maintenance. In Chapter 2, I demonstrated that Ngn2 can promote uniform differentiation of pMNs and increase the percentage of Hb9+ pMNs generated during in vitro

ESC to motor neuron differentiation. Presumably, if the model is correct, overexpression of

Olig2 will have an opposite affect and lead to repression of Hb9 and reduced motor neuron differentiation. Previously, our lab generated an Olig2-V5 inducible ESC line and verified

Olig2’s patterning role by demonstrating its ability to repress the p3 ventral domain marker

Nkx2.2 and the dorsal p2 marker Pax6 (Mazzoni et al., 2011). Furthermore, a genome-wide binding profile was generated by performing chromatin immunoprecipitation in Olig2-V5 induced pMNs using both native Olig2 antibody and V5 antibody. Olig2 binding sites were identified in both Nkx2.2 and Pax6, suggesting that Olig2 directly represses their transcription

(Mazzoni et al., 2011).

To test if constitutive Olig2 overexpression would prevent motor neuron differentiation, I treated cells with doxycycline from day 3 through day 6 of ESC to motor neuron differentiation.

Olig2 immunostaining revealed that Olig2 is still robustly expressed in the majority of cells on day 6 of differentiation after continuous doxycycline treatment (Fig. 4.1A). However, quantification of motor neurons by FACS analysis of Hb9-GFP indicated that, rather than inhibiting motor neuron differentiation, constitutive Olig2 expression results in a modest but significant increase in the percentage of cells that differentiated into Hb9-GFP+ motor neurons

(Fig 4.1B). The increase in motor neuron genesis is likely the result of an expansion of the pMN domain by Olig2 overexpression. Co-staining with Olig2 and Hb9 revealed that many cells co- expressed Hb9 and Olig2, indicating that maintained Olig2 expression is not incompatible with 119 motor neuron differentiation (Fig. 4.1A). We reasoned that while our result did not directly support the model proposed by Lee et al., it is possible that Ngn2 is able to outcompete Olig2 binding of motor neuron specific genes and thus drive their expression and motor neuron consolidation.

Ngn2 ChIP-Seq identifies genes directly regulated by Ngn2

To identify other potential motor neuron genes regulated by Olig2 and Ngn2, I profiled genomic binding sites of Ngn2 and compared them to the previously identified sites occupied by

Olig2. Taking advantage of the inducible expression of V5-tagged Ngn2, I pulled down Ngn2 for ChIP-Seq analysis using a V5 antibody. Consistent with the previously reported role of Ngn2 in inducing Dll1 expression during lateral inhibition, I found three highly significant Ngn2 peaks in close proximity (< 10Kb) to the Dll1 transcriptional start site (Fig. 4.2A). Comparisons of induced Ngn2 and control Dll1 mRNA levels by qRT-PCR showed a 3.7-fold increase in the expression of Dll1 (Fig. 4.2B), confirming Ngn2 as likely a direct transcriptional inducer of Dll1.

In agreement with my finding that Ngn2 is capable of promoting motor neuron differentiation, the previously identified Hb9 enhancer region also contains a highly significant

Ngn2 binding peak (Fig 4.2C). Interestingly, the Hb9 enhancer also contains an Olig2 binding peak, but this peak is much weaker than that for Ngn2 and is not statistically significant (Fig.

4.2C). This observation is consistent with the failure to repress Hb9 by overexpression of Olig2 in postmitotic motor neurons; thus, Olig2 is not a strong repressor of Hb9 in pMNs.

Olig2 and Ngn2 do not have similar binding profiles 120

The overall signal in the Olig2 ChIP-Seq experiments was significantly stronger than that for Ngn2, leading to the identification of 17,725 Olig2 peaks but only 5,361 Ngn2 peaks with

P<.001 (Fig. 4.3 B). Whether this reflects a true difference in binding properties between Olig2 and Ngn2 or is the result of technical variation is unclear.

Consistent with previous reports (Mazzoni et al., 2011; Seo et al., 2007), motif analyses using the top 1,000 Olig2 or Ngn2 peaks revealed E-Box (CANNTG) sequences to be the most highly represented motifs bound by Olig2 or Ngn2. Interestingly, Olig2 and Ngn2 show slightly different nucleotide preferences at the two central wobble positions. Ngn2 almost universally binds to sites where the first wobble position is guanine, and slightly prefers adenine over thymine at the second wobble position. In contrast, Olig2 displays roughly equal preferences for guanine or thymine at the first position and cytosine or adenine at the second position (Fig 4.3C).

This motif analysis indicates that Ngn2 primarily binds to two E-Box sequences (CAGATG,

CAGCTG), while Olig2 recognizes four different E-Box sequences (CAGCTG, CAGATG,

CATCTG, CATATG). The broader affinity of Olig2 for four different E-Box sequences partially explains the greater number of identified Olig2 peaks.

Considering the similarity in primary motifs bound by Olig2 or Ngn2, I was interested in examining the overlap between transcription factor recruitment to DNA. Surprisingly, I observed little correlation between Ngn2 and Olig2 binding sites in the genome-wide analysis

(Fig. 4.3A). In all, 1,925 sites, representing 36% of all Ngn2 sites and 10.6% of all Olig2 sites, were found to be co-occupied by both Olig2 and Ngn2 (Fig. 4.3B). The lower percentage of

Olig2 sites that are co-bound may be the result of the greater than threefold higher number of

Olig2 peaks identified in this study, relative to Ngn2. The finding that less than 30% of all sites 121 are shared between Olig2 and Ngn2 is surprising, as a similar degree of overlap has been reported between Hoxc9 and Olig2, two transcription factors that have completely distinct DNA binding sequences (Mazzoni et al., 2011). These data indicate that despite similar primary motif preference, Olig2 and Ngn2 are recruited to largely non-overlapping cis-regulatory regions, raising the possibility that their interaction with factor-specific DNA binding co-factors may underlie differences in their genome recruitment.

Olig2 and Ngn2 do not constitute a cooperative motor neuron binding program

As Ngn2 is capable of promoting differentiation of pMNs into motor neurons, I used microarray expression analysis to identify the direct Ngn2 downstream genetic network that programs motor neuron differentiation. In order to minimize indirect gene expression changes, we treated iNgn2 cultures with doxycycline on the evening of day 3, and isolated RNA 12 hours later on the morning of day 4. Overall, 226 unique genes are up- or downregulated by Ngn2 with a fold-change greater than 2.0 and P<.05. Reflecting Ngn2’s role as a transcriptional activator,

187 of the genes are upregulated in Ngn2-induced pMNs. However, 39 genes are downregulated, raising the possibility that, even at this early time point, some of the differentially regulated genes may not be direct targets of Ngn2.

If Olig2 and Ngn2 coordinate a motor neuron gene expression program, then Olig2/Ngn2 should be a better predictor of whether a gene will be upregulated during Ngn2-induced motor neuron differentiation. To test this hypothesis, I compared the percentage of Ngn2 only, Olig2 only, and Ngn2/Olig2 bound genes that are upregulated after Ngn2 induction (Fig. 4.4A). I defined a gene as bound by a transcription factor only if a significant binding peak is located within 10,000 bases of the transcriptional start site. Overall, 4.1% of Ngn2 only and 1.4% of 122

Olig2 only bound genes are upregulated after Ngn2 induction. Olig2/Ngn2 co-bound genes show an increase of activation to 5.8% from the 4.1% seen for Ngn2 only bound genes. As this represents only a modest increase, it is likely that Olig2 and Ngn2 do not cooperatively identify a motor neuron differentiation program.

I next asked whether Ngn2 or Olig2 binding is predictive of the directionality of gene expression after Ngn2 induction. Of the differentially regulated genes that are not bound by either of the transcription factors, 57 genes were upregulated while 22 were downregulated (Fig.

4.4B). The finding that 27% of unbound genes were downregulated indicates that many of these genes are indirectly regulated by Ngn2. Similarly, while 50 of the Olig2 bound genes are upregulated, a significant number (14 genes) are downregulated. In contrast, both Ngn2 only binding and Ngn2/Olig2 co-binding are highly selective for genes that are upregulated after

Ngn2 induction (Fig. 4.4B). This finding indicates that Ngn2 binding predicts genes that are directly activated by Ngn2 induction in pMNs. Furthermore, Ngn2/Olig2 co-binding is as predictive for Ngn2 activation as Ngn2 binding alone, indicating either that Ngn2 has stronger affinity for the promoters of these genes than Olig2 does, or that Olig2 is not an active repressor of these genes.

Genes directly regulated by Ngn2 define an early motor neuron differentiation program

To determine if genes directly regulated by Ngn2 defined a motor neuron gene expression program, we analyzed how these genes behave during normal ESC to motor neuron differentiation. In addition to the gene expression profiles for motor neurons after day 5 or 7 days of differentiation described used in Chapter 3, our lab has also generated gene expression profiles for days 0, 2, 3 and 4 of differentiation (Mazzoni and Wichterle, unpublished). 123

Together, this time series gene expression analysis describes the dynamic gene expression changes that occur throughout ESC to motor neuron differentiation (Fig. 4.4C). Strikingly, most genes directly regulated Ngn2 are not expressed at the ESC stage, but show an upward trend in expression from days 3-5 in culture after the onset of Ngn2 expression and motor neuron differentiation (Fig 4.4D). Ngn2/Olig2 co-bound genes display a similar profile (Fig. 4.4E).

Thus, genes directly regulated by Ngn2 define an early genetic program directing differentiation of pMNs into postmitotic motor neurons. Consistent with this idea, functional annotation clustering of directly regulated Ngn2 genes reveals that the three most enriched functional annotations are extracellular matrix, synapse formation, and neuronal projection. Additionally, five transcription factors, Heyl, Neurod1, Ebf2, and Nhlh1 and Zfp64 are direct Ngn2 targets, and several of them have previously been implicated in neurogenesis (Table 4.1).

Discussion

Genetic LOF and GOF studies have indicated that Olig2 and Ngn2 cooperatively specify motor neuron identity during CNS development. Combined with the fact that Olig2 and Ngn2 are both bHLH transcription factors, this has led to the hypothesis that Olig2 and Ngn2 also cooperate at the molecular level and regulate a similar set of genes during motor neuron differentiation. Sequential expression of Olig2 and Ngn2 combined with their opposing effects on gene expression suggest that a handover of enhancers from an Olig2-bound (repressed) to

Ngn2-bound (activated) state may regulate the precise onset of postmitotic genes required for motor neuron differentiation. In this chapter, I have provided two pieces of evidence to suggest that Olig2/Ngn2 competition does not play a central gene regulatory role during motor neuron differentiation. 124

Olig2 is not an in vivo repressor of Hb9

A key finding underpinning the bHLH competition model is that Olig2 is a direct repressor of Hb9. Lee et al. demonstrated this interaction by showing that Olig2 represses an

Hb9 enhancer element co-expressed in heterologous P19 cells and that Olig2 directly binds the

Hb9 enhancer element via ChIP-PCR in P19 cells and using in vitro EMSA assays ((Lee et al.,

2005). In contrast, my experiments using ESC-derived motor neurons show that Olig2 is incapable of repressing Hb9 or inhibiting motor neuron differentiation. This result is consistent with an independent study on the effects of Olig2 overexpression in postmitotic stem cell- derived motor neurons (Du et al., 2006). Moreover, genome-wide binding analysis of Olig2 in pMNs fails to find a significant Olig2 peak in the Hb9 enhancer element. These findings lead us to conclude that Olig2 is not a strong repressor of Hb9.

Ngn2 and Olig2 have disparate binding profiles

Casting further doubt on an Olig2/Ngn2 cooperative gene regulatory program, ChIP-Seq analysis of genome-wide binding for Olig2 and Ngn2 demonstrates surprisingly little overlap in the binding profiles of the two bHLH transcription factors. Since there is only a small difference in the binding preferences of Olig2 and Ngn2, we speculate that differential recruitment of the two bHLH transcription factors is mediated by differences in co-factors. Finally, the small number of genes co-bound by Olig2/Ngn2 did not exhibit a significantly higher degree of upregulation during motor neuron differentiation, as compared to genes bound by Ngn2 only.

Together these results indicate that it is highly unlikely that Olig2 and Ngn2 cooperatively regulate a cohort of motor neuron genes. Instead, Olig2 likely functions as a repressor for genes 125 that promote progenitor identity, such as Nkx2.2, Pax6, or Irx3, while Ngn2 activates a functionally distinct set of genes that promote motor neuron differentiation.

Genes directly regulated by Ngn2 represent the motor neuron differentiation program downstream of Ngn2 induction

Among the genes directly regulated by Ngn2, the most highly represented functional annotations are those associated with general processes in neuronal development, including synapse formation and neuronal projection. Additionally, several transcription factors that may transduce gene expressions changes further downstream of Ngn2 were identified. Perhaps the most interesting direct Ngn2 target is Early B cell factor 2 (Ebf2) (Table 4.1). A recent study in

C. Elegans identified the Ebf homologue UNC-3 as a gene both necessary for motor neuron identity and sufficient for inducing other neurons to adopt a cholinergic motor neuron cell fate

(Jalali et al., 2011). In chick spinal cord, Ebf1 and Ebf3 have been identified as downstream

Ngn2 effectors that are capable of promoting neuronal differentiation. However, while Ebf1 promotes general neurogenesis, it inhibits the differentiation of Isl1+ motor neurons, and instead promotes differentiation of spinal progenitors into Lim1 and CRABPI-expressing dorsal interneurons (Garcia-Dominguez et al., 2003). This highlights the intriguing possibility that

Ngn2 promotes differentiation into different neuronal classes based on contextual activation of different Ebf genes. Whereas Ebf1 results in specification of dorsal interneurons, Ebf2 may be a specifier of ventral motor neuron identity. 126

Day 6 A B Control iOlig2 70 * 60 50 GFP

- 40

Olig2 30

%Hb9 20 10 0 Control iOlig2 Hb9 Olig2 Hb9

Figure 4.1

127

Figure 4.1. Olig2 does not repress Hb9

To determine if Olig2 represses Hb9 during motor neuron differentiation, I constitutively overexpressed Olig2 from days 3-6 of differentiation and assayed the status of Hb9 and Olig2 on day 6 of differentiation. A. Continuous doxycycline treatment maintains high levels of Olig2 expression on day 6 of motor neuron differentiation. Despite prolonged Olig2 expression, many cells still differentiate into Hb9+ motor neurons. Olig2 and Hb9 are co-expressed in many cells

(yellow cells) suggesting that Olig2 is incapable of repressing Hb9, during motor neuron differentiation. B. FACS quantification of the percentage of Hb9-GFP positive cells indicates that constitutive Olig2 expression does not inhibit motor neuron differentiation. Instead, Olig2 overexpression results in a modest but statistically significant increase in Hb9-GFP positive cells from 49.6% ± 4.8% to 58.9% ± 1.9% of total cells (n=3, P=.01). 128

A B 4.5 iNgn2-V5 4 3.5 3 2.5 2 Chip Signal Chip

(Read Counts) (Read 1.5 1 0.5

iOlig2-V5 Relative Dll1 Expression 0 Control iNgn2 Chip Signal Chip (Read Counts) (Read

Dll1 C iNgn2-V5 Chip Signal Chip (Read Counts) (Read

iOlig2-V5 Chip Signal Chip (Read Counts) (Read

Hb9 enhancer Hb9 Figure 4.2

129

Figure 4.2. ChIP-Seq identifies Dll1 as a direct target of Ngn2

To determine if Ngn2-V5 ChIP-Seq identifies direct targets of Ngn2, the status of Ngn2 binding in known target Dll1 was assessed. A. Ngn2-V5 ChIP-Seq identifies three highly significant peaks near the Dll1 promoter region. Olig2-V5 also binds to the more distal Dll1 promoter sites.

B. qRT-PCR analysis of Dll1 expression finds that Ngn2 induction leads to a 3.65 ± .4 fold change in Dll1 expression, and confirms that Dll1 is directly regulated by Ngn2. C. Ngn2 ChIP-

Seq identifies an extremely significant Ngn2 bound peak directly over the previously identified motor neuron enhancer in the Hb9 promoter. In contrast, Olig2 did not significantly bind to the motor neuron enhancer. 130

A B Olig2 Ngn2 17,725 5,361 Peaks Peaks

1925 15,800 3436

C D Ngn2 Olig2

Figure 4.3

131

Figure 4.3. Comparative analysis of Ngn2 and Olig2 genome wide binding profiles

ChIP-Seq was used to probe genome wide binding for Olig2 and Ngn2. A. Scatter plot comparing Olig2 and Ngn2 reads at peaks identified for either gene. Surprisingly, despite their identity as bHLH transcription factors with affinity for E-Box motifs, Olig2 and Ngn2 binding profiles display very little correlation. B. 17,725 Olig2 and 5,361 Ngn2 peaks were identified throughout the genome. In total 1,925 peaks were co-bound by Olig2 and Ngn2, representing

10.9% of Olig2 sites and 36.0% of Ngn2 bound peaks. C. The most over-represented motif for

Ngn2(top) and Olig2(bottom) identified during motif analysis. 132

8 A C All Genes

7 4 6

Probes 5 0 4 3 -4 2 1 -8 Upregulated 0 8 Ngn2 Bound

% D All Probes Ngn2 Only Olig2 Only Ngn2/Olig2 and Bound Probesets 4 Upregulated

0 B -4 70 Upregulated 60

Normalized Signal Intensity Signal Normalized -8 Downregulated -8 50 Ngn2/Olig2 E Bound and 40 4 Upregulated 30

20 0

10 -4 0 Unbound Ngn2 Only Olig2 Only Ngn2/Olig2 -8

# of Differentially Regulated Genes Regulated of Differentially # Bound Genes 0 2 3 4 5 7 ES NP pMN yMN mMN Day of Differntiation Day 0 Expression

Figure 4.4

133

Figure 4.4. Genes directly regulated by Ngn2 constitute a motor neuron gene expression program

Microarray expression profiles were generated for Ngn2 induced pMNs for comparison with bHLH binding to identify genes that are directly regulated during motor neuron differentiation.

A. Comparison of the percentage of unbound (.6%), Ngn2 only (4.1%), Olig2 only (1.4%), and

Olig2/Ngn2 (5.9%) bound probesets that were unregulated after Ngn2 induction. B.

Comparison of the number of genes that were upregulated or downregulated after Ngn2 induction for unbound (57 upregulated, 22down) , Ngn2 only (40 upregulated, 2 downregulated),

Olig2 only (50 upregulated, 14 downregulated) and Ngn2/Olig2 bound genes (40 upregulated, 1 downregulated). C. Microarray expression profile time series generated for days 0, 2, 3, and 4 of differentiation as well as day 5 and day 7 sorted motor neurons. Red lines represent ESC gene expression program that is downregulated immediately after the initiation of differentiation.

Blue lines represent genes that are repressed at the ESC state that become highly expressed during differentiation. D. Ngn2 bound and enriched genes identify a motor neuron gene expression program that is initially not expressed in ES cells, and increase in expression from days 3-5 of differentiation just prior to the onset of motor neuron differentiation. E. Ngn2/Olig2 co-bound genes exhibit similar profile to Ngn2 bound genes. 134

Probe ID Gene Symbol Fold Change P-Value Neuronal Projection/Synapse Genes 1418950_at Drd2 2.1 6.6E-03 1450121_at Scn1a 2.3 9.2E-04 1439940_at Slc1a2 3.8 1.5E-02 1434089_at Synpo 3.0 3.3E-03 1423640_at Synpr 2.3 4.7E-02 1449866_at Syt2 2.8 4.0E-03 1415845_at Syt4 3.4 1.1E-03 Transcription Factors 1449102_at Ebf2 3.8 3.9E-03 1419302_at Heyl 6.4 2.5E-03 1426413_at Neurod1 2.6 6.5E-03 1419533_at Nhlh1 2.4 3.0E-04 1440251_s_at Zfp64 2.3 2.0E-02 Table 4.1

135

Table 4.1. Genes directly regulated by Ngn2 during motor neuron differentiation

Functional annotation clustering identifies neuronal projections and synapse as the second and third most highly overrepresented terms. Top: Neuron projections and synapse are largely represented by an overlapping set of genes involved in general neurogenesis and the generation of neuronal projects and synaptic function. Bottom: Five transcription factors are identified as direct targets of Ngn2 during motor neuron differentiation. With the exception of Zfp64, all of them have been previously identified in neurons and have putative roles in neuronal differentiation, and are candidate transcription factors for transducing Ngn2’s pro-Neural downstream gene expression program.

136

Chapter 5. Discussion and Perspectives

Lineage tracing experiments of pMN progenitors has revealed that throughout the vertebrate spinal cord, motor neurons, oligodendrocytes and astrocytes are all generated by

Olig2+ cells in the motor neuron progenitor domain (Masahira et al., 2006). Pioneering studies by figures such as George Romanes, Lynn Landmesser, and others have further demonstrated that motor neurons are not a uniform class of cells but rather can be further defined into subpopulations of motor neurons that have distinct cell body positions, trajectories, synaptic targets, and molecular profiles (Jessell et al., 2011; Landmesser, 1978, 2001). However, motor neuron diversity is established during developmental periods prior to axonal projection, arguing that motor neuron subtype is specified by signaling pathways affecting progenitors and nascent postmitotic motor neurons. While studies over the past few decades have implicated long-range patterning molecules (RA, FGF, Wnt, TGFβ) in the process of motor neuron subtype diversification (Dasen and Jessell, 2009), surprisingly little is known about the involvement of short-range regulation via the Notch signaling pathway. In this dissertation, I have identified an iterative role for Notch signaling in controlling both neuronal versus glial differentiation, and in selecting motor neuron columnar identity (Fig. 5.1). In doing so, these findings provide key insight into the lineage relationships between motor neurons and glia, as well as between different subclasses of motor neurons.

Lineage relationship between motor neurons, oligodendrocytes, and astrocytes

Although it is known that both motor neurons and glial cells are generated from Olig2+ cells in the pMN domain, whether they are produced within the same lineage, or by distinct

Olig2+ progenitors that are committed to only one cell type, has been a long standing question. 137

The experiments in Chapter 2 provide strong evidence that motor neurons and glia are generated from a common Olig2+ progenitor within the pMN domain. First, I demonstrate that cell intrinsic inhibition or activation of Notch signaling in a largely homogenous population of

Olig2+ pMNs leads to conversion of pMNs into motor neurons or glial progenitors, respectively.

This finding indicates that Olig2+ pMNs are plastic in regards to their cell fate and can be directed into either neurogenic or gliogenic lineages by Notch activity. Second, I show that inhibition of Notch signaling leads to increased motor neuron genesis and complete depletion of

Olig2+ progenitors from cultures. The complete loss of Olig2+ progenitors after increased motor neuron genesis is consistent with the idea that Notch signaling is required for preserving Olig2+ progenitors for later gliogenesis.

These findings are in sharp contrast to conclusions drawn by Wu et al., who found that in an Olig1-DTA mouse model, in which Olig2-expressing cells are continually killed off by diphtheria toxin driven by the Olig1 promoter, generation of Olig2+ cells continues past the time point at which motor neuron differentiation is normally complete (Wu et al., 2006). The authors conclude that late-born Olig2+ cells signify a separate gliogenic lineage. Instead, I propose that the continued generation of Olig2+ cells in Olig1-DTA mice is a response to injury and that, based on our results, Olig2+ cells within the pMN do indeed have the potential to differentiate into both motor neurons and glia.

I have also shown that, in addition to promoting progenitor maintenance, prolonged activation of Notch signaling induces the expression of glial genes Sox9 and Sox10. This suggests that Notch signaling plays an instructive role in specifying glial cell fate, and leads to the intriguing possibility that the motor neuron to glial cell fate switch is controlled by the 138 integration of lateral inhibition and Sox9/10 induction. In this model, Notch signaling maintains progenitor identity by inducing Hes5 and repressing pro-neural bHLH Ngn2 (Fig. 5.2A).

Concomitantly, NICD/CSL induces the expression of Sox9 and Sox10, which, at high levels, re- specify pMNs as glial progenitors (Fig. 5.2B). A feature of this putative model is that progenitor maintenance and specification of glial cell fate can be separated at the molecular level. A key prediction is that Notch induction in the absence of Sox9/10 would maintain progenitors and inhibit motor neuron differentiation, but not specify glial cell fate. Thus, the fundamental basis of this model can be tested by knocking down Sox9/10 during ESC to motor neuron differentiation and assaying whether motor neuron genesis persists in cultures beyond day 5 of differentiation.

The expression of Sox9/Sox10 in NICD induced Olig2+ progenitors indicates that they have likely adopted a glial identity, but whether they are oligodendrocyte or astrocyte progenitors remains unclear. The novel finding that Notch represses Nkx2.2 expression in glial progenitors suggests two possibilities. First, Notch may function to prevent precocious oligodendrocyte differentiation by repressing Nkx2.2, and subsequent attenuation of Notch signaling may allow Olig2+/Nkx2.2- glial progenitors to differentiate into Nkx2.2+ OPCs. While

Nkx2.2 mutant mice have reduced numbers of mature oligodendrocytes and myelination defects, they do not show changes in glial specification. This result is reminiscent of Ngn2 mutants, in which progenitors are unaffected, but motor neuron specification and maturation is impaired

(Scardigli et al., 2001). In the intestine, loss of Notch effector Hes1 leads to induction of Nkx2.2 and depletion of intestinal progenitors by precocious differentiation into postmitotic glucagon secreting cells (Jensen et al., 2000), indicating that Notch and Nkx2.2 are functionally related in intestinal progenitors. However, the functional relationships between Nkx2.2 and Notch in the 139 spinal cord are currently unknown. Moreover, demonstration of a Notch/Nkx2.2-centered lateral inhibition program in OPCs will require further characterization of the functional relationships between Notch effectors and Nkx2.2 in glial progenitors.

Alternatively, loss of Nkx2.2 may reflect that Olig2+/Nkx2.2- cells have adopted an astrocyte cell fate. Distinction between this possibility and the one described above will require further characterization of fate specification after attenuation of Notch signaling in

Olig2+/Nkx2.2- cells. Unfortunately, continued growth of embryoid bodies leads to widespread cell death after day 7 of differentiation under our current culture conditions, precluding the characterization of cell fates adopted by Olig2+/Nkx2.2- glial progenitors.

Although additional studies that define the pathways that direct Olig2+ progenitors to differentiate into motor neuron, oligodendrocyte, or astrocyte can elucidate the plasticity of pMN progenitors, they would not answer the question of whether a single Olig2+ progenitor can give rise to all three linages. Conceivably, these pathways can operate between distinct Olig2+ cells that separately differentiate into the three lineages. To definitively answer this question, clonal analysis following the progeny of individual progenitors must be performed. One potential approach to this problem would be to cross the Olig2-CreER mouse line with the Rosa26-

Confetti reporter line in which Cre-mediated recombination leads to the stochastic expression of one of four possible reporter genes (GFP, RFP, YFP, or CFP) (Snippert et al., 2010). Subsequent immunohistochemical and morphological analysis can determine whether or not individual progenitors are restricted in their potential for generating motor neurons, oligodendrocytes, and astrocytes.

Contextual Notch signaling 140

While Notch signaling controls which pMNs will differentiate into motor neurons, I have demonstrated that it also controls selection of MMC over non-MMC fates and may play a role in astrocyte versus oligodendrocyte fate as well. The ability of Ngn2 to recapitulate the increased neurogenesis but not changes in columnar cell fates after Notch inhibition, suggests that Notch signaling operates in a context-dependent manner and regulates independent molecular pathways during each of its functions. Through experiments showing that inhibition of Notch leads to both increased neurogenesis and conversion of MMC motor neurons into either HMC or LMC motor neurons, I inferred that Notch signaling preferentially selects MMC cell fate. However, manipulations that activate Notch signaling at the pMN stage completely inhibit motor neuron differentiation, and thus, I am unable to directly test whether activation of Notch promotes MMC identity after motor neuron specification. To complete our understanding of motor neuron subtype diversification, it will be critical to determine the distinct pathways controlled by Notch signaling during this selection of columnar cell fate.

My work in Chapter 4 highlights a potential approach to identify these downstream

Notch pathways. Several studies have generated gene expression and binding profiles for Notch in various cellular contexts (Hamidi et al., 2011; Li et al., 2012; Wang et al., 2011), but a detailed analysis of genome-wide occupancy and gene expression controlled by NICD has not been performed in pMNs. The inducible NICD transgene used in Chapter 2 is also tagged with a

V5 epitope and can be used in the same ChIP-Seq approach used for the Olig2/Ngn2 studies (as described in the Chapter 4) to generate binding profiles for NICD. Additionally, genome-wide occupancy analysis in NICD-induced glial progenitors (day of differentiation) and nascent motor neurons (day 5) can be used to determine if NICD binds distinct genes in each of these contexts.

Microarray gene expression profiling can be used to identify genes that are differentially 141 regulated by DnMaml1-eGFP inhibition of Notch signaling in pMNs. Comparing these two sets of data will reveal the gene expression network that is directly regulated by Notch signaling during DnMaml1-eGFP induced differentiation of pMNs into HMC motor neurons.

Furthermore, comparison with the downstream Ngn2 gene expression program identified in

Chapter 4, will distinguish genes that are involved in general neuronal differentiation by Ngn2 induction, from genes that directly regulate selection of alternative cell fate. This analysis will generate a candidate list of downstream Notch effectors that may regulate the selection of different motor neuron columnar cell fates and rescue MMC cell fate in Notch-inhibited conditions (DnMaml1-eGFP or DAPT).

Temporal progression

Similar to both the neocortex and retina, where temporal generation of neurons results in laminar organization of neurons into distinct layers (Bassett and Wallace, 2012; Desai and

McConnell, 2000), autoradiography experiments in spinal cord have indicated that there is a temporal sequence to motor neuron birth from medial to lateral (Fujita, 1964; Langman and

Haden, 1970). In contrast to these experiments, I found that inhibition of Notch signaling on both day 3 and day 4 of differentiation converts MMC motor neurons into HMC motor neurons, indicating that motor neurons are born in a synchronous fashion where columnar identity is established by heterogeneous activation of Notch signaling.

My results are incompatible with a cell autonomous model where temporal motor neuron genesis is achieved by progression of motor neuron progenitors through a series of motor column competency states. Instead, I propose that selection of MMC identity over other cell fates is the consequence of either spatial or temporal patterning of Notch signaling in the ventral spinal cord. 142

Assays of Hes5 and Dll1 expression, as wells as CSL reporter activity have demonstrated that

Notch signaling is strongly active in medial regions closest to the neural tube and is largely absent in the lateral aspect of the spinal cord (Basak and Taylor, 2007; Genethliou et al., 2009).

On account of their medial position, early motor neurons may adopt an MMC cell fate due to their higher postmitotic exposure to Notch signaling after prolonged contact with the Notch- active ventricular zone. In contrast, later motor neurons migrate further away from Notch active regions and thus adopt Notch-independent HMC and LMC cell fates.

Interestingly, this model also suggests a possible mechanism through which Notch and non-canonical Wnt cooperatively specify MMC identity through independent molecular pathways. Non-canonical Wnt signaling is frequently involved in regulating neuronal migration

(Clark et al., 2012), and in the peripheral nervous system, non-canonical Wnt interactions restricts neural crest migration to the center of somites (Banerjee et al., 2011). Thus, non- canonical Wnt potentially restricts migration of differentiation motor neuron progenitors to medial positions where they encounter higher levels of Notch ligand and adopt MMC identity.

Alternatively, Notch activity may change over time, where early high levels of Notch signaling results in adoption of MMC identity and subsequent lower levels of Notch signaling results in adoption HMC or LMC identity. This possibility is supported by multiple analyses that revealed Hes5 expression to be initially broad along the medial-lateral axis of all spinal progenitor domains at E10.5 but progressively more restricted, such that, by E12.5, it is expressed in a narrow region proximal to the neural tube (Basak and Taylor, 2007; Genethliou et al., 2009). Most likely, a combination of temporal and spatial organization of Notch signaling is required for controlling motor neuron columnar diversity. 143

The requirement of proper spatial and temporal regulation of Notch signaling in proper specification of motor columns may also explain the large number of Lhx3+ MMC motor neurons that are generated in vitro relative to in vivo. In spinal cord, late differentiating progenitors migrate further away from the Notch-activated ventricular zone and would thus differentiate into HMC or LMC motor neurons. Embryoid bodies do not have the same stereotypical ventricular, intermediate, and mantle layers exhibited by spinal cord and may have more uniform Notch signaling due to the prolonged intermingling of pMNs and motor neurons.

As a consequence, differentiating pMNs are exposed to higher levels of Notch signaling than they would be in vivo, and may selectively prefer Notch-dependent MMC identity. To distinguish between these possibilities, detailed analyses of motor column identity after altering the spatiotemporal pattern of Notch signaling during in vivo motor neuron development will have to be performed.

Conclusion

In conclusion, my studies have built upon the current understanding of neural diversity within the pMN domain by defining a role for Notch signaling during two crucial processes.

First, I demonstrated that Notch signaling controls the neuronal to glial temporal cell fate switch by both maintaining progenitors via lateral inhibition and specifying glial identity by promoting expression of Sox9/10. I also identified a novel potential role for Notch signaling in regulating glial cell fate specification through regulation of OPC marker Nkx2.2. Finally, I determined that

Notch controls intra-segmental motor neuron diversity by selecting motor neuron columnar identity. 144

These findings not only fill gaps in our understanding of neural diversity in the pMN domain but may provide key insights into other areas of developmental biology. While Notch is known to regulate neuronal diversity via lateral inhibition and selection of alternative cell fates in

Drosophilia, the distinct targets that Notch regulates during each of these activities are not known (Udolph, 2012). Our identification of the pMN domain as a region where Notch plays a contextual role in regulating motor neuron differentiation through both lateral inhibition and selection of alternative cell fates, provides an attractive model for studying the molecular targets of Notch during each of its contextual roles. Many of the tools generated in this dissertation can be used to probe the molecular pathways that are regulated by Notch signaling in each of its contextual roles.

While Notch signaling controls asymmetric division in all Drosophila neuroblast lineages, until recently there was very little evidence that Notch signaling plays a similar role in the vertebrate CNS. Our study demonstrates that Notch signaling does indeed control selection of alternative cell fates and bolsters the idea that Notch contributes to neuronal diversity within other neurogenic regions. In the neocortex, neural progenitors are temporally patterned and progress through a series of competency states, during which they give rise to different types of neurons that migrate radially, and occupy distinct layers of the neocortex (Desai and McConnell,

2000). However, each of these layers still exhibits a large degree of neuronal diversity, and the signaling pathways that control differentiation of neural progenitors into different neurons within a layer is not understood. A role for Notch signaling in controlling neuronal subtype identity in the neocortex would further our understanding of neuronal development in the neocortex and demonstrate that selection of alternative cell fates by Notch signaling is a conserved mechanism operating in multiples regions of the vertebrate CNS. 145

Olig2

Notch

Olig2 Olig2 Sox9 Sox10 Ngn2

Notch Notch

Olig2 OPC Sox9 + - Sox9 Sox10 Lhx3 Lhx3 Sox10 Nkx2.2

MMC HMC LMC

OPC? Astrocyte? Oligodendrocyte Figure 5.1

146

Figure 5.1. The iterative role of Notch signaling during motor neuron differentiation

Joint model describing cell fate decisions in the pMN domain controlled by Notch signaling. In the first step Notch both inhibits motor neuron differentiation and promotes glial progenitor identity. Subsequently, in differentiating pMNs, Notch signaling promotes differentiation of

MMC motor neurons over HMC or LMC motor neurons. In glial progenitors, Notch also inhibits oligodendrocyte differentiation by repressing Nkx2.2, and potentially promoting astrocyte cell fate. 147

A pMN B Glial Progenitor

NICD NICD Sox9/10

Hes Hes

Ngn2 Nkx2.2

C Selection of Columnar Identity

NICD

?

Lhx3

Figure 5.2

148

Figure 5.1. Genetic networks controlled by Notch signaling

Genetic regulatory pathways for Notch in each of its contextual roles. Solid lines indicate interactions demonstrated in this dissertation. Dashed lines represent putative interactions that have not been demonstated in the pMN domain A. In pMNs, Notch operates through the classic

Notch lateral inhibition pathways. Activated Notch induces the expression of Hes transcription factors that repress Ngn2 and inhibit motor neuron differentiation. B. Notch induces the expression of Sox9/10 to promote glial identity of Olig2+ progenitors. However, Notch also regulates glial differentiation by repressing Nkx2.2. C. During selection of alternative motor neuron columnar identities, Notch promotes MMC identity by inducing Lhx3 expression.

Induction of Lhx3 is likely indirect through a currently unknown factor.

149

Chapter 6: Materials and Methods

Chick electroporation

Electroporation of plasmid DNA into chick neural tube was performed as previously described (Kania). Experimental DNA was diluted to a final concentration of 1µg/µl, and vehicle plasmid without a promoter (PRSet) was used to raise the final DNA concentration to

2.5µg/µl in order to increase the viscosity of the DNA solution. 1.0mm capillary tubes (FHC) were pulled into needles (Sutter Instruments) and used to inject DNA into the neural canal of

Hamburger Hamilton Stage 12-15 developing embryos. Embryos were electroporated using a

BTX Electro Square Electroporator (BTX) at 25 volts, for 50ms, 1.0 second intervals for 8 pulses. Embryos were collected after 48 hours at approximately HH Stg 21 and were fixed in

4% PFA for one hour at 4°C and processed for immunohistochemistry.

Generation of inducible lines

Inducible ESC lines were generated as previously described (Mazzoni et al., 2011). Open reading frames of desired genes were first cloned using high fidelity Phusion polymerase (New

England Biolabs), and directionally inserted into the Gateway pENTR/D-TOPO vector (Life

Technologies). Subsequently, LR clonase was used to transfer open reading frames from the pENTR vector into a modified p2Lox plasmid where GFP was replaced with the L1-L2 cassette from the pDEST40 Gateway destination vector. Included in the cassette was a downstream V5-

His sequence that was in frame with the L2 recombination site and followed by a stop codon.

For generation of inducible lines with V5 tag, the initial PCR reaction was performed such that the stop codon was removed from the original open reading frame. 150

Inducible ESC lines were generated from a parental ESC line harboring the RTA transactivator at the Rosa-26 locus and the Tet-on inducible promoter at the HPRT locus

(Khyba). Downstream of the Tet-on promoter was a Cre-Recombinase gene flanked by loxp sites. ESC cells were pre-treated with doxycycline for 24 hours to robustly induce Cre expression, and p2Lox plasmids bearing the gene of interest were electroporated into the Cre induced ESCs. Recombination resulted in replacement of the Cre-recombinase gene with the desired construct and a neomycin resistance gene driven by the PGK reporter. Recipient ESC cells were treated with G418 at a concentration of 250 ng ml−1 to select for recombined colonies.

After more than 10 days of selection ESC colonies were picked and expanded for analysis.

Recombination was confirmed by inducing putative inducible ESC lines with doxycycline and immunostaining with V5 or other antibodies.

ESC to motor neuron differentiation

ESC to motor neuron differentiation was optimized from previously published protocols

(Peljto and Wichterle, 2011; Wichterle et al., 2002). To start differentiation, 250,000 ES cells were plated in 10cm adherent tissue cultures dishes (Nunc) in motor neuron differentiation medium consisting of: Advanced D-MEM/F-12 (Life Technologies) : Neurobasal Medium (1:1 ratio) (Life Technologies), 10% Knockout serum replacement (Life Technologies), 200mM L-

Glutamine (Life Technologies), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich). On day 2 of differentiation, embryoid bodies were collected and split into four 10 cm non-adherent tissue culture dishes (Corning) and supplemented with 1µM of RA (Sigma-Aldrich) and .50 µM of

Smoothened Agonist (SAG, Calbiochem) for Hb9-GFP ESC lines, or .25 µM for all other ESC lines. SAG and RA were washed out on day 5 of differentiation and cells were re-plated in fresh 151 motor neuron differentiation medium. For analysis of pMNs, embryoid bodies were collected at day 4 of differentiation. For analysis of motor neurons, embryoid bodies were collected on day 6 of differentiation.

Induction of transgenes and DAPT treatment

To assess the role of transgenes in pMNs during motor neuron differentiation, doxycycline was added to cultures at a concentration of 2 µg/ml. To inhibit Notch signaling in pMNs during motor neuron differentiation, DAPT was added to the cultures at a concentration of

5 µg/ml (Sigma-Aldrich). To prevent precipitation, DAPT was pre-diluted in DMSO to a concentration of 5mg/ml. To induce expression of transgenes in pMNs, 2 µg/ml of doxycycline

(Sigma-Aldrich) was added on day 3 of differentiation.

Embedding of embryoid bodies for immunocytochemistry

Embryoid bodies were collected at either day 4 (pMN) or day 6 (motor neuron) of differentiation and fixed in 4% PFA for 10 minutes at 4°C. After several washes Embryoid bodies were incubated in 30% sucrose until they equilibrated and sank by gravity. Embryoid bodies were then deposited in square molds (Polysciences) filled with optimal cutting temperature (OCT, Tissue-Tek) embedding media, frozen in dried ice, and stored at -80°C until cryosectioning.

Dissociation of embryoid bodies for motor neuron culture

Day 6 embryoid bodies were dissociated using 0.05% Trypsin-EDTA (Life

Technologies) and plated on 15mm coverslips (Carolina Biological) in four well dishes (Nunc).

Coverslips were first sterilized by incubation for 1 minute in a Plasma Cleaner (Harrick Plasma 152

Cat# PDC-32G). Following sterilization coverslips were first coated overnight in 0.001% poly-l- ornithine (Sigma-Aldritch) diluted in water and then overnight in mouse laminin diluted to

5ng/ml (Life Technologies). Dissociated motor neurons were plated in motor neuron medium consisting of Advanced D- MEM/Neurobasal(1:1) (Gibco), B27 supplement (Life

Technologies), 200mM L-Glutamine (Gibco), and 10% Knockout serum replacement (Life

Technologies). Medium was supplemented with GDNF (R&D Systems) at a concentration of

5ng/ml. On day 9 of differentiation, cells were fixed in 4% PFA for 15 minutes and room temperature and processed for immunocytochemistry.

Dissection of chick and mouse spinal cords

Mouse and chick embryos were dissected into cervical, brachial, thoracic and lumbar segments, and deposited into OCT filled plastic molds with the rostral side facing down.

Cryosectioned transverse sections of spinal cord were collected on frost free glass slides for immunostaining.

Immunocytochemistry/Immunohistochemistry

Immunostaining of tissues was performed as follows. Tissues were first permeabilized by incubation in a .5% solution of Triton X-100 (Sigma-Aldrich) for 2 minutes. Blocking was done in 10% FBS at room temperature for one hour, followed by primary staining overnight in

2% FBS. After 4 washes at 10 minutes, secondary antibody was applied in 2% FBS for 2 hours at room temperature. After four additional washes, coverslips were mounted and imaged.

Antibodies 153

Commercial antibodies used in this thesis include: rabbit anti-Olig2 (Chemicon), rabbit anti-NICD (Abcam), mouse anti-Isl1/2 (Developmental Studies Hybridoma Bank, DSHB), mouse anti-Hb9 (DSHB), mouse anti-Lhx3 (DSHB), mouse anti-Nkx2.2 (DSHB), mouse anti-

Ki67 (BD Biosciences, BD)mouse anti-Esrrg (Perseus Proteomics, PPX), sheep anti-Onecut2

(Research and Development Systems, R&D), rabbit anti-Onecut1 (Santa Cruz Biotechnologies,

SCB), rabbit anti-Sox9 (Abcam), rabbit anti-Sox10 (Chemicon) . Non-commercial antibodies use in this thesis include: mouse anti-Ngn2 (generous gift of David Anderson), guinea pig anti-

Hb9 (generous gift of Project A.L.S). The following antibodies were generous gifts from Tom

Jessell: guinea pig anti-Olig2, rabbit anti-Hoxa5, mouse anti-Hoxc8, rabbit anti-Lhx3, sheep anti-GFP, goat anti-Sp8, guinea pig anti-Foxp1, rabbit anti-Foxp1.

Imaging

All images were acquired using a confocal laser microscope (LSM Zeiss Meta 510).

Quantification of Chick spinal cord electroporations

For quantification of Olig2 and Hb9 in H1B-eGFP, DnMaml1-eGFP, and NICD electroporated spinal cord, Olig2 and Hb9 were co-immunostained with either anti-GFP or anti-

NICD antibodies. The number of GFP or NICD positive cells that were Olig2, Olig2/Hb9, or

Hb9 positive was counted in three sections of spinal cord in three independent electroporations.

Quantifications were limited to the pMN domain as marked by the dorsal/ventral limit of Olig2 and Hb9 expression.

Quantification of embryoid bodies 154

The multi wavelength sorting application in MetaMorph (Meta Imaging Series Software

7.1, Molecular Devices) was used to perform quantification of nuclear markers in embryoid bodies. For quantification of Olig2/Ki67 as a percentage of total cells, Olig2 and Ki67 were counter stained with nuclear marker TO-PRO and MetaMorph was used to calculate the percentage of TO-PRO stained cells that were immunoreactive for Olig2 and Ki67. Similarly, for quantification of the percent of MNs that were positive for a particular transcription factor,

Hb9 or Isl1/2 was co-stained with antibodies targeting the desired transcription factor.

MetaMorph multi wavelength sorting was used to determine the percentage of Hb9+ or Isl1/2+ nuclei that were also positive for the given transcription factor. At least 3 frames (>1000 cells) were quantified for each sample and n ≥ 3 unless otherwise noted.

Quantification of dissociated cells

Day 9 motor neurons were co-stained with antibodies against Hb9, Lhx3, and Sst. The number of Lhx3+, Sst+, and Lhx3+/Sst+ Hb9 MNs was counted. For each sample, at least 10 fields (>100 cells) were quantified for each experiment (n=3).

Statistical analysis

Quantifications were reported as average ± standard error of mean (SEM). Statistical significance was calculated using two-tailed Student’s t-test with unequal variance. Relevant p values were as follows: *p=0.01-0.05, **p=0.001-0.01, ***p<0.001.

RNA purification

RNA purification was performed using Trizol or Trizol LS RNA extraction (Life

Technologies) to separate DNA, RNA and protein into aqueous fractions. RNA supernatant was 155 then further purified using RNeasy (Qiagen) spin column mini kits. RNA concentration and quality was determined by Nanodrop spectrophotometer and stored at -80° C. qRT-PCR

cDNA was first prepared using Superscript III Reverse Transcriptase per the manufacturer’s instructions (Life Technologies). cDNA was then mixed with .5 µM of desired primer and 2X SYBR green qRT-PCR mix (Stratagene), and analyzed using an MX3000P QPCR

System (Stratagene). Primers used are indicated in Table 6.1.

Microarray preparation and analysis

Amplified cDNA was generated from RNA using OvationTM WTA Pico v2 cDNA preparting kit (Nugen, cat# 3302-12), and subsequently labeled and fragmented using the

EncoreTM Biotin module (Nugen, cat.# 4200-12). Labeled cDNA was hybridized on Mouse

Genome 430 2.0 Array microarrays (Affymetrix), and analyzed using GeneSpring 11.0

(Agilent). For the DAPT and aged MN analysis, differentially expressed genes were identified by greater than fourfold enrichment in either DAPT versus untreated or day 7 versus day 5 MNs.

In the Ngn2/Olig2 binding analysis, differentially expressed genes were identified by greater than two fold change and P<.05.

ChIP-Seq preparation

Chromatin immunoprecipitation was performed as previously described (Mazzoni et al.,

2011). Subsequently, DNA fragments from ChIP were sequenced using a Solexa Genome

Analayzer II (Solexa).

ChIP-Seq analysis 156

Chip-Seq data was analyzed using GPM software (Guo et al., 2010). Significant peaks were identified by those with a P<.01 using a binomial distribution. Consensus sequences were generated using GimmeMotifs software (van Heeringen and Veenstra, 2011).

157

Gene Symbol 5' Forward Sequence Primer 3' Reverse Sequence Primer Dll1 CCAGTCGGTGTATGTTCTGTCT GTTTCCTCTCCCTTCCTCTCTC Hes5 AGATGCTCAGTCCCAAGGAGAA GGCTTTGCTGTGTTTCAGGTAG Ngn2 AGACAACCACGCACGAGAAC ACCTCCTCTTCCTCCTTCAACT Table 6.1

158

Table 6.1. qRT-PCR primers.

Sequences of qRT-PCR primers used in this study. In all cases, designed primers span exon- intron boundaries.

159

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