INACTIVATION OF ERK1 AND ERK2 DISRUPTS CORTICAL

PROGENITOR PROLIFERATION LEADING TO ABNORMAL

CYTOARCHITECTURE, CIRCUITRY AND BEHAVIOR, MODELING

HUMAN NCFC AND RELATED SYNDROMES.

by

JOANNA PUCILOWSKA

Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Gary E. Landreth

Department of Neurosciences

CASE WESTERN RESERVE UNIVERSITY

August 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Joanna Pucilowska

candidate for the PhD degree*.

(signed) Robert Miller (chair of the committee)

Gary Landreth

Jerry Silver

Stephen Maricich

(date) 5/29/2012 *We also certify that written approval has been obtained for any proprietary material contained within.

2

TABLE OF CONTENTS List of figures……...……………………………………………………………….….6

ABSTRACT…...………………………………………………………………….…..9

CHAPTER 1:

INTRODUCTION………………………………………………………….11

MAP KINASE Signaling Pathway………………………………………………...11

MAPK Specificity: The Right Place at the Right Time………………………..…16

ERKs and Isoform Specificity………………..…………………………………….23

ERKs in Learning and Memory………………………..………………………….26

ERKs and their FGF Ligands………………………..…………………………….28

CORTICAL DEVELOPMENT:

Forebrain Regionalization and Morphogenesis………..…………………31

The Role of Cell Cycle in Corticogenesis……..…………...………………33

ERKs and the Cell Cycle Progression…………………..…………………36

Progenitor Proliferation and Neurogenesis………………..……………...39

Migration…………………..………………………………………………..45

Gliogenesis…………………………………………………………..………47

Differentiation……………………………………………………..………..48

Synaptogenesis…………………………………………………..………….50

Pathology Associated with Cortical Development……………………..…………52

Developmental Disorders of the MAP Kinase Pathway…………………..……...57

ERK and Autism……………………..……………………………………………..63

Research Goals……...………………………………………………………..……..65

3 CHAPTER 2:

Disrupted ERK signaling during cortical development leads to abnormal progenitor proliferation, neuronal and network excitability and behavior, modeling human NCFC and related syndromes……………………………..…...67

Abstract…………………………………………………………………..….68

Introduction………………………………………………………..………..69

Materials and Methods………………………………………………..……71

Results…………………………………………..……………………...……82

Discussion………………………………………..………………………….95

Literature Cited……….……………………………………………..…....101

Figures…………………………………………..……………………….....106

CHAPTER 3:

The Role of ERKs in Interneuron Migration………………………………..…..134

Introduction………………………………..………………………………134

Results…………………………………………..………………...………..139

Discussion……………………………………………..……...……………146

CHAPTER 4:

DISCUSSION…………………………………………………….……………….149

FGF Signaling, ERKs and Cortical Development……………………....152

ERKs and Cortical Progenitor Dynamics……………………………..…157

ERKs and Cognition…………………………………...…………..……...160

Disorders of the Ras/MAPK pathway and Cognitive Deficits…….....…166

4 The contribution of MAPK/ERK signaling to Autism Spectrum Disorders

associated with copy number variation of 16p11.2……………….…..…171

Potential Impact on Autism Research…………………..………………..176

Towards a Treatment for Developmental Disorders of the MAPK

Pathway……………..………………………………………..…………….177

Conclusion……………………………………………………………..…..180

Literature Cited……………………………………………………..…….181

5 LIST OF FIGURES

Figure 1-1 Mitogen Activated Protein Kinase cascade………………………..….12

Figure 1-2 Basic signal transduction pathway mediating ERK activation…..….15

Figure 1-3 Progression through the cell cycle is controlled by ERKs………..….39

Figure 1-4 Division modes of the expansion of the cerebral cortex in mice:

Lateral and Radial………………………………………………………………….41

Figure 1-5 Disorders of the MAP Kinase Pathway………………..……………...60

Figure 2-1 ERK activity is abrogated in the dorsal telencephalon of ERK2 CKO

and ERK1/2 DKOs……………………………………………………………..….106

Figure 2-2 Loss of ERK1 and ERK2 leads to layer perturbations in the adult

cortex……………………………………………………..………………………...109

Figure 2-3 The frequency of neurons generated during mid-neurogenesis is

altered in mutant mice lacking ERK1 and/or ERK2…………………………....111

Figure 2-4 ERK2 CKO mice show reduction in Pax6+ progenitors, mitotic radial

glia and transition to intermediate progenitor cells…………………………..…113

Figure 2-5 Loss of ERK2 disrupts basal progenitor frequency and generation,

resulting in premature progenitor pool depletion…………………………….....115

Figure 2-6 ERK2 CKO neural progenitors exhibit premature cell cycle exit and

lengthening of the cell cycle during mid-neurogenesis………………..………...117

Figure 2-7 Loss of ERK2 alters expression of key cell cycle regulators cyclin D1

and p27Kip1 in the developing cortex………………..……………………………120

6 Figure 2-8 Cell-intrinsic electrophysiological parameters are altered in ERK2

mutant mice………………………………………………………………..………122

Figure 2-9 Network-level electrophysiological parameters are altered in Erk2

mutant mice…………………………………………………………………..……124

Figure 2-10 Loss of ERK2 leads to behavioral alterations and cognitive

impairment in adult mice…………………………………………………….…...126

Supplementary Figure 2-1 The cortical size and body weight is reduced in the

ERK2 CKO and DKO mice………………………………………………..…..…128

Supplementary Figure 2-2 Apical and basal mitosis are reduced in the CKO and

DKO mice during mid-neurogenesis…………………………………..…………130

Supplementary Figure 2-3 Cell death in not significantly altered in the

developing dorsal telencephalon of CKO mice……………………..…………...133

Figure 3-1 Do laminar alterations in the dorsal telencephalon caused by ERK1/2

deletion affect the migration and final positioning of the sub-pallially derived

interneurons?...... 138

Figure 3-2 The migration pattern but not the total number of SST+ interneurons

is altered in the ERK2 deficient dorsal cortex at E13.5……………………...….141

Figure 3-3 The migration pattern but not the total number of SST+ interneurons

is altered in the ERK2 deficient dorsal cortex at E16.5……………...………….143

Figure 3-4 Loss of ERK2 from dorsal telencephalon results in an increase in

SST+ interneurons in dorsal but not lateral postnatal cortex……………...…..145

Figure 3-5 Loss of ERK2 from dorsal telencephalon results in a decrease in

Calretinin+ interneurons in the cortex of P10 mice……………………….……146

7 Figure 4-1 Cell cycle progression during cortical development……………...... 151

Figure 4-2 The Wnt and Notch developmental pathways that are important in

corticogenesis are not altered in ERK deficient mice…………………..……….156

Figure 4-3 ERK deficient mice exhibit changes in hippocampal volume and

severely reduced Tbr2+ progenitor population in the dentate gyrus……...…..164

Figure 4-4 Mutations in upstream and downstream elements of the MAPK/ERK pathway lead to NCFC and other developmental syndromes………..……...…167

Figure 4-5 General schematic illustrating changes in cortical development in

ERK deficient mice………………………………………………………………..170

Figure 4-6 The cell cycle dependent mechanism of ERK action during cortical

development……………………………………………………………………….171

8 The Role of ERKs in Cortical Development

ABSTRACT

by

JOANNA PUCILOWSKA

Genetic disorders arising from copy number variations in the ERK1 and

ERK2 MAP kinases and mutations in their upstream regulators result in Neuro- cardio-facial cutaneous (NCFC) syndromes that are a significant genetic cause of mental retardation in humans. Furthermore, these disorders are associated with developmental abnormalities, cognitive deficits and psychiatric disease. Deletions or duplications of the human ERK genes provide the strongest genetic linkage to autism spectrum disorders and to distal 22q11 syndrome. Our findings demonstrate that deletion of one or both ERK isoforms at the beginning of neurogenesis disrupts apical and basal progenitor generation and proliferation, altering cortical cytoarchitecture of the adult brain in a gene-dose dependent manner. The changes in proliferation dynamics were due to ERK-dependent regulation of the potent cell cycle progression regulators, cyclin D1 and p27Kip1. Loss of ERK signaling in dorsal telencephalic progenitors resulted in altered levels of cyclin D1 and p27Kip1 leading to premature

elongation of the cell cycle, which favors neurogenic over self-renewing divisions.

The precocious neurogenesis caused premature progenitor pool depletion,

subsequently altering the number and distribution of cortical pyramidal neurons in the

postnatal cortex. Importantly, loss of ERK2 alters the intrinsic excitability of cortical

9 neurons and contributes to global perturbations in network activity. Together, these molecular and physiological changes observed in the ERK-deficient mice may contribute to severe anxiety and cognitive deficits observed in these mice. This study describes in detail the functional mechanisms through which ERKs act to regulate cortical development, providing a novel insight into their important role in normal brain function as well as why deregulation of this pathway may contribute to the pathology associated with autism and related cognitive disorders.

10 INTRODUCTION:

All eukaryotic cells possess multiple mitogen-activated protein kinase

(MAPK) pathways, which coordinately regulate diverse cellular functions. The mammalian family of MAPKs is divided into five distinct subfamilies, based on sequence homology, activity and substrate specificity. The MAPK family includes the

Extra cellular signal Regulated Kinases (ERK1 and ERK2, also known as p44 and p42, respectively), the stress activated protein kinases p38MAPKs (α, β and γ) and c-

Jun NH2-terminal kinases (JNK1, 2 and 3), as well as ERKs 3, 4 and 5. Each member of this family is part of a core module of classical three-tier kinase signaling cascade which is activated by diverse intracellular and extracellular stimuli such as growth factors, hormones and cytokines as well as oxidative stress (Figure 1). The activity of these enzymes is tightly regulated by their phosphorylation status. They undergo sequential phosphorylation leading to their enzymatic activity as well as dephosphorylation by their phosphatases. All members of the MAPK superfamily are activated by dual phosphorylation on tyrosine and threonine residues within a highly conserved Thr-Pro-Tyr (TPY) motif and upon phosphorylation translocate from the cytoplasm to the nucleus (Mizukami et al., 1997). Aberrant activation of the JNK or p38 stress pathways has been linked to neuronal apoptosis in context of neurodegenerative diseases including Parkinson’s, Alzheimer’s and amyotrophic lateral sclerosis. On the other hand, the ERK signaling pathway plays a crucial role in cell proliferation, migration and tumorigenesis (O’Neill and Kolch, 2004). The ERKs are best studied in the context of cancer due to their role in growth-factor-stimulated

11 cell-cycle progression. The JNK and p38 signaling pathways are activated by

genotoxic, osmotic, hypoxic or oxidative cellular stressors and pro-inflammatory

cytokines such as tumor necrosis factor (TNF)-alpha and interleukin 1β (IL-1beta).

The ERKs are preferentially activated by growth factors and phorbol esters (Pearson

et al., 2001). The MAPK cascades are subject to multiple levels of regulation that includes scaffold proteins, inhibitors, and compartmentalization mechanisms as well

as numerous positive and negative feedback loops. At present, there are nearly 70

genes encoding close to 200 distinct components of the entire MAPK system. This

vast number of different components allows for the diverse functions, cell type

specificity and tight regulatory control, which are hallmarks of these highly conserved

signaling cascades.

Figure 1-1.

Mitogen Activated Protein Kinase cascade.

12

The canonical Mitogen Activated Protein kinases (MAPK), ERK1 and ERK2, are the central elements of three tier protein kinase cascade. During development they are primarily activated by mitogens such as growth factors, which act through the extracellular part of receptor tyrosine kinases (RTKs). Receptor tyrosine kinases are imbedded in the plasma membrane and upon ligand binding homodimerize, achieving full transactivation through autophosphorylation on several tyrosine residues which leads to conformational change of the intracellular domain. Subsequently, these changes allow for recruitment of scaffolding proteins which bind to the newly phosphorylated tyrosine residues. The activated signaling pathway leads to recruitment of other signaling proteins with characteristic domains (homology 2

(SH2), phosphotyrosine (PTB) and PDZ domains) that recognize and bind to

13 phospho- tyrosine residues. Together with adapter proteins, such as Shc and Grb2, signaling complexes are formed near the plasma membrane which bind additional scaffolding proteins such as FRS2, Crk and CrkL. This ultimately leads to activation of guanine nucleotide exchange factors (SOS and C3G) which facilitate the activation of the RAS family of small G-proteins which include RAS and RAP1 (Figure 1-2),

(Kerkhoff et al., 2001). The activation of these small G proteins, in turn, initiates a downstream chain of phosphorylation events where serine/threonine MAP kinase kinase kinases, (MAP3Ks: B-raf, A-raf and c-raf) phosphorylate and activate the dual specificity MAP kinase kinases (MAP2Ks: MEK1/2), the only known activators of the extracellular signal regulated protein kinases (ERK1 and ERK2) (Hallberg et al.,

1994). Incidentally, activation of only 5% of RAS is necessary for full activation of

ERKs (Hallberg et al., 1994). ERK1 and ERK2 are proline-directed serine/threonine kinases which are phosphorylated by MEKs on Threonine 183 and Tyrosine 185, which are located in the activation loop (Thr-Glu-Tyr motif) of these enzymes (Roux and Blenis, 2004; Rubinfeld and Seger, 2005). Two genes, located on different chromosomes, are known to encode the ERKs, and those are designated as ERK1

(MAPK3) and ERK2 (MAPK1). These two genes encode two main proteins, p44 and p42, respectively. The ERKs are ubiquitous Ser/Thr kinases that phosphorylate hundreds of substrates either in the cytosol (e.g., PLA2, RSK), or upon translocation into the nucleus (e.g., Elk1, c-fos, c-myc).

The activated ERK kinases transduce extracellular environmental cues to nuclear and cytoplasmic effectors, thus influencing gene regulation and many cellular

14 activities including cell proliferation, differentiation, survival and metabolism (Pages et al., 1993; McCubrey et al., 2006; Torii et al., 2006; Dhillon et al., 2007). They are highly expressed within the brain and are essential for normal development as well as learning and memory (Sweatt et al., 1996). In addition, the ERKs induce phosphorylation of a staggering number of proteins, as it was reported that ERKs have at least 160 known substrates (Yoon et al., 2006).

Figure 1-2.

Basic signal transduction pathway mediating ERK activation.

15 MAPK Specificity: The Right Place at the Right Time

Although MAP Kinases ERK 1 and ERK2 become activated during a wide

range of biologically diverse processes, how they achieve their specificity has not been studied until recently. Nearly all growth factors and cytokines can bind to cell-

surface receptors such as receptor tyrosine kinases, initiating a cascade of signaling

events which promote the activation of RAS and, ultimately, dual phosphorylation

and activation of the ERKs. In turn, activated ERKs phosphorylate downstream

targets such as the family of ~90-kDa ribosomal S6 kinases (RSKs), which consists of

RSK1 to RSK4 and mitogen- and stress-activated kinases 1 and 2 (Roux and Blenis,

J. 2004). When activated, ERKs can also translocate into the nucleus where they

activate major transcriptional regulators such as Elk-1, CREB and H3, which in turn

induce transcription of immediate early genes (IEG). IEGs, such as c-fos, c-jun and c-

myc are potent regulators of cell cycle progression and are implicated in many

malignancies (Sharrocks, A.D. 2001). The strength and duration of their expression,

which depends strictly on the dynamics of the ERK signal, serves as a potent cellular

sensor mediating cell cycle dynamics, apoptosis and differentiation.

It is very clear that ERKs are involved in a diverse range of biological

functions that are very distinct or even mutually exclusive. This can only partially be

explained by cell type specificity, because ERK activation has distinct outcomes even

in the same cell type (Schaeffer and Weber, 1999; Tan and Kim, 1999). Presently,

five different mechanisms which determine ERKs’ specificity have been proposed.

They include: a) signal dynamics with distinct duration and strength; b) interaction

16 with various scaffold proteins that direct elements of the MAPK cascade to specific upstream and downstream substrates; c) interactions with other signaling pathways that are activated or inhibited simultaneously with the MAPK pathway; d) specific subcellular localization of different components and targets of the MAPK pathway which aids in compartmentalization of the signal; e) presence of multiple components with distinct specificities in each level of the cascade.

The specificity of ERK signaling can be regulated by the strength and the duration of the ERK signal. This can be at least partially modulated by factors such as cell-surface receptor density and expression and localization of scaffolding proteins and other kinases and phosphatases as well as the surrounding extracellular matrix.

Upon stimulation, ERK activity increases within minutes (2-30 min) and then declines back to basal levels. This fast decline (within 15-40 minutes) is associated with activation kinetics named “transient activation,” whereas slower decline lasting up to 180 minutes is termed “sustained activation.” The duration of ERK activity has been implicated as a critical factor in cell fate decisions. Experiments in PC12 cells showed that the duration of the ERK signal alone can lead to differential cellular responses determining cell fate. For example, PC12 cell data clearly demonstrates that sustained activation always leads to ERKs translocation into the nucleus whereas transient activation does not result in nuclear translocation (Traverse et al., 1992,

1994; Dikic et al., 1994). Therefore, sustained ERK activation will consequently result in nuclear accumulation of activated ERKs and subsequent phosphorylation or activation of many transcription factors. In addition, different growth factors can influence the biological outcomes in a specific cell type. Treatment of PC12 cells

17 with nerve growth factor (NGF) induces sustained ERK activation and differentiation.

On the other hand, epidermal growth factor (EGF) stimulates transient ERK activation and causes cell proliferation (Gotoh et al., 1990; Nguyen et al., 1993;

Marshall, 1995). Therefore, during development subtle changes in ligand concentration dictated by morphogenic gradients can contribute to sustained versus transient ERK activation and therefore influence nuclear translocation and alter gene expression (Dikic et al., 1994; Traverse et al., 1994).

Differences in receptor number can affect the duration of the ERK signal as well. This can be achieved by variable rate of receptor internalization and post activation receptor down-regulation. In PC12 cells, overexpression of the EGF receptor leads to sustained ERK activation, which results in differentiation instead of proliferation (Traverse et al., 1994). In contrast to PC12 cells, sustained, but not transient, ERK activation is necessary for quiescent fibroblasts to enter S phase and proliferate (Dobrowolski et al., 1994; Balmanno and Cook, 1999). In addition, differential ERK kinetics may be achieved by variable use of elements in the ERK pathway. For example, activation of RAS can be achieved by multiple pathways, some of which include additional scaffolding proteins (Syp-Grb2-Sos; Shc-Grb2-Sos;

Grb2-Sos) which result in differential amplitude and duration of the ERK signal

(Buday and Downward, 1993; Obermeier et al., 1994). The differences in kinetic profiles of inactivation are also mediated by the activity of phosphatases which counterbalance the phosporylation events and therefore regulate the strength and duration of the signal.

18 The mechanism of how ERKs stimulate proliferation in fibroblasts has been elucidated in recent years (Murphy et al., 2004). In fibroblasts, nuclear translocation is required for cell cycle progression (Brunet et al., 1999). The strength and duration of the signal can significantly alter the qualitative and quantitative aspects of ERKs’ downstream signaling targets such as immediate early genes. Although both sustained and transient ERK activation can trigger transcription of the IEGs such as Fos, Jun,

Myc and Egr1, only sustained activation allows for phosphorylation and stabilization of the proteins they encode. For example, c-Fos protein is very unstable (half-life, t1/2=30 min) and will accumulate only if its C- terminus is phosphorylated under conditions of prolonged and sustained ERK activation. Indeed, the induction of cell cycle re-entry requires sustained ERK signaling and the subsequent activation of interdependent waves of gene expression such as c-fos which only is stable enough to induce transcription of other genes when hyperphosphorylated. This stabilization then allows for a sequential upregulation of a set of delayed early genes that include potent cell cycle regulators such as cyclin D1 (L.O. Murphy, J. Blenis, 2006; Meloche et al.,

1992; Roovers et al., 1999). Numerous studies showed that cyclin D1 expression is not activated by transient ERK signaling but is only triggered after sustained activation of the ERKs (Lavoie et al., 1996; Weber et al., 1997; Balmanno et al.,

1999). Importantly, recent studies reveal that only sustained ERK activation is necessary for downregulation of anti-proliferative genes such as JunD, Sox6 and

MEF2C (Yamamoto et al., 2006).

In PC12 cells the transient and sustained ERK activation can also be mediated by the small GTPases, RAS and Rap1, respectively (York et al., 1998). In fact,

19 mutated Rap1 blocks the ability of NGF to stimulate sustained ERK activation

whereas a dominant-negative form of Ras blocks only the transient ERK signal. In

mature DRG neurons, the NGF–pTrkA–Rap1 signaling endosome contributes

critically to sustained ERK activation which is thought to be necessary for neuronal

survival and maintenance (Kao et al., 2001).

In addition, scaffolding proteins play an important role in regulation of the

magnitude of ERK activity. KSR (kinase suppressor of RAS) interacts with Raf,

MEK and ERK, potentiating ERK activation (Therrien et al., 1996; Morrison, 2001).

In dormant cells the E3 ubiquitin ligase, IMP (impedes mitogenic signal propagation)

binds to KSR thus maintaining KSR in an inactivated state (Matheny et al., 2004).

KSR null mice exhibit reduced ERK activation, which results in blocked T-cell

activation and inhibition of tumor development (Nguyen et al., 2002). Other scaffold

proteins for the ERK pathway also contribute to signal modulation (Morrison and

Davis, 2003). MP1 (MEK partner 1) couples ERK to MEK (Schaeffer et al., 1998), where decreased MP1 expression results in reduced ERK activation (Teis et al.,

2002). MEKK1 (MEK kinase 1) and β-arrestin also bind to Raf, MEK and ERK, enhancing ERK activation (Karandikar et al., 2000; Luttrell et al., 2001).

Negative feedback inhibitors of the ERK pathway such as Sprouty, provide another level of temporal regulation of ERK activity (Hanafusa et al., 2002). There are four Sprouty isoforms (Sprouty 1-4) in mammalian cells. Sprouty1 and Sprouty4 exhibit the most potent inhibitory effect on ERK signaling by suppressing the association of Grb2-Sos1 complex with FRS2 (Ozaki et al., 2005). Overexpression of

Sprouty in PC12 cells results in only transient ERK signal and suppresses

20 differentiation. By contrast, overexpression of a dominant-negative form of Sprouty results in sustained ERK activation promoting differentiation. The duration of ERK signaling is also precisely controlled by temporal induction of the MAP kinase phosphatases, MKP1-3 and their compartmentalization (Pouyssegur et al., 2002).

MKP1-2 induction is activated and strictly controlled by MAPK cascade activation by an autoregulatory mechanism and can be enhanced by the ability of ERKs to phosphorylate MKP1 and MKP2, which in turn stabilizes them (Brondello et al.,

1999). In addition, cross-talk between the MAPK cascade and other signaling components provides another important regulatory mechanism of signal specificity.

Downstream effectors of the MAPK cascade, such as transcription factors, may also be influenced by components of other pathways, resulting in a combinatorial effect on their ultimate activation status.

In summary, ERK activation can generate opposite biological outcomes depending on the situation: cell proliferation versus cell-cycle arrest and differentiation, cell survival versus cell death. Questions of how one signaling molecule transduces multiple signals from extracellular stimuli to specific cellular responses remain under investigation. Accumulating experimental data has demonstrated that differences in the duration, magnitude and subcellular compartmentalization of ERK activity can influence signaling specificity. These differences can be achieved by many temporal, strength and spatial regulators which influence changes in receptor numbers, ligand concentration, specific adapter proteins and their affinity for the receptor, and specific phosphatases. All of these components

21 influence the amplitude and duration of the signal which can then impact specific biological functions in a cell specific manner.

The ERK pathway is often studied in the context of oncogenesis, where the magnitude of ERK hyperactivity appears to influence the survival of cancer cells.

Mutations in key elements of the MAPK signaling cascade which cause sustained hyperactivation of the ERKs are highly correlated with cancer. In fact, ~30% of cancers carry mutations in the family of RAS proto-oncogenes: K-Ras, H-Ras and N-

Ras (Repasky et al., 2004), but this number goes up to ~90% in pancreatic cancers, which have mutations in K-Ras. Individuals with Costello syndrome have a high incidence of H-Ras mutations which predisposes them to tumor formation (Aoki et al., 2005). Therefore, small-molecule inhibitors of the RAS/ERK pathway have been tested as potential therapies in hopes of limiting the tumorgenic potential of the cancer cells (Solit, D.B. et al. 2005). In summary, sustained activation of ERK signaling is involved in cell cycle progression, cellular transformation and differentiation. However, these responses are context dependent and cell type specific since regulation of signal strength and duration of the ERK activity is context dependent and may be interpreted differently resulting in proliferation or differentiation.

22 ERK Isoform-specific Functions

ERK1 and ERK2 share 84% sequence identity and are evolutionarily conserved

across all species (Boulton et al., 1990; Lloyd et al., 2006). They are among the most studied

protein kinases and have been implicated in a wide range of biological functions (Pearson et

al., 2001; Roux and Blenis, 2004). Whether ERK1 and ERK2 possess individual targets or

functions remains one of the most controversial questions that surrounds the biology of these enzymes. There are compelling data addressing both sides of the question. Although both isoforms are ubiquitously expressed, the expression levels of ERK2 are much greater in most tissues, including the brain. Specifically, in the cerebral cortex, ERK2 is expressed at thirteen times the level of ERK1, a fact that contributed to misinterpretation of many initial studies attempting to address the isoform specific function question. ERK1 and ERK2 have identical substrate specificity and kinetics of activation, however, new studies point to a slightly different kinetics of nuclear entrance (Marchi et al., 2008; Marchi et al., 2010). A recent and controversial study has also reported that ERK1 and ERK2 act antagonistically to regulate cellular proliferation, where ERK1 actively suppresses the growth promoting actions of

ERK2 (Vantaggiato et al., 2006). Ablation of ERK1 in embryonic fibroblasts and NIH3T3 cells resulted in enhancement of ERK2 activity and significant growth advantage but in vitro knock down of ERK2 resulted in a drastic decrease in proliferation. It is also interesting that the total level of ERK2 protein was not changed, only its phosphorylation status (Pages et al.,

1999; Mazzucchelli et al., 2002). Additionally, ectopic expression of ERK1, but not ERK2, resulted in inhibition of proliferation and colony formation and was not dependent on kinase activity. Ectopic expression of ERK1 but not ERK2 was sufficient to attenuate tumorgenesis

23 in nude mice. On the other hand, studies by Lefloch et al. concluded that ERK1 and ERK2

signal proportionally to their expression levels and are functionally indistinguishable in their

ability to promote nuclear gene expression and that their isoform pseudo-specificity is a

result of skewed expression patterns and protein levels in different tissues (Lefloch et al.,

2008). Indeed, the overall gene dosage is important for the proper development of the neural crest and its derivatives in mice (Newbern et al., 2008). Analysis of mice in which one of the

ERK isoforms has been inactivated reveals a compensatory mechanism by which the remaining isoform becomes hyper-activated in some, but not all, tissues (our unpublished data). To what degree this compensatory mechanism is able to compensate for the deleted isoform remains largely unknown, as does the sensor that triggers this process.

The notion that ERKs may have isoform specific functions is supported by the fact that germline ERK2 knockouts are embryonically lethal; the embryos die around E6.5 due to defects in trophoblast development where the developing embryo fails to form proper placenta (Hatano et al., 2003; Saba-El-Leil et al., 2003; Yao et al., 2003). In contrast, data by

Yao et al. suggest that the ERK2 null mice die much later, at around E11.5, because of failure to induce mesoderm formation.

More than 10 years ago several labs including ours generated the ERK1 knock out mice, which were described as normal, viable and fertile with no overt phenotype (Selcher et al., 2001; 2003; Nekrasova et al. 2005 and Pages et al., 1999). Although the characterization

of these mice was thorough at the time of the investigation, due to recent advances in our

understanding of intricate details of cortical development it may not have been adequately

explored. In addition, Pages et al. reported that ERK1 mice exhibit reduction in T-cell

differentiation whereas Nekrasova et al. (2005) and Dillon et al., (2004) showed that these

24 animals exhibit greater sensitivity to experimental encephalitis and defective immune

responses, respectively. Selcher et al. reported that the ERK1 null mice do not exhibit any

abnormalities in working and long term memory and have no deficits in induction of

hippocampal LTP or passive avoidance (Selcher et al., 2001; Mazzucchelli et al., 2002).

However, Brambilla et al. reported that these animals are hyperactive showing a paradoxical

enhancement in striatal- based long term memory and facilitation of LTP in the nucleus

accumbens, a major part of the ventral striatum (Mazzucchelli et al., 2002). The nucleus

accumbens has been classically studied in the context of reward learning behaviors but is also

implicated in learning socially relevant information (Aragona et al., 2003). Other limbic

structures including the caudate, thalamus and putamen are also part of this circuit. Deficits

in both reward learning and social behaviors have been associated with Autism Spectrum

Disorders (ASD) in humans (Dichter et al., 2010).

The ERK1 null mice have not been examined behaviorally in a

comprehensive set of assays utilized to mimic ASD in mice such as social interaction,

communication and repetitive behaviors. On the other hand, ERK2 inactivation

within the telencephalon results in profound deficits in contextual memory and

learning. The recent generation of transgenic mice, which are ERK2 hypomorphs has

further supported a dominant role for this isoform for memory and behavior. These

two findings seem to be mutually exclusive, one pointing to a compensatory

mechanism by which one ERK isoform compensates for the absence of another ERK

isoform. The second suggests specific functions for each of the ERK isoforms.

Protein-DNA interactions (PDIs) are involved in many biological functions essential for cellular differentiation, function, and survival. In addition to classic,

25 kinase mediated, functions of MAP kinases, recent data by Hu et al., revealed that

ERK2 may also act in an unconventional way as a DNA binding protein. Microarray-

based PDI analysis showed that ERK2 can bind to a G/CAAAG/C consensus

sequences. In vitro and in vivo experiments demonstrated that ERK acts as a

transcriptional repressor for interferon gamma-induced genes (Hu et al., 2009).

ERKs in LTP, Learning and Memory

The MAPK cascade was originally discovered as a critical regulator of cell

division and differentiation. Regulation of cell cycle progression is the best-studied

function of these kinases (Gotoh et al. 1991; Posada and Cooper 1992). Although the

ERKs are expressed in proliferating cells of all types, they are also abundantly

expressed in neurons in the mature central nervous system including terminally

differentiated neurons. It has been postulated that the ERK signaling system has been

co-opted in mature neurons to function in synaptic plasticity and memory. The

formation of memory and learning in mammals is at least partly accomplished by a

form of long lasting synaptic plasticity known as long term potentiation (LTP). A significant body of work points to the idea that ERK activation plays a critical role in

LTP induction. Initial in vitro studies used hippocampal slices and focused on NMDA receptor-dependent LTP in the CA1 region (English and Sweatt 1996, 1997; Atkins et al.1998; Impey et al. 1998; Winder et al. 1999; Wu et al. 1999). ERK activation is also necessary for induction of NMDA receptor-independent LTP (Coogan et al.

1999) and LTP in the dentate gyrus in vitro (Coogan et al. 1999). These LTP findings

26 were also recapitulated in vivo (McGahon et al. 1999; Davis et al. 2000; Rosenblum

et al. 2000). Inhibitors of MEKs, the only known activators of the ERKs, successfully

block LTP (Liu et al. 1999). The in vitro data implicating ERK in synaptic plasticity

led to further investigation of the role ERKs may play in learning in vivo. These

experiments used contextual (hippocampus-dependent, associative learning) and cued

(tone; amygdala-dependent, associative learning that is hippocampus independent)

fear conditioning, as well as Morris water maze (MWM) (spatial learning) tests.

Atkins et al. showed that contextual and cued fear conditioning results in the

activation of MAPK in the hippocampus which is NMDA-receptor dependent and can

be blocked by MEK inhibitors (Atkins et al.1998). Furthermore, using the MWM behavioral test, Blum et al., with hippocampal infusions of MEK inhibitors, clearly demonstrated the necessity of ERK activation for spatial memory formation. These observations were later confirmed by Selcher et al. (1999). Therefore, hippocampal

ERK activation is critical for LTP and various forms of hippocampus-dependent memory formation. Which signaling mechanisms control ERK activation in the hippocampus? Hippocampal ERK activation can be regulated by a variety of neurotransmitter receptors: NMDA receptors, adrenergic receptors, DA receptors, muscarinic acetylcholine receptors and metabotropic glutamate receptors coupled to either PKA or PKC. Additionally, ERKs in the hippocampus can be activated by brain-derived neurotrophic factor (BDNF) (Gottschalk et al. 1998, 1999; Pozzo-

Miller et al. 1999). Since ERKs play a crucial role in integrating signals in the hippocampus, do they implement downstream effectors which contribute to the induction of synaptic plasticity and learning? CREB (cAMP response element

27 binding protein) and the potassium channel Kv4.2 have both been implicated as the

downstream effectors of the hippocampal activation of ERKs in learning and

memory. The current working model postulates that CREB plays a key role in

modulating the induction of gene expression necessary for LTP, and that Kv4.2 regulates the calcium influx necessary for generating forms of PKC and CaMKII that are needed for early LTP (Impey et al. 1998; Sweatt et al., 1996). ERKs have also been implicated in other forms of synaptic plasticity including LTP in the amygdala, which is associated with fear-dependent learning (Huang et al., 2000), and cortical

LTP (Di Cristo et al., 2001).

The MAPK signaling pathway, in a stimulus-dependent fashion, participates in regulation of neuronal excitability, synaptic potentiation, nuclear signaling and memory formation. One of the cellular mechanisms involved in various forms of synaptic plasticity, in learning and memory, involves activity-dependent remodeling of dendritic spines and filopodia. Repeated stimulation of hippocampal neurons induces activation of the MAPK pathway, which when sustained is essential for formation of new dendritic spines and filopodial extensions, synapse remodeling and induction of long-term synaptic changes. The finding that the MAPK pathway helps control dendrite shape has additional implications for activity-dependent neural development.

ERKs and their regulation by FGFs

28 An extensive body of literature has documented that much of the ERK

signaling during early development is dependent upon the action of family of fibroblast growth factors (FGFs) (Eswarakumar et al., 2005; Mason et al., 2007). FGF

family consists of 22 members, which play a crucial role in brain development and

patterning through their control of cell behaviors such as cell proliferation, migration

and differentiation, axon pathfinding and synaptogenesis. Furthermore, FGFs are also involved in organogenesis, tissue repair and cancer (Eswarakumar et al., 2005;

Mason, 2007). FGF molecules signal from the extracellular matrix by interacting with their high affinity tyrosine kinase receptors (RTKs). FGF receptors undergo homodimerization and autophosphorylation upon binding of the FGF ligands

(Eswarakumar et al., 2005). In vertebrates there are four FGF receptors each encoded by its own gene (Itoh and Ornitz, 2004). They are single spanning transmembrane proteins which in addition to FGFs can also bind heparin sulfate proteoglycans and cell adhesion molecules (Bottcher and Niehrs, 2005). In the developing mammalian forebrain only FGFR 1-3 are transcriptionally expressed, thus modulating and propagating the signal (Mason 2007). The FGF signaling which occurs downstream of the Shh-Gli3 signaling and Foxg1 is crucial for proper brain patterning and generation of ventral as well as dorsal telencephalic structures (Gutin et al. 2006). In mice, FGF8 is expressed in the ANR (anterior neural ridge) starting at E8.5 and then upon neural tube folding in the commissural plate (CoP) which are both part of the rostral signaling center (Crossley and Martin 1995; Ohkubo et al., 2002) and also express several other FGFs. Among them, FGF8, FGF15 and FGF17 are involved in specification of the frontal cortex (Sur and Rubenstein 2005; Mallamaci and

29 Stoykova 2006; Rash and Grove 2006; O’Leary et al., 2007). Genetic studies show that FGF ligands play an important role in arealization of the cortex. In addition, inactivation of FGF8 signaling during the neural crest development leads to severe cardiac and craniofacial defects. In contrast to the drastic changes to the telencephalic development observed in mice with altered FGF expression or genetic ablations, deletions of any single FGF receptor generated only mildly altered cortical structure.

This is most liked due to compensation from other FGFRs. Recently, triple FGFR knock out mice were generated where all three receptors were inactivated during early stages of corticogenesis. By E12.5 severe deficits in surface area were reported due to accelerated neurogenesis and premature loss of radial progenitors.

Remarkably, little change in cortical layering occurred (Rash et al., 2011).

The FGFs and their receptors activate the MAPK/ERK signaling cascade which in turn activates or enhances the expression of many transcription factors including Ets proteins, AP1, GATA, c-myc and CREB (Yordy and Muise-

Helmericks, 2000). In addition, they induce multiple feedback mechanisms through inhibitors such as SEF, MKP3 and Sprouty. Sef and the Sprouty proteins closely mirror the FGF8 expression pattern and fine tune the MAPK/ERK signal during brain development (Faedo et al., 2010; Labalette et al., 2011). The PI3kinase/AKT pathway can also mediate FGF action in other tissues, but it is not involved in neural development downstream of the FGFRs.

30 Cortical Development

Forebrain Regionalization and Morphogenesis

The development of the cerebral cortex represents one of the most intricate and complex processes in CNS development. It can be divided into two distinct events: patterning and neurogenesis. The neurogenic phase can be further subdivided into three stages: proliferation, expansion of the precursors and neurogenesis. During early forebrain patterning three main telencephalic regions are established: the dorsal, dorsomedial and ventral telencephalon (Sur and Rubenstain, 2005). The dorsal primordium of the cortex forms excitatory, pyramidal neurons; the dorso-medial telencephalon becomes the hippocampus; the ventral region gives rise to inhibitory interneurons, which then tangentially migrate into the cortex and the hippocampus.

This regionalization is in part due to several morphogenic molecules secreted from signaling centers during early development. The most rostrally located is the anterior neural ridge (ANR) which later becomes the commissural plate (CoP), the cortical hem which influences the dorsomedial region and the anti-hem located at the pallial- subpallial boundary of the lateral telencephalic region (Sur and Rubenstain 2005;

Mallamaci and Stoykova 2006; Rash and Grove 2006; O’Leary and Sahara 2008).

These signaling hubs express morphogens such as FGFs (FGF8, FGF15, FGF17 and

FGF18 (CoP); bone morphogenic proteins (BMP), and Wnt in the cortical hem; as well as FGF7, FGF15, transforming growth factor-α (TGF-α) and secreted frizzled- related protein 2 (Sfrp-2) in the anti-hem. In addition, Sonic hedgehog (Shh) is expressed from the ventral signaling center. These morphogenic molecules induce or

31 regulate the expression of global transcription factors such as Pax6, Emx2, Coup-Tf1

and Sp8 which form unique gradients establishing the area specific aspects of the

nascent cortex which can be functionally subdivided into motor, somatosensory,

auditory and visual areas (Schuurmans et al., 2002). Emx2 and Pax6 are especially

important in areal patterning as demonstrated by experiments, which alter their

gradients (Bishop et al., 2000; Hamasaki et al., 2004).

Corticogenesis begins in the early stages of development upon closure of the

neural tube and formation of the neural axis, where a single layer of multipotent,

highly polarized neuronal progenitor cells (neuroepithelial cells) divide symmetrically

to produce more committed neural precursors. These neuroepithelial cells ultimately

transform into Pax6+ radial glia, which divide at the apical surface of the ventricular

zone (VZ) and together with neuroepithelial cells are collectively referred to as apical

progenitors. Apical progenitors populate the early telencephalic ventricular zone and

give rise to more restricted progenitors, the intermediate progenitor cells (IPCs, also known as basal progenitors), as well as to neurons. The newly generated IPCs populate the subventricular zone and can be delineated by their expression of Tbr2

transcription factor (Gotz and Huttner 2005, Dehay and Kennedy 2005, Kowalczyk et

al., 2009). The process of neurogenesis in the mouse takes place between day 11 and

day 17 of mouse embryonic development. During this time the proliferating cells

undergo 11 precisely orchestrated cell cycles carefully balancing proliferation and

cell cycle exit. It is of interest that the length of the cell cycle, specifically of the G1

phase, becomes longer as neurogenesis progresses (Caviness et al. 1995; Takahashi et

al., 1995; Dehay and Kennedy 2007).

32 The proliferating radial glia contribute to the expansion of the cortical surface area whereas the exiting cells mature as excitatory projection neurons and later glia.

During this crucial, time-dependent process, all cortical pyramidal neurons as well as other cortical cells are generated, migrating radialy to appropriate cortical lamina following an inside out pattern, where later born neurons migrate past earlier born neurons and settle in more superficial layers (Gupta et al., 2002; Cooper 2008). The radial migration contributes to the thickness of the mature cortex. On the other hand, most of the inhibitory, GABAergic interneurons are generated from the ganglionic eminences of the ventral telencephalon and migrate tangentially into appropriate cortical layer (Corbin et al., 2001; Marin and Rubenstein 2001). The roles that cortical environment and proper lamination plays in tangential migration of interneurons will be discussed in detail in chapter 3.

The Role of Cell Cycle in Corticogenesis

In the developing cortex, excitatory pyramidal neurons can be generated as a result of asymmetric divisions of radial glia in the ventricular zone or symmetric divisions of subventricular progenitors (IPCs), which themselves are derived from radial glia. It is interesting to note that about 80% of all cell divisions in the VZ are asymmetric, suggesting their primary role in progenitor pool expansion, whereas almost 100% of divisions within the subventricular zone are symmetric. In fact, it has been postulated by Pontious and others that Tbr2 positive IPCs divide once or twice, generating the vast majority of radially migrating cortical projection neurons. The

33 majority of these cells are destined for more superficially located upper neuronal layers (Pontious et al 2008; Tarabykin et al., 2010).

Neurogenesis is initiated when a fraction of mitotically active cells exits the cell cycle (the quitting - Q fraction) at the expense of continuous proliferation (the proliferative - P fraction) during early cortical development. The proliferating cells are the p fraction, which by surveying the external environment decide whether or not to commit to another division. The G1 phase of the cell cycle is the most critical, since it is at this time that the cells make a decision to either undergo another round of division or differentiate. The switch from proliferative to neurogenic cell division during cortical development is important since it determines the final number of neurons and overall adult brain size. In recent years, an accumulating body of in vitro and in vivo data has lead to the proposal of the “cell cycle length hypothesis”

(Lukaszewicz et al., 2002; Calegari and Huttner 2003; Gotz and Huttner 2005). This hypothesis states that the lengthening of the G1 phase during corticogenesis causes a switch in progenitor mode of division from proliferative to neurogenic. Huttner and

Caligeri have proposed “the cell cycle length hypothesis” whereby the progressive lengthening of the G1 phase of each cell cycle during corticogenesis, may influence the change from proliferative to neurogenic cell divisions. For example, selective lengthening of the G1 phase alone by knock down of cdk4/cyclin D1 (known components of regulatory mechanism of G1 check point) results in a switch from proliferative to neurogenic divisions and is sufficient to induce premature neurogenesis (Calegari and Huttner, 2003). It has also been noted that neuron generating, differentiative divisions inhibit G1 progression (for example, Tis21 (also

34 known as Btg2) and BM88 (also known as Cend1) pointing to a link between G1 duration and the mode of division (Iacopetti et al., 1999; Tirone et al., 2001;

Georgopoulou et al., 2006). The idea that the lengthening of the G1 phase is a predominant cause, rather then a consequence of neurogenesis, has also been supported by studies which implemented artificial shortening of G1 by in utero overexpression of positive G1 regulators during embryonic development. Loss of G1 regulators inhibited neurogenesis, expanding the neural progenitor pool, which resulted in a thicker germinal zone and a three-fold increase in cortical surface area of the postnatal brain (Lange et al., 2009). Because the transcriptional machinery involved in the neurogenic switch may require more time, a short G1 may simply not allow the time that is necessary for a cell fate change to take place whereas a longer

G1 will.

Furthermore, mitogens such as the FGFs are known regulators of the G1 phase of the cell cycle. Indeed, addition of FGF2 to a primary cortical culture at

E14.5 resulted in shorter G1 length, which correlated with an increased number of proliferative divisions (Lukaszewicz et al. 2002). In vivo data from Storm (Storm et al. 2006) indicates that FGF8 plays an important role in cell proliferation and apoptosis during rostral cortical development. However, the downstream mechanisms activated by FGFs during cortical development remain largely unknown and are the subject of this work.

In summary, the decision to exit the cell cycle or remain proliferative is partially influenced by the balance between mitogenic and anti-proliferative signals from a cell’s external environment, which influence intracellular signal transduction

35 systems. Together they provide a general plan governing number of cell divisions and ultimately establishing telencephalic size of the developing brain. The neurogenic phase is followed by the gliogenic phase during which progenitor cells in proliferative

hubs generate astrocytes and subsequently oligodendrocytes. In mice, at the time of

birth, cortical neurogenesis is fully completed, but a few small populations of neural stem cells in the hippocampus and subependymal region still remain.

ERKs and Cell Cycle Progression

The development of the mammalian cerebral cortex requires strictly controlled spatial and temporal regulation of cellular proliferation, which depends on a precisely timed and controlled division of neuronal progenitors within the germinal zones of the developing cortex. Therefore, cell cycle length, cell cycle exit and terminal differentiation are important for establishing the final number of pyramidal neurons and ultimately the size of the adult brain. During corticogenesis, perturbations in any of the strictly orchestrated proliferative events lead to many developmental disorders and impaired cognition.

The ERK1/2 MAP kinases were initially identified because of their ability to promote cell cycle progression and cell proliferation. The ERKs become phosphorylated by the MEKs as a result of mitogenic stimulation and exhibit sustained phosphorylation through the G1 phase leading to cell cycle entry and progression. On the other hand, the requirement for ERKs for the S phase entry has been controversial (Korotayev et al., 2008).

36 Parameters of total cell cycle length (Tc) as well as length of G1 phase (TG1) are regulated by precisely orchestrated actions of cyclins, cyclin-dependent kinases

(cdk) and their inhibitors, some of which are regulated by ERKs. Cyclin D1 is one of the most prominent downstream effectors of the ERKs and its expression is the critical and rate limiting step in growth factor-induced cell cycle initiation. Recent work from Elizabeth Ross’ lab elegantly demonstrates the preferential expression of cyclin D1 in the VZ, and cyclin D2 in the SVZ (Glickstein et al., 2007). It has been postulated, but not proven, that cyclin D1 may be important in cell cycle progression of apical progenitors whereas cyclin D2 may be important in intermediate progenitor proliferation. However this data is controversial and has been questioned by others

(Glickstein et al., 2009). In response to growth factors, such as FGF, ERK1/2 activation leads to phosphorylation of the Ets transcription factor Elk-1 and c-fos, where c-fos and c-jun dimers form the AP-1 complex, which then binds with greater affinity to the serum response element of the cyclin D1 promoter (Figure 1-3).

ERK1/2 dependent cyclin D1 expression allows for its binding to cdk4/6, which stimulates the cell to exit G0 thus promoting cell cycle entry. Once the cell is in G1, it requires continuous ERK activity in order to progress into the S phase. This step is accomplished by ERK-dependent phosphorylation of pRb and p27Kip1. p27Kip1, a

cyclin dependent kinase (CDK) protein inhibitor, is a member of the Cip/Kip family

which also includes p21Cip1 and p57Kip2 (Elledge and Harper 1994). P27Kip1 broadly

binds to and blocks the activity of cyclin/cyclin dependent kinase (CDK) complexes

and therefore critically modulates the G1-to S-phase transition (Cheng et al., 1999;

Sherr and Roberts, 1999). It is interesting that p27Kip1 is also implicated in

37 mechanisms involved in neuronal specification (Ohnuma et al. 1999, 2001, 2002,

Vernon et al., 2003; Nguyen et al., 2006) and maintenance of differentiated neurons

in a postmitotic state (Zindy et al., 1999). P27Kip1 is phosphorylated by the ERKs in

the cytosol which targets this protein for proteosomal degradation, allowing for cell

cycle progression (Ahamed et al., 2001). Recent work of Caviness and others elegantly shows that induction of P27Kip1 expression during neurogenesis resulted in

an increased Q fraction, premature cell cycle exit and reduction in number of upper

cortical neurons (Mitsuhashi et al., 2001; Tarui et al., 2005). The Kip family has also

been implicated in fate specification and differentiation of glial cells, including oligodendrocytes (Durand et al., 1997; Zezula et al., 2001).

It has also been reported that ERK1/2 may be involved in p27Kip1translation

(Han et al., 2005) and mRNA stability (Bagui et al., 2009). In addition, our lab and others reported that ERK1/2 inhibition correlates with elevation of p27Kip1 and cell cycle arrest during neurogenesis (Matsui et al., 2010; unpublished data).

Figure 1-3

Progression through the cell cycle is controlled by ERKs.

38

Progenitor Proliferation and Neurogenesis

In mammals, all pyramidal neurons of the developing cerebral cortex are derived either directly or indirectly from the neuroepithelium, a specialized layer lining the lumen of the lateral ventricle. As early as the end of the 19th century, His,

Cajal and others observed that most of the mitotic activity in the entire nervous

system was restricted mainly to the ventricular surface of the neuroepithelium. At this

time, the theory that the VZ contained two distinct types of progenitors: neuronal and

39 glial was widely accepted (Cajal, 1911). In 1970, for the first time, the Boulder

Committee agreed that there was only a single type of progenitor cell in the cortical

VZ. In the 1980, Rakic and others revived the original idea of distinct types of

progenitors: GFAP+ and GFAP-, generating glia and neurons, respectively (Levitt et

al., 1981, 1983). This view prevailed until very recently. In 1973, Smart et al.

described a population of non-surface mitotic progenitors in the SVZ which came

from VZ progenitors and generated mostly neurons (Smart, 1973). This view was

subsequently challenged by Takahashi and colleagues (Takahashi et al., 1995). With

the advent of technology and new molecular markers the idea that cortical progenitors

might in fact generate both neurons and glia was revisited. Many labs, using different

approaches, confirmed that radial glia in the VZ, in fact, generate neurons (Malatesta

et al., 2000; Noctor et al., 2001; 2002; Miyata et al., 2001). Furthermore, by 2004

three other labs confirmed that the SVZ progenitors are also capable of generating

neurons (Noctor et al., 2004; Haubensak et al., 2004 and Kawaguchi et al., 2004).

The neuroepithelial cells (NE) include neural stem cells and neural progenitor

cells, which can divide symmetrically or asymmetrically. At the initial stages of

cortical development, during the expansion phase, NE cells divide symmetrically to

expand their population and enlarge the two hemisphere anlagen. The NE cells are

highly polarized and undergo mitosis at the apical ventricular surface. They are

characterized by the cell-cycle dependent interkinetic nuclear movement (INM) with

apical to basal (G1) and basal to apical trajectory (G2), which leads to pseudostratification of the neuroepithelium. After the closure of the neural tube and

the onset of cortical neurogenesis around E9–10 in the mouse, NE cells acquire glial-

40 like markers such as Pax6, Hes5, GLAST and BLBP, and switch to an asymmetric mode of division generating radial glia (RG), basal progenitors, (BPs also known as intermediate progenitor cells) and neurons (Campbell and Gotz, 2002). Just like NE cells, radial glia assume apical-basal polarity spanning the entire VZ with their basal end-foot, the basal process, touching the pia. Interestingly, recent data indicates that basal contacts of RG with the meninges and retinoic acid signaling are essential for the transition from neuroepithelial symmetric proliferation to asymmetric neurogenic proliferation (Siegenthaler et al., 2010). In addition, RG nuclei also undergo INM.

Since radial glia retain some properties of embryonic stem cells, they are regarded as such and together with NE cells are often referred to as apical progenitors (Gaiano et al., 2000; Malatesta et al., et al., 2000; Noctor et al., 2001). During neurogenesis the progressive growth of the cortical thickness is accompanied by lengthening of the pial-directed radial processes of RG, which acquire 24-nm microtubules and 9-nm intermediate filaments as well as glycogen storage granules within the radial fiber and in the end feet, respectively (Choi & Lapham 1978, Bruckner & Biesold 1981,

Gadisseux & Evrard 1985).

Figure 1-4

Division modes of the expansion of the cerebral cortex in mice: Lateral and

Radial.

41

The next type of progenitor cell appearing at the early stages of neurogenesis are the basal progenitors, also known as intermediate progenitor cells (IPCs) or non-surface dividing progenitors (Smart et al., 1973; Noctor et al., 2004; Malatesta et al., 2004).

They delaminate from the apical surface of the neuroepithelium and travel to the basal edge of the VZ where they form a second germinal layer, the SVZ. Their molecular footprint includes Tbr2 transcription factor and a non-coding RNA, Svet1

(Englund et al., 2005). Curiously, they retract their apical and basal process, assuming multipolar morphology during mitotic division away form the ventricle and can migrate slowly in radial and tangential directions (Noctor et al., 2004). In rodents,

IPCs undergo mostly symmetric, self-consuming divisions which generate two immature neurons (Noctor et al., 2004). However, about 10% of IPCs are capable of self-renewing symmetric divisions which expand their pool in the SVZ (Noctor et al.,

2004; Noctor et al., 2008; Kowalczyk et al., 2008). It has been postulated by

42 Kreigstein and others that the small number of self-renewing IPCs may be

evolutionarily responsible for the rapid cortical expansion of the secondary

proliferative zone observed in higher mammals (Kreigstein 2005, Kennedy and

Dehay 2005, 2007). Therefore, the dual neurogenic pathways, direct and indirect, are

still heavily investigated. Interestingly, the question of whether each radial glia is

committed to either neuronal or glial lineage from early corticogenesis or whether

they are all truly multipotent remains unanswered.

The choice to become a neuron takes place in the progenitor cell, where radial

glia and IPCs commit to neurogenic divisions (as indicated by Tis21 expression)

during G1 phase of the cell cycle, whereas the laminar fates are established during the

S/G2 transition period (Haubensak et al., 2004; McConnell et al., 1991). On the other

hand, the neuronal laminar fate and neuronal subtype is strictly linked to the temporal birth day of the neuron. The series of fate decisions are coordinated by complex, multifactorial cell-intrinsic and cell-extrinsic molecular cues. The intrinsic transcription factors are modulated by signals from other cells and morphogenic centers, but can also be influenced by asymmetrical cellular inheritance of other factors such as Numb, EGF and apical plasma membrane components (Petersen et al.,

2004; Kosado et al., 2004; Sun et al., 2005). During neurogenesis, the decision to self-renew or become a neuron is influenced by several transcription factors including

Pax6, Tlx, and Ngn1/2, where Ngn2 has been implicated in IPC production and is directly regulated by Pax6 (Miyata et al., 2004; Heins et al., 2002). In fact, Pax6

deficient mutant mice show decreased levels of Ngn2 and fewer cortical neurons

(Heins et al., 2002). The most significant counterbalance to neurogenesis comes from

43 inhibitors such as Hes and Id, which belong to bHLH superfamily. Hes and Id mutant mice show precocious neurogenesis and increased IPC production (Ohtsuka et al.,

2001, Cau et al., 2000; Yun et al., 2004). The perfect balance of proliferative and neurogenic divisions will ultimately shape the cortical surface area as well as cortical thickness. To fully appreciate the complexity of the developing cortex, one must also take into account the cell cycle dynamics which play an important part in executing the regulatory decisions made by the transcription factors and are often themselves influenced by transcriptional and morphogenic machinery.

In the cortex, direct or indirect neurogenesis results in production of young pyramidal neurons which then have to assume a proper laminar fate. Several transcription factors have been linked to the laminar fate decision. Er81 is expressed in layer 5 projection neurons whereas Cux2 may specify upper layer neurons. Other transcription factors which have been implicated in layer-related patterning include:

Tbr1, Foxp2, Foxp1, Nurr1, Fez1 and others (Hevner et al., 2001; Ferland et al.,

2003; Arimatsu et al., 2003; Inoue et al., 2004). In addition, FGF signalling plays an important role in accurate development of young neurons. For example, FGF18, secreted by young neurons, signals back to the progenitors controlling the migratory behaviour and laminar distribution of the next neuronal progeny (Hasegawa et al.,

2004). FGFs are also involved in the assembly of circuitry, where they act as chemoatractants aiding in target recognition, navigation and branching. In fact, analysis of FGF8 hypomorphs reveals aberrant formation of axonal projections between cortical areas of the telencephalon (Huffman et al., 2004).

44 Migration

Cell migration is one of the most critical steps during brain development,

where immature neurons travel from their germinal zones to their specific locations

and targets forming intricately complex synaptic brain circuitry. Postmitotic neurons

and sometimes their precursor cells migrate long distances along the dorso-ventral

(DV) and anterior-posterior (AP) axes of the developing embryo. Higher mammals evolved a radial migratory pathway which aids postmitotic neurons in their migration.

A number of genes have been implicated in the process of neuronal migration. They mediate a wide range of cellular functions, including chemoattraction/repulsion, cell adhesion, cell motility, and cytoskeletal dynamics.

During corticogenesis DV migration occurs in the forebrain, where proliferating progenitor cells in the proliferative zone of the basal ganglia, the lateral and medial ganglionic eminences (LGE and MGE, respectively), migrate in a ventral to dorsal direction. These GABAergic interneurons can be divided into an early population migrating dorsally from the MGE into the cortex, and a later population which migrates along the same pathway from the LGE.

In the dorsal telencephalon, the radial migration pattern which is unique to higher vertebrates takes place at the same time as the tangential migration from the ventral pallium. The radial migration utilizes the processes of radial glial cells as a

guide, and, importantly, provides the basis for the radial unit hypothesis, which

proposed that cells generated in the VZ are projected along radial glia into a columnar

arrangement of the cortex (Rakic, 1988). First evidence for this came from cell-based

migration assays utilizing easily purified cerebellar granule cells showing salutatory

45 movements and formation of an adhesion junction along the length of the soma and

the extension of a leading process, which projects short filopodia along the glial fiber

(Edmondson and Hatten, 1987). This hypothesis has since been supported by studies

showing that some pyramidal neurons can utilize somal translocation and are not

dependent on glial processes for locomotion (Morest, 1970; Hawthorne et al., 2010).

In the mouse embryo, the initiation of radial migration takes place around embryonic

day 11, generating a layer of postmitotic neurons known as the preplate, which is

composed of Cajal-Retzius and subplate cells. This region splits as more cells become

postmitotic in the outer layer of Cajal-Retzius cells and the underlying subplate which

forms the cortical plate. Subsequent populations of cells migrate past the subplate

settling in the area right underneath the Cajal-Retzius cells, which forms layer 1 of

the developing cortex, and cells of the cortical plate form layers 2 to 6. The layer 6 neurons are born the earliest and those of layers 5 to 2 will migrate past them, thus generating the “inside-out” arrangement described by Sidman. Newly born neurons undergo a dynamic, multiphasic migration comprised of 4 stages: radial migration from the ventricle to the SVZ, dynamic extension/retraction of processes with lateral and tangential movement during which cells may become multipolar, downward migration toward ventricular surface followed by final radial migration of truly polar neurons toward upper layers of developing cortex. The precise radial and tangential migration of cortical neurons can be disrupted by a large number of genetic mutations as well as physical (e.g., ionizing radiation, ultrasonic waves, and heat), chemical

(e.g., drugs and alcohol), and biological (e.g., viruses) agents. Minute changes or even

slowing down of neuronal migration can lead to gross and drastic but also subtle

46 abnormalities in neuronal positioning that can significantly alter the pattern of

synaptic circuitry (Algan and Rakic, 1997).

Gliogenesis

In 1846, Rudolf Virchow was the first person who suggested the presence of

supporting cells in the CNS. He assumed that, as the other support cells, they were

derived from the mesenchyme. It wasn’t until 40 years later that Wilhelm His

demonstrated glial cells are derived from the CNS, leading to the rejection of the mesenchymal hypothesis (Jacobson, 1991). In modern science, the term glia can be

somewhat confusing because it is used to describe both a progenitor population as

well as a differentiated population of parenchymal astrocytes, oligodendrocytes, and

ependymal cells.

Neurons and glia are both generated from a pseudostratified neuroepithelium

of ectodermal origin that lines the cerebral ventricles during early cortical

development.

In all vertebrates, neurogenesis takes place before gliogenesis, where neuronal

connections are established first and then aided by an appropriate number of glia. In

vitro culture experiments show that growth factors such as FGF2, induce cortical

progenitors to adopt gliogenic fate (Morrow et al., 2001; Qian et al., 2000), where

newly born cortical neurons secrete FGF2, acting through a negative feedback loop,

provoke the switch from neurogenesis to gliogenesis. FGF9 may also follow a similar

regulatory pattern and may participate in the timing of astrogliogenesis in the

developing cortex (Seuntjens et al., 2009). The mechanism by which this switch

47 occurs is not fully understood but in vitro evidence suggests that FGF promotes astrogenesis by instigating changes in histone methylation of the Glial Fibrillary

Acidic Protein (GFAP) promoter. In turn this change facilitates the activation of other gliogenic pathways such as CNTF-JAK-STAT (Song and Ghosh 2004). Furthermore,

FGFs signalling through FGFR1 and FGFR2 are involved in translocation of astroglia from the neurogenic hubs to the pia (Smith et al., 2006).

FGF signalling is also important in specification of oligodendrocytes, specifically the non-Shh dependent dorsally derived population via induction of Olig2 and Sox9 (Chandran et al., 2003; Esain et al., 2010; Gabay et al., 2003; Kessaris et al., 2004; Naruse et al., 2006).

Differentiation

The maturing cerebral cortex is composed of excitatory glutamatergic projection neurons (about 80%) and inhibitory GABAergic interneurons (about 20%).

It has been long appreciated that neurons within a single lamina share a common birthdate but even within a single layer, different neuronal subtypes with distinct anatomical and molecular profiles coexist (Bayer et al., 1991; Rakic, 1974). How these neurons acquire their subtype-specificity is a subject of ongoing research. The excitatory projection neurons fall into three subclasses: the corticofugal projection neurons (CFuPN); the colossal projection neurons (CPN); and ipsilateral circuit connection neurons. CFuPN are early born, deep layer neurons (layer 5-6), which project away from the cortex and do not cross the midline. Their projections target deeper brain areas such as the thalamus, and also project subcerebrally to the

48 brainstem and spinal cord. In contrast, CPNs in layer 2-3 form cortico-cortical

connections, cross the midline, project interhemispherically and integrate information

between the two hemispheres. They are aided by populations of glia and local neurons forming a sub-colossal bridge (Silver et al., 1982). CPN are more abundant than CFuPN, comprising the largest class of commissural neurons in placental mammals (Aboitiz et al., 2003). In the developing mouse embryo, CPNs in layer VI are born around E12.5 along with corticothalamic projection neurons whereas layer V

CPN are born around E13.5 along with corticospinal motor neurons (Molyneaux et al., 2007; Angevine et al., 1961). Neurons in layer 1 and 4 extend their axons locally.

How neurons assume their specificity and become committed to their laminar fate has been addressed by transplantation studies. These studies demonstrated that even before the completion of the final division, nascent neurons assume the molecular footprint and migrate to the layer appropriate to their birthdays (McConnell et al., 1991; Frantz et al. 1996; Desai et al., 2000). It is not the case as with earlier

stages of differentiation where more multipotent progenitors are able to generate

neurons according to their transplanted environment (McConnell et al., 1991).

However, the competence to respond to specific fate-inducing cues is lost as

development progresses. For example, progenitors which normally generate layer 4

neurons can differentiate into later born neurons when transplanted to the appropriate

environment but can no longer generate neurons of layer 6, despite earlier

transplantation time (Desai et al., 2000). On the other hand, in vitro clonal analyses

point to a cell-intrinsic clock as an important part of the mechanism guiding cell fate

specification and differentiation (Shen Q et al., 2006). Recent data suggests that

49 precisely orchestrated patterns of gene expression guide changes in cortical

progenitor behavior and ultimately neuronal specificity. For example, transcription

factors such as Fezf2, Otx1, Sox2 and Emx2 are expressed in early progenitors in the

VZ and are maintained in their postmitotic progeny of the deeper cortical layers

(Bani-Yaghoub et al., 2006; Chen B et al., 2005; Frantz et al., 1994; Leingartner et

al., 2003) but SVZ progenitors mimic patters of gene expression of upper neuronal

layers including Svet1 and Cux1/2 (Nieto et al., 2004; Tarabykin et al., 2001). It is

evident that intricate interplay among transcriptional gene profiles defines the fate of

cortical neurons. During early development, generation of deep cortical neurons is

promoted by Fezf2 expression in the VZ progenitors, which together with Ctip2 confers subcortical neuronal fate. More subtle changes in the expression levels of

Sox5, Tbr1 and Ctip2 further fine-tune the identity of these deep layer and subplate

neurons. The upper colossal neuronal fate is actively repressed by Fezf2, which

actively blocks the expression of Satb2. During later stages of corticogenesis Fezf2

expression is downregulated, relieving the expression of Satb2, which then represses

Ctip2 hence promoting colossal identity. Not all the factors involved in neuronal differentiation and their interplay are currently understood and more data needs to be acquired to fully comprehend this important developmental step.

Synaptogenesis

Highly polarized new neurons make thousands of synapses with other neurons in a strictly regulated fashion, eventually integrating into a functional neuronal

50 network. The generation of synapses requires coordinated assembly of pre- and postsynaptic entities, where axons and dendrites make contacts and establish nascent synapses which become stabilized or eliminated due to synaptic activity (Waites et al., 2005; McAllister et al., 2007). Synapsins (syns) are neuronally expressed phosphoproteins that are thought to be involved in neuronal development and synaptic function (De Camilli et al., 1983, Huttner et al., 1983). They are phosphorylated by many kinases, including MAPKs with conserved, specific binding sites, which modulate the association of syns with synaptic vesicles, actin filaments and other synaptic proteins (Jovanovic et al., 1996; Matsubara et al., 1996).

Differential phosphorylation of syns influences synapse formation and function during development and in mature synapses and is specifically important in fine tuning of neural networks. Syns knock out mice develop epilepsy and behavioral abnormalities, and human mutations in these proteins have been linked with autism, epilepsy and mental disability.

ERKs and their effectors are highly expressed in mature neurons (Boulton et al. 1991). The MAPK pathway phosphorylates synapsins upon BDNF and NGF stimulation in neuronal and PC12 cells (Jovanovic et al. 1996, 2000) where MAPK phosphorylation caused a redistribution of syns from synaptic vesicles to cytosol (Chi et al., 2003). Therefore, it has been postulated that MAPK signaling contributes to establishment of functional synapses through activity dependent modulation of vesicle cycling and neurotransmitter release (Kushner et al., 2005; Vara et al., 2009;

Giachello et al., 2010). Furthermore, members of the FGF family including FGF22,

FGF7 and FGF10 are expressed in neurons during synapse formation and can

51 promote synaptogenesis in cultured chick motor neurons by inducing synaptic vesicle

aggregation (Umemori et al., 2004). Importantly, analysis of hippocampal synapse

formation in FGF22 and FGF7 mutant mice revealed that FGF22 is necessary for

presynaptic differentiation of excitatory synapses whereas FGF7 is required for

GABAergic synapses (Terauchi et al., 2010).

Decrease in spine number and morphology have been linked to many

neuropsychiatric disorders such as schizophrenia, depression and mental retardation.

Recent work by Gao et al. demonstrates that ERK signaling is important for dendritic

spine formation in the hippocampus and the lateral amygdala through their

association with a scaffolding protein, IQGAP1. Although, ERKs are activated by

stimulation of NMDARs in vitro, or learning in vivo, constitutive deletion of IQGAP1 scaffolding protein failed to elicit ERK phosphorylation despite normal ERK levels.

Importantly, the failure to elicit proper ERK signaling resulted in significant impairments in memory formation and long term potentiation but not in emotional or motivational behavior (Gao et al., 2011). The potential mechanism involved in the ineffective ERK signaling or why spine density is important for selective behavior has yet to be determined.

Pathophysiology Associated with Cortical Development

The development of the central nervous system is one of the most complicated biological processes, requiring an orchestrated amalgamation of many cellular and environmental cues. Our understanding of the intricate mechanisms which form the

52 brain and the cerebral cortex has progressed exponentially in the last decade.

However, the regulation of the molecular and cellular mechanisms and how they

influence cortical function and subsequently behavior are still not well understood.

Pathology associated with aberrant cortical development is a major health problem

because of its implications in epilepsy, cerebral palsy and most importantly severe

cognitive disability (Ross and Walsh, 2001).

The term malformation of cortical development (MCDs) was first introduced

in 1996 in order to describe a common group of disorders in children presenting with

developmental delay and epilepsy (Barkovich et al. 1996; Barkovich and Kuzniecky,

1996). The MCDs are a major source of mental retardation, motor dysfunction, and

epilepsy in children where 10% of children with epilepsy harbor an MCD (Larsson K,

et al., 2006) but the degree of motor or cognitive disabilities varies. In addition, less

obvious cortical malformations may contribute to complex cognitive disorders such as autism and schizophrenia.

The advent of high-resolution MRI techniques has facilitated the in vivo

identification of a large group of cortical malformation phenotypes. In 2005 a

classification system for MCD was developed which divided the MCDs into disorders

of cell proliferation, migration, and cortical organization. This system has been used

for guiding the diagnostic approach in individual patients. In addition, advances in human and molecular genetics further contributed to this classification.

The generation of a large number of intermediate progenitors has been linked to increased cortical complexity and brain size in higher mammals (Martinez-Cerdeno et al., 2006; Kriegstein et al., 2006). The complex primate and human subventricular

53 zone is composed of an outer subventricular zone, which contains radial glia-like

cells as well as intermediate progenitor cells. Both types of cells lack contact with the

neuroependymal layer of the ventricular zone but are capable of symmetric, self-

renewing divisions, allowing for further expansion of the progenitor pool (Hensen et

al., 2010). This evolutionary expansion of the dividing progenitors translates to

significantly increased surface area and gyrification of the cortex (Zecevic et al.,

2005). Mutations in genes regulating cell proliferation, centrosome maturation,

mitotic spindle formation and DNA repair have been implicated in severe congenital

microcephaly (Thornton and Woods, 2009; Castiel et al., 2011, Kalay et al., 2011).

Other mutations have been linked to macrocephaly and polymicrogyria (Mirzaa et al.,

2004). Malformations due to abnormal neuronal migration are associated with

periventricular nodular heterotopia (Ferland et al. 2009), other heterotopias,

lissencaphaly and dysplasias (Kumar et al. 2010; Barkovich, 2000), whereas

polymicrogyria and schizencephaly are associated with postmigrational organization

of the cortex and may occur from disruptions in glial attachment to the pia (Jaglin and

Chelly, 2009). It is important to note that many aberrantly placed neurons can not be

easily visualized by modern histopathologic techniques, but only DNA labeling in

mutant animals, therefore little is known about their origin. It has been postulated that

these subtly misplaced neurons are implicated in idiopathic neurological disorders

ranging from childhood epilepsy and mental retardation to autism and developmental

dyslexia. One of the classic examples of genes required for neuronal migration came

from the “reeler” mouse, named for its abnormal motor coordination. The "reeler" mouse was first discovered in 1951 by D.S.Falconer as a spontaneous mutation

54 arising in a colony of mice maintained by geneticist Charlotte Auerbach. It wasn’t

until the 1970s that the inversion of the cortical layers was appreciated attracting

more attention to the reeler mutation (Caviness VS, 1976) and its association with

“upside down” cortical layering and anomalous neuronal migration (Caviness and

Rakic, 1978; Rakic and Caviness, 1995). Reelin is a large extracellular matrix molecule, synthesized by the earliest generated neurons (the Cajal-Retzius cells), that binds to receptor classes including VLDLR and ApoER2 (Trommsdorff et al., 1998).

Furthermore, abnormalities associated with misprojections of CPN lead to

behavioral pathology. For example, absence of CPN connectivity between the two hemispheres in humans is associated with defects in abstract reasoning, problem solving and generalization (Paul et al., 2007). In addition, CPN dysgenesis or reduced corpus collosum connectivity has been reported in patients with autism spectrum disorders (Minshew et al., 2007; Freitag et al., 2009).

FGF signaling plays an important role in neural development therefore it is nor surprising that that alterations in this pathway lead to multiple neurological disorders. Studies from postmortem human prefrontal cortex and hippocampus show

downregulation of many FGF ligands and their receptors in depressed patients (Evans

et al., 2004; Riva et al., 2005). In fact, acute or chronic administration of FGF2

reduced anxiety and depressive-like behavior in rats (Evans et al., 2004; Turner et al.,

2008; Perez et al., 2009). It has also been postulated that defects in FGF signaling

increase vulnerability to neuropsychiatric disorders and could account for

neuropathology associated with ASD (Rubenstein 2010; Vaccarino et al., 2009).

Studies have shown that mice with altered FGF signaling during early brain

55 development show structural and functional deficits in cortical development which

can be linked to abnormal behavior. For example, loss of FGF2 and subsequent

reduction in cortical pyramidal neurons resulted in much longer loss of lightening reflex after GABA receptor agonist (Korada et al., 2002). Furthermore, the FGF17

null mice have abnormalities in social interaction behaviors such as isolation-induced

ultrasonic vocalization, social recognition and social interaction tests (Cholfin and

Rubenstein, 2007a; Scearce-Levine et al., 2008). The FGFR knock out mice do not

develop a proper olfactory bulb, mimicking human Kallmann syndrome patients that

present with anosmia due to olfactory bulb dysgenesis (Kim et al. 2008). In fact

many patients with developmental cortical malformations experience anosmia. Mice

with a dominant-negative FGFR1 allele show altered neurogenesis and develop

spontaneous and locomotor hyperactivity by the second postnatal week (Shin et al.,

2004). The mechanistic explanation for this abnormal behavior is now known.

Hyperactivity was also reported in mice with deleted FGFR1 (hGFAP-Cre) despite

their normal monoaminergic and catecholaminergic signaling (Muller Smith et al.,

2008). However, most mouse models with altered FGF signaling do not show

impairments in learning and memory. Non-lethal thanatophoric dysplasia (TD) is

associated with a gain of function mutation in the FGFR3 gene where patients present

with severe motor and intellectual limitations (Bellus et al., 1999; 2000). New

approaches to treatment of cortical malformation disorders may become possible due

to greater understanding of cortical development. The generation of new neurons

from transplanted stem cells must take into consideration the intricate transcriptional

56 footprint required, so they can impart appropriate regional, areal and global

information necessary to guide them into the location of desired repair.

Developmental Disorders of the MAP Kinase Pathway

Disorders of cognition affect 1-3% of the general population in the United

States. They are caused by genetic predisposition, but can also be sporadic and

together account for more than half of all intellectual disability patients (formerly

termed mental retardation). A class of human genetic disorders with gain or loss of function mutations in different genes upstream of the RAS/MAPK signalling pathway has recently emerged. The proper regulation of this pathway is essential for normal development and even minor alterations result in neurocognitive disorders and sometimes an increased risk for developing cancer. These syndromes are genetic disorders of cognition with varying degrees of cognitive impairment and neuro- cardio-facio-cutaneous abnormalities, termed NCFC syndromes. In humans this disorders are caused by heterozygous mutations in elements of the MAP kinase signaling pathway as well as their modulators and downstream signal transducers.

The majority of these syndromes manifest with increased signal transduction of the

RAS/MAPK pathway, with an incidence of 1/3000. These include Noonan Syndrome

(mutations in SHP2, K-Ras, Sos1), LEOPARD Syndrome (mutations in SHP2 and c- raf), Costello Syndrome (mutations in H-Ras) and cardio-facio-cutaneous (CFC)

Syndrome (mutations in B-Raf, MEK1/2 and H-Ras). In addition, mutations in Rsk2, a downstream target of ERK1/2, lead to Coffin-Lowry Syndrome (CLS) and

57 mutations in Rsk-4 are associated with X-linked mental retardation.

Neurofibromatosis Type 1 is an autosomal dominant disorder affecting 1 in 3000

newborns with mutations in the NF1 gene, which negatively regulates RAS, resulting

in an increase in signalling in the MAPK pathway (Tidyman and Rauen, 2009;

Williams, 2009).

Noonan syndrome, Costello syndrome and CFC syndromes exhibit an

autosomal dominant mode of inheritance and are characterized by multiple congenital

anomalies including distinctive facial appearance, heart defects, musculo-cutaneous

abnormalities, and intellectual disability. Children with Costello syndrome also have

an increased risk of malignancy by approximately 17% (Gripp et al., 2002). Young

children, under the age of 4, are at risk for rhabdomyosarcoma or neuroblastoma

whereas patients older then 10 are at risk of bladder carcinoma (Gripp et al., 2002;

Gripp, 2005). Mutations in the PTPN11 gene, that encodes the SHP2 protein, are associated with Noonan as well as LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary

stenosis, abnormal genitalia, retardation of growth, andsensorineural deafness)

syndrome. Gain-of-function mutations in PTPN11 have been identified in

approximately 50% of individuals with clinically diagnosed Noonan syndrome

(Tartaglia et al., 2001; Musante et al., 2003; Niihori et al., 2005). On the other hand,

loss of- function or dominant negative mutations in PTPN11 are associated with

patients diagnosed with LEOPARD syndrome (Hanna et al., 2006; Kontaridis et al.,

2006; Tartaglia et al., 2006). Mutations in KRAS, BRAF, and mitogen-activated

protein kinase kinase 1 and 2 (MAP2K1/MAP2K2) have been identified in patients

58 with CFC syndrome (Niihori et al., 2006; Rodriguez- Viciana et al., 2006). Coffin–

Lowry syndrome (CLS) is a rare syndromic, X-linked form of mental retardation

caused by heterogeneous loss-of-function mutations in the RSK2 (90-kDa ribosomal

S6 kinase) gene. RSK2 is a growth factor-regulated serine-threonine protein kinase

that is activated and phosphorylated directly by ERK1/2 (Hauge et al., 2006). Major

clinical features of CLS patients include growth and psychomotor retardation,

characteristic facial and digital abnormalities, and progressive skeletal alterations.

Mutations in RSK2 are also associated with reduction in brain volume, specifically in

the hippocampal and cerebellar regions, and severe intellectual disability (Kesler et

al., 2007; Poirier et al., 2007).

The developmental syndromes of the MAPK pathway are caused by germline

mutations in many different genes and the diversity of mutations within each gene are

reflected in the variety of phenotypic features associated with this class of syndromes.

However, recent studies revealed that all the mutations share a common molecular

mechanism, which centers on the dysregulation of the MAPK cascade. The functional

studies of the mutated genes determined that most of the mutations increase the

downstream signaling of this pathway, and may even result in constitutive

hyperactivation. Therefore, in addition to the overlapping clinical manifestations of

these developmental syndromes they also share a strong predisposition to

malignancy. Although many of the germline mutations are similar to somatic

activating mutations seen in cancer, overall, they tend to be less hyper-activating.

This is most likely due to the fact that stronger somatic mutations in these genes are

59 not tolerated as germline mutations. This strongly highlights the importance of the

MAPK pathway in normal brain development and function.

Figure 1-5

Disorders of the MAP Kinase Pathway.

It has only recently been appreciated that haploinsufficiencies in elements of

the ERK1/2 cascade also present with similar clinical abnormalities. Interstitial

deletions of chromosome 22q11.2 are one of the most common deletions in humans

with frequency 1:4000-1:8000 births (Scambler 2000). The classical 22q11.2 deletion

60 syndrome is typically associated with two clinical phenotypes: velocardiofacial syndrome and DiGeorge syndrome (Ryan et al., 1997). Patients with 22q11.2 deletions present with a range of findings similar to NCFC syndromes including cardiovascular malformations (conotruncal heart defects), palatal abnormalities, velopharyngeal insufficiency, facial dysmorphia, learning difficulties and immune deficiency (Ryan et al., 1997; Emanuel et al., 2001). This deletion syndrome is also characterized by congenital hypocalcemia and urogenital defects. Nearly 90% of patients with 22q11.2 deletions harbour a common ~3Mb deletion, but a small subset carries a non-overlapping 1.5 Mb microdeletion which is clinically indistinguishable from the larger deletion. Similar features can be observed in reciprocal duplication of

22q11.21 region but the clinical phenotypes are more variable, ranging from multiple congenital defects to mild learning difficulties or even a normal presentation

(Ensenauer et al., 2003). A cluster of recurrent 1.4 and 2.1Mb rearrangements was also found distal to the classic 22q11.2 deletion (Rauch et al., 1999; Saitta et al.,

1999). The 22q11.2 distal deletion does not overlap with the proximal deletions and encompasses the MAPK1 locus, which encodes ERK2 (Stankiewicz et al. 2002).

Interestingly, all patients harbouring distal or proximal deletions share some common clinical characteristics including mild to moderate developmental delay and disability, cleft palate, and microcephaly as well as a variety of congenital heart defects (Lee et al., 2006; Verhoeven et al., 2011). In addition, both distal and proximal chromosomal rearrangements display common etiology, occurring as a result of nonallelic homologous recombination due to low copy repeats flanking the 22q11.2 region

(Shaikh et al., 2000; Edelmann et al., 1999; Saitta et al., 1999). However, new data

61 from patients with 22q11.2 distal deletions points to a more distinct set of characteristics not reported in the classical deletion patients. Most of the patients with distal deletion show prenatal and postnatal growth restrictions, more common developmental delay and intellectual disability, skeletal abnormalities and more distinct dysmorphic features. In addition, many are born prematurely with a mean gestational age of 35.2 vs. 38 weeks in other chromosomal syndromes (Goc et al.

2006).

While considerable attention has been paid to the craniofacial and cardiac phenotypes, little is known about how these mutations lead to neurological disorders of cognition. Automated gene prioritization identified candidate genes most likely responsible for the congenital heart defects associated with both, proximal and distal

22q11.2 syndromes. The highest ranking genes were CRKL and MAPK1/ERK2

(Breckpot et al., 2012). Importantly, CRKL and MAPK1/ERK2 interact within a common genetic pathway involved in craniofacial and outflow tract morphogenesis.

Pharyngeal ERK1/2 activation in response to FGF8 signaling was found to be CRKL dependent (Moon et al., 2006). Moreover, FGF8 hypomorphic mutant mice mimic many clinical features of 22q11.2 syndromes (Vitelli et al., 2002; Hu et al., 2004).

Deletion of ERK1/2 from the neural crest phenocopies the craniofacial and cardiac phenotypes as well (Newbern et al., 2008). In summary, both computational methods and biological evidence suggests that common craniofacial and cardiovascular abnormalities observed in patients with either the 3Mb deletion or with the distal

22q11 deletion arise from perturbation of a common pathway, the MAPK pathway

(Newbern et al., 2008).

62 Furthermore, our research as well as others’ noted that a deletion on chromosome 16

that includes MAPK3 gene, encoding ERK1, has been linked to autism, intellectual

disability, cardiac and craniofacial anomalies. An accruing body of evidence suggests

that the 16p11.2 deletion which results in loss of ERK1 among other genes may

contribute to autism and mental disability associated with these disorders. This

hypothesis is explored further in the discussion part of this work.

ERK and Autism

Autism spectrum disorders (ASD) are characterized by impairments in

reciprocal social interaction, communication, and the presence of stereotyped repetitive behaviors as well as highly restricted interests. In recent years, data from copy-number variation (CNV) studies characterized submicroscopic chromosomal deletions and duplications as a significant cause of ASD. (Iafrate et al., 2004; Sebat et al., 2004). The highly heterogeneous allelic architecture points to many different chromosomal loci including: 1q21.1, 15q11.2-13.1, 15q13.2-13.3, 16p11.2, 17q12, and 22q11.2 (Bucan et al., 2009; Glessner et al., 2009; Kumar et al., 2008, Marshall et al.,2008; Moreno-De-Luca et al., 2010; Noor et al., 2010, Pinto et al., 2010; Weiss

et al., 2008). Noticeably, both ERK1 and ERK2 are implicated in the ASD

association studies through their genetic linkage to 16p11.2 and 22q11.2. In fact, the

single most common genetic linkage to autism spectrum disorders is associated with

deletions and duplications of a region on chromosome 16p11.2 (Sanders et al., 2011

63 and Levy et al., 2011) and is associated with more than 1% of all ASD patients

(Zoghbi and Schaaf, 2011). The 16p11.2 most commonly deleted locus contains 27 genes including the ERK1 gene (MAPK3) and Major Vault Protein (MVP) gene.

MVP is reported to regulate signaling through the ERKs (Liang et al., 2010; Kolli et al., 2004). Recent data suggests that MVP functions as a novel scaffold protein for both SHP-2 and ERK, forming a complex which is constitutively activated in MCF-7 breast cancer cells (Kolli et al., 2004). In addition, data from MVP deficient fibroblasts demonstrates that MVP cooperates with Ras for optimal EGF-induced

Elk-1 activation. Furthermore, MVP has been found to be overexpressed in many chemoresistant cancer cells and tumors and may play a role in cell differentiation and subcellular transport (Mossink et al., 2003). Furthermore, a smaller deletion of the

16p11.2 locus associated with ASD contains only 5 genes including the MVP gene.

Behaviorally, probands carrying a 16p11.2 de novo CNV were indistinguishable from the larger group of ASD probands with regard to IQ, ASD severity, or categorical autism diagnosis.

Our current hypothesis explores the possibility that altered expression of one or more genes in the putative region on chromosome 16 may converge onto the

ERK/MAP kinase signaling pathway resulting in pathology and cognitive deficits associated with ASD. Since perturbations in ERK signaling are implicated in a number of neurodevelopmental disorders and have a prominent role in neurogenesis, it is important to consider ERKs as possible contributors to the ASD phenotype.

64 Research Goals

The evidence described above clearly demonstrates that MAP kinases,

ERK 1 and ERK2, are involved in multiple stages of brain development and are

crucial for its proper function. Independent genetic linkage of both of these

kinases to developmental disorders hallmarked by aberrant brain function and

intellectual disability strongly suggest that ERKs play an important role in

normal development and cognition, providing a strong rationale for studying

their biology and mechanism of action. Furthermore, ERKs are involved in

regulation of many signaling components and machinery necessary for accurate

cell cycle control which is important for most steps of cortical development.

ERKs are also uniquely positioned to receive a wide range of extracellular

stimuli but have the ability to interpret these signals in a concrete and cell

specific context. The goal of this dissertation is to discern the precise mechanism

by which ERK1 and ERK2 control progenitor proliferation during

corticogenesis and thereby contribute to proper cortical development and

function.

There is considerable interest in the role of ERK MAP kinases in the

development of the brain because genetic disorders arising from copy number variations in the ERK genes or mutations in their upstream regulators result in

Neuro-cardio-facial cutaneous (NCFC) syndromes and are associated with

developmental abnormalities, cognitive deficits and autism. In the present study

65 we have shown that ERK activity is confined to the ventricular zone during corticogenesis and peaks at E14.5. Therefore, we developed murine models of these disorders by deleting the ERKs at the beginning of neurogenesis utilizing an Emx-1 promoter, ablating ERK activity before neurogenesis.

We report that deletion of one or both ERK isoforms leads to disrupted cortical progenitor generation and proliferation and altered cytoarchitecture of the postnatal brain. Importantly, these changes occurred in a gene-dose dependent matter. We present convincing evidence that the deficits in progenitor proliferation dynamics are due to ERK-dependent dysregulation of cyclin D1 and p27Kip1, resulting in cell cycle elongation and favoring of neurogenic over self-renewing cell divisions. The precocious neurogenesis caused premature progenitor pool depletion, altering the number and distribution of pyramidal neurons in the mature brain. Furthermore, we provide evidence that loss of

ERK2 alters the intrinsic excitability of cortical neurons and contributes to perturbations in global network activity. These changes are associated with elevated anxiety and impaired working and hippocampal-dependent memory. In conclusion, we provide new mechanistic insight into the basis of cortical malformation and propose a potential link to cognitive deficits in individuals with altered ERK activity.

66 CHAPTER 2

Disrupted ERK signaling during cortical development leads to abnormal

progenitor proliferation, neuronal and network excitability and behavior,

modeling human NCFC and related syndromes.

Joanna Pucilowska, Pavel A.Puzerey, J. Colleen Karlo, Roberto F. Galán and Gary E.

Landreth

Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio

44106-4928

Correspondence: Dr. Gary Landreth Alzheimer Research Laboratory, E649 Department of Neurosciences School of Medicine Case Western Reserve University 10900 Euclid Ave. Cleveland, OH 44106-4928 216 368 6101 FAX 216 368 4650 Email: [email protected]

67

Abstract:

Genetic disorders arising from copy number variations in the ERK MAP

kinases or mutations in their upstream regulators that result in Neuro-cardio-facial

cutaneous (NCFC) syndromes are associated with developmental abnormalities, cognitive deficits and autism. We developed murine models of these disorders by deleting the ERKs at the beginning of neurogenesis and report disrupted cortical

progenitor generation and proliferation which leads to altered cytoarchitecture of the postnatal brain in a gene-dose dependent manner. We show that these changes are due

to ERK-dependent dysregulation of cyclin D1 and p27Kip1, resulting in cell cycle elongation, favoring neurogenic over self-renewing divisions. The precocious neurogenesis causes premature progenitor pool depletion, altering the number and distribution of pyramidal neurons. Importantly, loss of ERK2 alters the intrinsic excitability of cortical neurons and contributes to perturbations in global network activity. These changes are associated with elevated anxiety and impaired working and hippocampal-dependent memory in these mice. This study provides a novel mechanistic insight into the basis of cortical malformation which may provide a potential link to cognitive deficits in individuals with altered ERK activity.

68 Introduction:

The ERK MAP kinases, ERK1 and ERK2, are the central elements of a prominent signaling pathway governing neural development (Samuels et al. 2009).

Chromosomal duplications/deletions of 16p11.2, which includes the MAPK3 gene encoding ERK1, are the most common genetic cause of autism and are associated with cardiac and craniofacial abnormalities (Fernandez et al. 2010; Campbell et al.

2008). Saitta and others identified a cohort of patients with a 1 Mb micro-deletion on chromosome 22 which encompasses the MAPK1 (ERK2) gene (Saitta et al. 2004).

These individuals exhibit neurodevelopmental deficits indistinguishable from

DiGeorge, or 22q11 deletion syndrome, although this deletion is distal to the recurrent deletion associated with classical DiGeorge. Similarly, gain or loss of function mutations affecting ERK signaling lead to a number of clinically related disorders termed Neuro-cardio-facial cutaneous (NCFC) or “Ras/MAPK syndromes”, including Noonan, LEOPARD, Costello, and Cardio-Facio-Cutaneous syndromes

(Tidyman and Rauen 2008; Samuels et al. 2009). Affected children exhibit overlapping phenotypes including deficits in brain, heart, face, and skin development.

Importantly, these syndromes are characterized by a high incidence of cognitive impairments and psychiatric disease. Thus, mutations affecting ERK activation are a significant genetic cause of neurodevelopmental disorders in humans. To elucidate the precise mechanisms underlying the structural and functional deficits resulting from perturbations in the ERK signaling in the brain, we developed murine models of

69 NCFC and related syndromes in which ERKs are genetically inactivated within the

developing dorsal telencephalon.

The events coordinating the temporal and spatial organization of the

developing cortex are tightly coupled to cell cycle dynamics within the proliferating

progenitors. The molecular cues linking the cellular composition of the cortex to

factors regulating proliferation are not well understood. The size of the adult cortex is

determined by strict control of progenitor divisions in the ventricular (VZ) and

subventricular zones (SVZ), the hubs of neurogenesis. The cerebral cortex is

generated from a single neuroepithelial layer that become radial glia which

subsequently give rise to basal progenitors (also termed intermediate progenitor cells)

in the SVZ and all projection neurons, astrocytes, and oligodendrocytes in the mature

cortex. Radial glia divide through symmetric, self-renewing divisions or

asymmetrically, generating a neuron or a basal progenitor. In the SVZ, basal

progenitors undergo predominantly symmetric divisions, most commonly generating

two neurons. The total cell cycle length, G1 length and cells exiting the cell cycle

increase as neurogenesis proceeds (Rakic and Caviness 1995; Takahashi et al. 1996).

Huttner and colleagues demonstrated that elongation of G1 favors neurogenic

divisions (Lange et al. 2009). Two potent G1 regulators are directly controlled by the

ERKs: cyclin D1 and p27Kip1. Cyclin D1 is highly expressed in the proliferative zones and transcriptionally controlled by the ERKs (Glickstein et al. 2007). p27Kip1, a cdk

inhibitor, modulates the G1 to S-phase transition and is phosphorylated by the ERKs,

allowing cell cycle progression (Sherr and Roberts 1999).

70 Little is known about the exact roles of ERK1/2 during brain development.

We previously reported that basal progenitor proliferation was impaired when ERK2

was inactivated at mid-neurogenesis (Samuels et al. 2008). However, ERK activity

strongly mirrors the neurogenic gradient of the VZ during earlier development.

Therefore, we targeted its deletion to this specific and pivotal time to examine its

exact functions and mechanism of action within the developing cortex. We generated

a novel mouse model in which ERK2 is inactivated at E9.5 using Emx1-cre either on

wild type (CKO) or an ERK1 null background (double knock out; DKO). We report

that; 1) loss of ERK2 results in microcephaly with perturbations in the number of

projection neurons and these effects are more profound in DKO mice; 2) these

deficits arise from ERK-dependent dysregulation of progenitor generation and

proliferation; 3) altered cytoarchitecture disrupts cortical circuitry and network

dynamics; 4) loss of ERK2 alters the intrinsic excitability of cortical neurons; 5) these

effects are associated with anxiety and cognitive deficits. Thus, these data suggest

that loss of ERK activity may underlie the broad spectrum of clinical deficits

associated with NCFC syndromes, distal 22q11 deletion syndrome and a subset of

autism spectrum disorders.

Methods

Mice: All mice were bred on C57BL/6J background as previously described

(Samuels et al., 2008). Emx1-cre mice (Jackson Laboratory, Maine, USA) were

crossed with ERK2 floxed mice. The Emx-1 allele induces recombination in

progenitors and stem cells of the dorsal telencephalon by E9.5 (Gorski et al., 2002).

71 The day of vaginal plug was designated as 0.5 (E0.5). Experiments were performed in accordance with the CWRU Institutional Animal Care and Use Committee.

Immunohistochemistry: Postnatal mouse brains were fixed in 4% PFA in 1X PBS at

4°C overnight whereas embryonic brains were dissected in PBS and immersion fixed in 4% PFA for 1hour. 10 micron cryostat sections were rehydrated in PBS and antigen retrieval using 1X Reveal Decloaker (Biocare) was performed for 10 minutes at 95°C. Next, sections were blocked in 10% (embryonic) and 2% (postnatal) normal goat or donkey serum for 1 hour at room temperature with 0.1% (vol/vol) Triton X-

100 in PBS. Slides were incubated with primary antibodies overnight at 4 °C, washed and incubated with appropriate secondary antibodies for 1-2 hours at room temp. The primary antibodies used were as follows: polyclonal rabbit anti-pERK (Cell

Signaling, 1:100); rabbit polyclonal anti-Pax6 (Covance, 1:300); mouse monoclonal anti-Pax6 (DSHB, 1:100); rat-anti-BrdU (Abcam, 1:100); mouse anti-BrdU (BD,

1:100); mouse anti-NeuN (Chemicon, 1:500); mouse anti-PH3 (Upstate, 1:250); rabbit anti-PH3 (Upstate, 1;500); rabbit anti-Tbr1 (Chemicon,1:1000); rabbit anti-

Tbr2 (1:300); goat anti-Brn1 (Santa Cruz Biotechnology, 1:50), rat anti-Ctip2

(Abcam, 1:500) and rabbit anti-caspase 3 (1:200; Cell Signaling Technology), rabbit anti-Cux1 (Santa Cruz Biotechnology, 1:100), rabbit anti-SatB2 (Abcam, 1:300).

Secondary antibodies used were Alexa Fluor 488 (1:1000), 546 or 593 (1:1000) conjugated to goat or donkey anti-mouse, anti-rabbit or anti-goat (Invitrogen). DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI) for 5 min (DAPI, Molecular

Probes).

72 Western analysis: Cortices of E14.5 brains were washed with ice-cold HBSS and sonicated in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 1.5 mM MgCl2, 20 mM NaF, and 20 mM β- glycerophosphate) in the presence of protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM PMSF, and 1 mM Na3VO4). Lysates were centrifuged, and protein concentration was calculated using the bicinchoninic acid assay (Pierce) with BSA as a standard. Equal amounts of protein were boiled in sample buffer, separated by SDS-

PAGE gels, and transferred to Immobilon-P polyvinylidene difluoride membranes

(Millipore). Membranes were blocked in 3% BSA [or 5% skim-milk powder for β- tubulin (Santa Cruz) in TBS and 0.1% Tween 20 (TBS-T) for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies used were: rabbit anti-pERK (Cell Signaling, 1:1000), mouse anti-ERK2 (BD Biosciences

Discovery Labware, 1:3000), mouse anti-ERK1 (Zymed Laboratories, 1:1000), anti-

β-tubulin and (1:5000; Santa Cruz Technology). Membranes were washed with TBS-

T, incubated with HRP-conjugated secondary antibodies: goat anti-mouse or anti- rabbit (1:5000; GE Healthcare) in TBS-T with 5% milk for 2 h at room temperature.

Detection was performed using either Millipore chemiluminescence using BioMax

MR X-ray film (Eastman Kodak). Densitometry was performed using Adobe

Photoshop histogram function, and statistical analysis was done with GraphPad Prism software.

BrdU analysis: Timed pregnant dams were injected with BrdU (50mg/kg body weight, Sigma B5002) by i.p and processed for BrdU immunohistochemistry.

73 Standard BrdU labeling was utilized with an addition of antigen retrieval (10 minutes

in sodium citrate buffer). For pulse-chase labeling of deep cortical neurons, pregnant dams were given a single BrdU injection at E12.5 and embryos were fixed and co- labeled with a lamina specific marker at P2 and P10. Superficial cortical neurons

were pulsed with BrdU at E14.5 and co-labled with a layer specific marker at P2 and

P10. Cell cycle exit was analyzed by injecting pregnant mice at E13.5 and fixing embryos at E14.5.

Quantification: Anatomically matched sections of littermate control and mutant mice were analyzed. At least 3 sections per animal were analyzed alongside the rostro-caudal axis of the telencephalon. Embryonic coronal sections were evaluated at the level of the mid-ganglionic eminences and analyzed at medial and lateral positions by counting all cells in designated 100µM wide boxes. In postnatal brains, coronal sections were used to count cells in 400µm boxes in somatosensory cortex.

Behavioral analysis: All tests were performed in conjunction with the Case Western

Reserve University Rodent Behavior Core. Three-month-old male mice were

evaluated using standardized behavioral tests: elevated plus maze, place preference,

open field, continuous alterations and delayed T-maze, rotarod, and contextual and

cued-fear conditioning. Tests were performed in order listed above. All testing was

completed in the same room during the light cycle between 9:00 A.M. and 6:00 P.M.

Mice were single housed with ad libitum access to food and water with a 12 h

light/dark cycle. All equipment used was sterilized with 70% ethanol after each use in

order to remove odor cues. The elevated plus maze test was conducted first to avoid

74 its sensitivity to prior experience. Each test was performed at least 48h apart, so the mice could rest. The tester was blinded to the genotype of each animal.

The elevated plus maze: the two open and two closed arms forming a cross were placed approximately 1 m above the floor and fitted with infrared grid and video tracking system (Med Associates Inc.). Individual mice were placed in the center facing the open arm and their activity was analyzed for 5 minutes. The total time spent in open vs. closed arms, and the number of entries into each arm was analyzed, the number of head dips and frequency of urination/defecation were noted.

The place preference test: two square chambers with dark gray flooring were used.

The translucent, lit chamber (20cm x 20cm) was illuminated with 100-W light placed

40cm above the floor. The dark chamber (15cm x 15cm) had a dark cover. Each mouse was placed in the open chamber facing away from the dark chamber. The movement was recorder for 5 minutes. Latency to enter into the dark chamber and the number of re-entries into light were scored.

The open field test: A box (40cm x 40cm) was placed in a dimly lit environment.

EthoVision XT 5.0 (Noldus) was used to digitally subdivide the box area into a 20 cm

× 20 cm center area and a periphery. The peripheral area was further divided into a middle (inner 10 cm) and an outer area (outer 10 cm) to determine thigmotaxic behavior. Mice were placed in the center and allowed to explore the area freely for 15 min. Locomotor parameters such as total distance moved, velocity, angular velocity and heading degrees were measured to determine basic locomotor activity and presence of stereotypies. Frequency and duration in the center, periphery and outer

75 quadrants were collected to determine anxiety-like behavior. In addition, data were nested into 5-min bins and distance moved during each of these 3 periods was recorded to evaluate habituation differences across groups.

Continuous Alternation and Delayed T Maze: A T-shaped box was marked with different tape designs. The entry arm was perpendicular to the other two arms. All experiments were recorded with EthoVision XT 5.0 (Noldus). For continuous alteration test, the percent of alterations was recorded for each animal during a 3 minute interval. Each mouse was placed in the entry arm not facing the other two arms. For delayed T maze, the mice were habituated to the environment for two consecutive days. The closed arm was chosen at random for each animal. Time in novel arm, number of entries and total number of entries were scored for each mouse.

Cortical slice preparation: Parasagittal thalamocortical slices (350 µm) from somatosensory cortex were prepared as described previously (Agmon and Connors,

1991) using young (P13-P18) C57/BL6 wild-type mice and ERK2 CKO mice. Mice were anaesthetized with isoflurane and then decapitated using a guillotine. The brain was cut on a vibratome (Leica VT1200) while immersed in ice-cold pre-oxygenated artificial cerebrospinal fluid (aCSF) containing: 125 mM NaCl, 2.5 mM KCl, 1 mM

MgCl2, 2 mM CaCl2, 25 mM NaHCO3, 1.25 mM NaH2PO4 and 25 mM glucose.

Salts and reagents were from Fisher Scientific (Pittsburgh, Pa). Slices were then transferred to an incubating bath containing normal aCSF at room temperature and allowed to equilibrate for 20-40 minutes before being transferred to the recording chamber.

76 Electrophysiology: Whole-cell patch-clamp recordings were executed in layer II/III

PCs from thalamocortical slices of mouse somatosensory cortex. Slices were

constantly perfused (4mL/min) with oxygenated ACSF while pyramidal cells (PCs) in

layer II/III were identified visually at high magnification (63×) with Kohler

illumination using an upright microscope (Zeiss Axioskop 2). Whole-cell recordings

in voltage-clamp and current-clamp mode were established in PC somas using

pipettes (6-10 MΩ) filled with the following (in mM): 120 potassium gluconate, 2

KCl, 10 HEPES, 10 sodium phosphocreatine, 4 MgATP, 0.3 Na3GTP, 25 QX314,

and adjusted to pH 7.4 with KOH. Experiments in which the transient outward K+

current was measured required the addition of 20 mM BAPTA to eliminate

contamination with currents generated by Ca2+-activated K+ channels. The transient

outward K+ current was measured using previously described protocols under

voltage-clamp (Eder and Heinemann, 1994). Measurements of miniature postsynaptic currents were carried out using a modified internal pipette solution containing (in mM): 120 cesium gluconate, 2 CsCl, 10 HEPES, 10 sodium phosphocreatine, 4

MgATP, 0.3 Na3GTP, 25 QX314, 20 mM BAPTA and adjusted to pH 7.4 with

CsOH. Current and voltage recordings were obtained using a Multiclamp 700B

amplifier (Molecular Devices, Foster City, CA) digitized at 10 KHz using a Digidata

1400 data acquisition interface. Currents in voltage-clamp were filtered at 1KHz and voltage traces were band-pass filtered in the frequency domain with a custom-made

filter (band pass: 0.05 - 300 Hz; stop band: 0 - 400 Hz). Experiments in voltage and

current clamp configuration were used to assess passive membrane properties. The

neuronal input resistance was determined by stepping the membrane voltage across a

77 range of potentials (-100 to -60 mV) for 500 ms in the voltage-clamp configuration

and plotting the subsequent mean steady-state current against the holding potential.

Using linear regression, we fit the resulting current-voltage (I-V) plot and obtained the input resistance by taking the inverse of the I-V slope. Membrane resistance and

capacitance were obtained directly from the pClamp acquisition software (Molecular

Devices, Foster City, CA) in voltage-clamp at a holding potential of -65 mV. The

membrane time constant, τ, was measured by first injecting a brief (100 ms)

hyperpolarizing square current pulse (-250 pA) in the current clamp configuration.

The discharge of the membrane potential after current step termination was plotted on

a semi-log scale and the membrane τ was taken as the slope of the linear fit of the second slow phase of the membrane potential decay (Iansek and Redman, 1973).

Firing frequency-input current (F-I) curves were generated by injecting 1 s current pulses ranging from 0 to 1000 pA in 30 pA increments. The total number of spikes at each current step was then plotted against the input current. Then the slope of the individual F-I curves were determined by a linear fit of the region between firing onset and maximal firing frequency. This region of the x-axis was defined as the input dynamic range. Averaged F-I curves were normalized along the x-axis to the input current that produced maximal firing rate (Imax). Ramp current injection in

current-clamp was used to assess: 1) spike latency, defined as the time elapsed between onset of stimulation and the first action potential; 2) number of spikes per

ramp; 3) the firing window which is the time elapsed between the first and the last

spike, and 4) the resting potential which was taken as the membrane voltage

78 preceding ramp current injection. Ramp velocity and duration were set at 225 pA/s and 2 s, respectively, injecting a total of 450 pA.

Recordings made in current-clamp mode were also used to assess spontaneous network activity. Gabazine (5 µM) was bath applied to elicit spontaneous network activity in the form of high conductance events (HCEs; ~10 nS). This activity is characterized by an initial plateau depolarization (~ 60 mV from baseline) lasting ca.

400 ms followed by decay (tail) of the membrane potential lasting ca. 500 ms. The membrane voltage was then recorded for 10 minutes with no current injection. EPSPs were detected using the criterion that the rate of change of the membrane potential must be in the interval 0.05 < dV/dt < 10 mV/s. We quantified and compared the number of EPSPs 2000 ms before and after burst onset (pre- and post-burst EPSPs, respectively) between WT and CKO populations. Miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) were recorded under voltage-clamp using cesium-modified internal solution and 1μ tetrodotoxin (TTX) in the extracellular solution. mEPSCs and mIPSCs were recorded at the reversal potentials of mIPSCs and mEPSCs , respectively (EIPSC = -80 mV; EEPSC = +15 mV), and detected using a custom algorithm written in Matlab (Mathworks). Drugs were obtained from Sigma (St. Louis, MO).

Biocytin labeling and histology: Layer II/III PCs were filled with 20 mM biocytin during patch-clamp experiments in thalamocortical slices (350µm) of mouse somatosensory cortex. Slices were preserved in 0.1 M PBS containing 4% PFA over

24 hours then washed 3x in 0.1 M PBS containing 0.5% triton-X 100 for 15 minutes and left to incubate for 24 h in the same solution containing avidin-biotin complex

79 (ABC Staining Kit – Elite Vector Labs). Next, slices were washed 3x in PBS-triton

and stained with a diaminobenzedine (DAB Peroxidase Substrate Kit – Elite Vector

Labs) solution until desired staining intensity was achieved. A series of alcohol and

xylene washes were then performed to dehydrate the tissue, after which it was kept in

methyl salycilate. Stained neurons were visualized under bright field illumination

using a Zeiss 63× oil immersion objective and reconstructed in 3D using Neurolucida

software (MicroBrightField, Colchester, VT). Neurons with weak or non-uniform

staining intensity were excluded from analysis.

Reconstruction analysis: Sholl analysis of dendritic structures was carried out

automatically with NeuroExplorer software (MicroBrightField, Colchester, VT). 1) a

series of concentric spheres with radii increasing at 10 µm intervals were drawn

around the centroid of the soma at distances 5 to 165 µm away from the soma; 2) the number of dendritic intersections crossing the boundary of each sphere was quantified

and was not cumulative, i.e., number of dendritic intersections in any given sphere

did not include dendritic intersections contained within smaller spheres; 3) the

number of dendritic intersections for WT and CKO mice was averaged and plotted as

a function of distance away from soma; 4) bootstrap statistical analysis (see

Electrophysiology Methods) was carried out to test for significant differences in the

number of dendritic intersections at each concentric sphere boundary. Other

morphological parameters, such as axon length, were automatically determined by

NeuroExplorer software and analyzed using bootstrap methods.

80 Statistical analysis. Miniature post-synaptic currents were analyzed using a two-

sample Kolmogorov-Smirnov goodness-of-fit test to compare distributions of

amplitude and inter-event intervals of recorded events between WT and CKO groups.

P-values below 0.05 were considered to be significant. All other significance tests were run in Matlab (version 2010a) using bootstrap analysis. Parametric (e.g. t-tests) and non-parametric (e.g. Wilcoxon ranksum) tests were not applicable for our data sets, as the implicit assumptions of these tests were not satisfied. Bootstrap analysis was performed as follows. For given quantity (e.g. membrane resistance), we measured n values from WTs and m values from CKOs. We wanted to test whether the difference of the means of both groups was significantly different. To this end, we

first gathered the n + m data points into one group and drew n points randomly to

form a new group, allocating the remaining m data points to form a second group.

This way we created random surrogate data sets from which the difference of the

means was calculated. We then iterated this process at least one million times to build

a probability distribution of the difference of the means for surrogate data. If the

difference of the means from the actual data was larger than the 95 percentile of this

distribution, then that value was considered to be statistically significant. The p-value

(p) was calculated as the integral of the distribution from left end (−∞) up to the

actual value. Significant values were indicated in the figures with one asterisk if 0.01

< p < 0.05, and with two asterisks if p < 0.001. This method was applied for all

parameters of interest and was the final measure of statistical significance.

81 Microscopy and image analysis. Images were acquired with a Zeiss LSM 510 confocal laser microscope with argon and helium–neon lasers and analyzed with LCS confocal software, Prism and Photoshop (Adobe). Images were collected approximately at the midpoint between the top and bottom of the two planes of focus.

Student’s t-test and one-way ANOVA with post-hoc Tukey test were used to establish statistical significance.

Results:

ERK1/2 activity is confined to the VZ/SVZ of the developing cortex and mirrors the neurogenic gradient.

To investigate the precise role of ERK1 and ERK2 during corticogenesis we first evaluated its activity during crucial early stages of telencephalic development.

We found that ERK activity in the embryonic cortex was confined to the proliferative zones with a low-medial to high-lateral expression pattern, mirroring the neurogenic gradient (Fig.1A,A’). We detected phosphorylated, active ERK1/2 in apical and basal neural progenitors, but not in postmitotic neurons, suggesting that ERKs act selectively within neural progenitors (Fig.1C-E). To test this, we utilized Emx1-cre to conditionally delete ERK2 (CKO) from the dorsal telencephalon at the beginning of the neurogenic period, E9.5 (Fig.1A,B). To evaluate ERK1 contributions to corticogenesis we bred the ERK2 CKO mice to ERK1 null mice of the same genetic background, thus generating double knockout mice (DKO), (Fig.1A,B). Evaluation of

CKO and DKO E14.5 embryos showed little ERK activity within the neurogenic region of the developing dorsal telencephalon (Fig.1A-E), which we also quantified by Western analysis (Fig.1B). Furthermore, we also examined the CKO mice for

82 potential changes in other developmentally important pathways, which are known to play a role in early cortical development and patterning. We did not see any significant changes in downstream elements of the Wnt or Notch pathways (data not shown).

Cortical size and thickness is reduced in ERK2 CKO and DKO mice.

Diminished ERK activity early in cortical development resulted in changes in brain size and cortical cytoarchitecture. The overall brain weight of mutant CKO mice at P2 and P10 was reduced by 11% when compared to WT littermate controls

(Fig.I,J; supp. Figure 1). However, brain weight of DKO mice was reduced by 32% at

P2 (data not shown) and 25% at P10 (Fig.1J). Most DKO mice died within the first postnatal week. The few animals that survived to P21 exhibited even more severe brain weight reduction (38%) than those measured at earlier ages (supp. figure 1), indicating that the loss of ERK1 and ERK2 within dorsal telencephalon is allele dose- dependent and critical for survival of these mice. In addition, we found that the CKO and DKO mice showed a significant decrease in cortical area, A-P length and cortical length, when compared to WT littermate control mice (Fig. 1F-J), and that the severity of the cortical deficits correlated with the number of ERK alleles deleted. In addition, we measured the overall body weight of P21 CKO and DKO mice as compared to sex matched, WT, littermate controls (Fig. 1F,H). We found an allele- dependent decrease in total body mass at P21 by 12% in CKO and 66% in DKO mice

(Fig. 1F,H). At P21 DKO mice were not mobile or responsive when handled.

83 Loss of ERK1/2 leads to perturbations in the laminar composition and neuronal

morphology of the postnatal murine cortex.

To address whether deletion of ERK1/2 alters the cellular composition of the

postnatal cortex we quantified the number of neurons using lamina-specific markers

in P2 and P10 mutant and WT cortices (Fig.2A,B, data not shown for P10). We

divided the cortex into 10 equivalent bins and visualized neurons using the pan

neuronal marker, NeuN. We found a significant deficit in the number of neurons in

bin 2-3 which correspond to the cortical layer II/III (Fig.3A) and can be delineated by

expression of Brn1. We then counted the number of Brn1+ neurons in CKO and WT

mice at P2 and observed a 35% loss of pyramidal neurons of layer II/III (Fig. 2A-B).

This deficit was greater (58%) in the DKO mice (Fig.2A,B). These pyramidal neurons are predominantly generated from SVZ progenitors during later stages of cortical development.(Wu et al. 2005; Tarabykin et al. 2001) Surprisingly, we observed an increase in NeuN+ neurons in bin 7 which corresponds to cortical layer

V (Fig.3B). CKO and DKO mice both showed a 28% and 29% increase, respectively in layer V sub-cortical projection neurons, which express the transcription factor

Ctip2 (Chen et al. 2005; Arlotta et al. 2005). These cells are derived mostly from VZ progenitors during early neurogenesis (Fig.2A-B). Interestingly, the number of neurons in layer V/VI, as marked by Tbr1, remained unchanged (Fig.2A,B).

Studies in several model systems demonstrated the importance of ERKs in neuritic development (Xiao and Liu 2003; Perron and Bixby 1999; Karasewski and

Ferreira 2003). We examined neuronal morphology in mice lacking ERK2 by

84 generating 3-D reconstructions of DAB-labeled layer II/III PCs from cortical slices.

Analysis of neuronal processes revealed a significant 50% reduction in the mean total

axonal length in ERK2 CKO mice (WT: 1019 ± 507 µm, n = 6; CKO: 496 ± 304 µm,

n = 7; p = 0.024, p=0.0244) (Fig. 2C,D) without significant changes in total number

of axon collaterals (WT: 2.8 ± 2.2; CKO: 1.9 ± 1.9; p > 0.05). Additionally, Sholl

analysis of basal dendrites revealed 35% and 30% less dendritic intersections at

distances 25 and 35 µm away from the soma, respectively (Fig.2C,E), suggesting

abnormal dendritic ramification in CKO animals. Importantly, this spatial range

includes a large proportion of synaptic contacts from neighboring layer II/III PCs,

(Feldmeyer et al. 2006; Hooks et al. 2011; Shepherd et al. 2005) and is therefore

likely to affect network function within layer II/III.

To test whether the postnatal changes in layer composition are a consequence

of developmental perturbations in progenitor proliferation, we determined the

frequency of Brn1+ and Ctip2+ neurons born at E14.5 and E12.5, respectively (Fig.

3). In CKO mice, we found a 40% decrease in Brn1+ neurons pulsed with a single

BrdU dose at E14.5 (Brn1+/BrdU+) and a more severe, 53% reduction in DKO mice

at P2 (Fig.3C,D). In contrast, the number of Ctip2 positive neurons co-labeled with

BrdU after a single pulse at E12.5 was increased by 26% and 30% in CKO and DKO

mice, respectively (Fig.3C, D). The Ctip2 labeled neurons in mutant mice appeared

larger and more dispersed (Fig. 3C). However, no significant difference was observed in frequency of BrdU/Tbr1 double- labeled neurons (data not shown). Similar results were obtained when mice were analyzed at P10 (data not shown). These data demonstrate that early loss of ERK activity in the CKO and DKO mice leads to

85 alterations in specific cortical lamina, which we directly link to perturbations of progenitor proliferation dynamics during cortical neurogenesis.

Apical and basal progenitor proliferation is altered in ERK2 mutant mice leading to premature progenitor pool depletion.

To establish whether the perturbations in cortical cytoarchitecture are due to altered progenitor proliferation dynamics, we examined both apical progenitors

(including neuroepithelial and radial glia) and basal progenitors. We identified radial glia by their expression of Pax6, a transcription factor which becomes downregulated in most basal progenitors expressing the T-domain transcription factor, Tbr2

(Englund et al. 2005). Post-mitotic neurons were identified by Tbr1 expression (Götz et al. 1998; Bulfone et al. 1999; Englund et al. 2005). We first examined the mitotic index of apical and basal progenitors at E14.5 and found reduced frequency of basal mitoses and a small reduction of apical mitosis in CKO mice, an effect that was exaggerated in DKO mice (Supp. Figure 2). In addition, the number of cells in S- phase, marked by BrdU, was also reduced by 40% in the mutant animals (Fig. 5C,D).

Furthermore, in the CKO mice the number of Pax6+ radial glia was reduced by 33%

(Fig.4A,C); (p=0.0002), whereas the number of mitotic radial glia was reduced by

44% (p=0.0014);(Fig.4A,D). Moreover, the number of progenitors co-expressing

Pax6+ and Tbr2+ (a marker of basal progenitors) was reduced by 49% in the CKO mice (Fig.4B,E), suggesting mutant Pax6+ radial glia do not appropriately differentiate into basal progenitors. This effect was more pronounced in the embryonic lateral cortex but still present in dorso-medial section of the developing

86 cortex. We also noticed that the intensity of Pax6 expression was often diminished in

mutant mice when compared to WT (Fig.4A).

Since the number of transitioning Pax6+/Tbr2+ progenitors was reduced, we

reasoned that the number of basal progenitors may be affected. We found that the

number of Tbr2+ basal progenitors was reduced by 30% in the CKO mice

(Fig.5A,B). Interestingly, the reduction in Tbr2+ cells was particularly evident in the cortical VZ, suggesting that the frequency of basal progenitor generation may be decreased when ERK2 is deleted. Therefore, we investigated the number of cycling basal progenitors by administering an acute BrdU pulse and counting the number of

Tbr2+/BrdU+ double labeled cells. We found that the number of cycling basal progenitors was reduced by more than 35% in the mutant mice (Fig.5E,F). In order to further confirm the defect in basal progenitor generation, we co-labeled BrdU+ cells with the basal progenitor marker Tbr2 16 hours after BrdU injection at E13.5. Since this period allows for S-phase apical progenitors to acquire Tbr2 expression, the double labeled cells represent newly generated basal progenitors. We found that in the ERK2 CKO mice the number of newly generated basal progenitors was decreased by 44% (Fig.5G,H). Thus, the ERKs are important regulators of apical and basal

progenitor dynamics in the developing cortex during neurogenesis.

Loss of ERK2 results in premature cell cycle exit, excessive neurogenesis and elongated cell cycle length.

One of the most likely explanations for the altered progenitor generation dynamics in the CKO mice is premature progenitor cell cycle withdrawal. This leads to an increase in the fraction of differentiated cells, coupled with a premature

87 decrease in progenitor pool size. We examined the proportion of cells leaving the cell

cycle by utilizing a standard BrdU labeling protocol as previously described

(Ormerod 1997; Quinn et al. 2007). In the developing cortex, a single BrdU injection

generates two distinctly labeled cell populations. Lightly labeled BrdU+ cells that re-

entered the cell cycle and subsequently divided, generating two daughter cells.

Heavily labeled cells that are in S-phase during BrdU administration and

subsequently undergo terminal division (del Rio and Soriano 1989; Gillies and Price

1993; Price et al. 1997; Roy et al. 2004; Cubelos et al. 2008). The lightly labeled

population accumulates with increasing length of BrdU exposure until a maximum is

reached, as some cells reenter S-phase and subsequently incorporate more BrdU

(Fig.6A,B). We predicted that if more cells exit the cell cycle, then the proportion of

heavily labeled cells would be greater in mutant mice 24 hours after BrdU

administration. We confirmed that there were significantly more darkly labeled cells

throughout the mutant cortex (at least 85% of cell marked by BrdU) (Fig.6A,B) when

compared to WT controls. We also co-labeled BrdU+ cells with the mitotic marker

Ki67. The number of BrdU+/Ki67- cells was used to establish the leaving cell

fraction. In the mutant embryos the fraction of all BrdU+ progenitors which exited the

cell cycle (BrdU+/Ki67-) was significantly (30%) greater when compared to wild

type controls (Fig.6C,D). Thus, loss of ERK2 resulted in premature cell cycle withdrawal in the developing cortex.

The premature cell cycle exit of neuronal precursors raised the question regarding the fate of the exiting cells. The newly postmitotic cells could either differentiate into post-mitotic neurons or undergo cell death. We did not observe

88 changes in caspase-3 activity during corticogenesis and only a few apoptotic cells were observed postnatally (Supp.Fig.3) but could not account for the much more substantial leaving fraction observed in CKO mice. Since many more cells were exiting the cell cycle, we predicted a corresponding increase in the proportion of differentiating neurons. To test this, we utilized the expression of the early neuronal marker, Tbr1, (Englund et al. 2005) which indeed was greater in the mutant mice in the period between E12.5-E16.5 when compared to controls (Fig.6G). To test our hypothesis that progenitor cells prematurely exit the cell cycle and become neurons due to defects in progenitor proliferation, we examined the birth frequency of Tbr1+ postmitotic neurons. We pulsed cycling progenitors with a single BrdU injection at

E13.5 and examined their fate by co-labeling with the post-mitotic marker Tbr1 24 hours later. During mid-neurogenesis this period of time is sufficient for a fraction of the progenitors to undergo terminal division and differentiate (Englund et al. 2005;

Takahashi et al. 1996). We found that the number of Tbr1+/BrdU+ neurons was increased by 35% in the mutant mice (Fig.6E,F). In addition, we performed cell cycle analysis, as previously described by Martynoga et al., and found the length of total cell cycle (Tc) significantly elongated in the CKO mice at E14.5 (Fig.6H-K)

(Martynoga et al. 2005). Our findings suggest that altered cell cycle kinetics of the proliferating progenitors result in premature cell cycle exit, generating an abnormally elevated number of postmitotic neurons at the expense of basal progenitors. This ultimately results in premature progenitor pool depletion reflecting postnatal deficits in cortical cytoarchitecture.

89 ERK2 regulates cell cycle progression through cyclin D1 and the cell cycle

inhibitor, p27Kip1.

Cell cycle regulators and transcription factors have been implicated in the

control of cell cycle dynamics in the developing cortex (Calegari and Huttner 2003;

Roy et al. 2004). Cyclin D1 is expressed in the developing cortex and acts to regulate

the G1/S cell cycle transition and thus the length of G1 (Lukas et al. 1996; Glickstein

et al. 2007). Cyclin D1 is transcriptionally regulated by the ERKs. Cyclin D1 protein

levels were significantly diminished at mid-neurogenesis in the cortical lysates

evaluated by Western analysis (Fig.7B) or immunohistochemistry (Fig.7A) in mutant

mice. The ERKs also regulate the levels of p27Kip1, a cyclin dependent kinase inhibitor and a potent G1 to S checkpoint regulator. We found that p27Kip1 protein levels are elevated in the developing cortex of CKO mice (Fig.7B). Specifically, we observed that p27Kip1 aberrantly accumulates in Tbr2+ basal progenitors as well as

postmitotic Tbr1+ neurons (Fig.7C-F) when compared to wild type controls. We

report a 36% increase in the number of basal progenitors that show accumulation of p27Kip1 when compared to controls (Fig. 7C,C’,F). Furthermore, the number of

Tbr1+/p27Kip1+ postmitotic neurons was significantly increased in the CKO mice

(Fig.7D,E). Therefore, loss of ERK activity contributes to altered levels of these

potent cell cycle regulators and provides a mechanistic explanation for perturbation of

the cell cycle dynamics in cortical progenitors during the critical neurogenic period.

Cell-intrinsic electrophysiological properties are altered in ERK2 mutant mice.

90

Given the cortical abnormalities observed in the CKO mice, we hypothesized

that single cell and network excitability of cortical layer II/III pyramidal cells (PCs)

may be altered in ERK2 deficient mice. We performed whole-cell patch-clamp

experiments in PCs from acute brain slices to examine cell-intrinsic measures of

excitability. Examination of frequency-input (F-I) curves revealed that the sensitivity

of neuronal firing to changes in input (gain) was unchanged between WT and CKO

animals (Fig.8A,B). Despite having similar input sensitivity, the maximal action

potential (AP) firing rate was significantly lower in the CKO mice (Fig.8C-E).

Moreover, the range of input amplitudes able to change the neuronal firing rate (input

dynamic range) was significantly reduced in CKOs (Fig.8F). The diminished intrinsic

neuronal excitability is further demonstrated by experiments in which ramp current

injection into layer II/III PCs elicited firing with longer response latencies in the CKO

mice (Fig.8G-I). AP latency in response to current injection is in part regulated by the

transient outward potassium current (A-current) [Hille, 2001]. We found that despite

some variability across cells, larger A-current values can be attributed to PCs from

ERK2 CKO mice (Fig.8J). This is consistent with previous work showing that ERK2 negatively regulates the A-current mediated by Kv4.2 channels [Schrader, 2005]. It is likely that neurons lacking ERK2 may be compensating for the increase in response latency by broadening their firing window (defined as the time elapsed between onset and termination of firing) and maximizing the number of generated APs (Fig.8K,L).

In addition, deletion of ERK2 led to a significant reduction in membrane resistance R and an increase in the membrane time constant τ while leaving input resistance,

91 membrane capacitance, AP threshold and resting potential unchanged (Table 1).

These data demonstrate that loss of ERK2 significantly alters the intrinsic excitability

of cortical PCs.

Network-level electrophysiological properties are altered in ERK2 mutant mice.

We hypothesized that the significant loss of PCs in layer II/III, altered cell

morphology, and changes in intrinsic neuronal excitability may be associated with a

change in network activity. Therefore, we compared network activity between WT

and CKO mouse cortex in vitro. Spontaneous network events were elicited by

disinhibiting slices with 5 µM gabazine, a GABAA receptor antagonist. Network

bursts (high-conductance events, HCE) were less frequent in the CKO mice (Fig.9A-

C), demonstrating the loss of ERK2 dramatically affects overall network activity. We

reasoned that diminished network activity could be explained in part by a reduction in

recurrent excitability within the local circuit, which we tested by analyzing individual

HCEs. The HCE has a characteristic shape consisting of an initial plateau depolarization lasting about 400 ms with an average magnitude of 60 mV and a succeeding decay (tail) lasting about 500 ms . Although the area under the HCE

plateau shows no differences, the area under the tail is significantly reduced in CKO

mice (Fig.9D,E). We predicted that a decrease in recurrent network excitation in ERK

CKO mice would result in a reduction of HCE tail area. We quantified recurrent

excitation by comparing the number of excitatory postsynaptic potentials (EPSPs)

before and after HCE onset, motivated by the assumption that a HCE drives recurrent

92 activity among neighboring layer II/III PCs. Indeed, we found that the EPSP count

during the HCE tail was significantly higher than during a 2-second period preceding

the HCE onset in both WT and CKO mice (Fig.9F, G). Furthermore, the EPSP count

in CKO mice was significantly lower during the pre- and post-HCE periods as

compared to WT mice. We ruled out the contribution of the membrane τ to tail area

as it is two orders of magnitude lower than the HCE tail duration. The reduction in

recurrent excitation may be attributed to a decreased number of functional

connections resulting from the loss of layer II/III PCs or altered neuronal

morphology. We therefore measured spontaneous miniature postsynaptic currents in

the presence of tetrodotoxin and observed a decrease in the frequency, but not

amplitude, of miniature excitatory postsynaptic currents (mEPSCs) (Fig.9H,I), suggesting a decrease in the total number of functional excitatory synapses.

Interestingly, the inhibitory mIPSCs revealed a decrease in both frequency and

amplitude (Fig.9J,K), suggesting that the cortical network may be compensating for

lack of excitation by reducing the quantal content of inhibitory synaptic vesicles and

the total number of inhibitory synapses. These results demonstrate for the first time

that eliminating ERK2 within the telencephalon effectively alters pyramidal cell

excitability and network activity.

Deletion of ERK2 leads to behavioral deficits and cognitive impairment in adult

mice.

Individuals with NCFC syndromes and copy number variations of ERK1/2

exhibit cognitive impairment, a broad spectrum of behavioral abnormalities and

93 psychiatric disease. Therefore, we examined 3 month old CKO male mice (DKO animals do not survive to this age) by implementing a battery of standardized behavioral tests. An important outcome of this analysis was the discovery that ERK2

CKO mice exhibited robust anxiety-like phenotypes with altered behavior on the elevated plus maze, light-dark exploration and open field test (Fig.10A-C), which is different from other ERK knock outs. In this latter test, general locomotor activity, measured by total distance traveled and velocity, was not affected (Fig. 10C).

We next evaluated CKO mice for deficits in working and hippocampus- dependent memory using continuous alteration and delayed T-maze tests, respectively. The delayed T-maze assay was used in order to circumvent the high levels of anxiety and freezing behavior seen in ERK2 CKO mice. The CKO mice spent less time in the novel arm and did not consistently pick the novel arm when compared to wild type mice; however the total number of entries was not significantly altered, although there was a trend to explore less (Fig. 10E). We also tested the ERK2 CKO mice using contextual fear conditioning, however, due to the strong anxiety-like behavior the animals continuously froze above normal threshold levels (data not shown). ERK2 CKO mice were indistinguishable from WT age matched controls in measures of motor coordination and sensitivity to pain

(Fig.10D,F). In conclusion, deletion of ERK2 during early neurogenesis results in anxiety-like behavior with deficits in working and hippocampal memory components.

94 Discussion:

The phenotypes of children with copy number variations of ERKs or NCFC syndromes clearly illustrate that dysregulation of ERK signaling dramatically affects

CNS development and function. ERKs are uniquely positioned to influence the magnitude and timing of corticogenesis. The present study describes a mechanism by which ERKs, in a dose dependent matter, act to regulate neural progenitor proliferation and consequently progenitor pool size. Furthermore, it addresses the potential consequences of the embryonic deficit on postnatal brain cytoarchitecture, global cortical circuitry and behavior. During a limited embryonic period two mitotically active layers, the VZ and the SVZ, generate the complex cytoarchitecture and the ultimate size of the adult cortex across species (Götz and Huttner 2005;

Kriegstein et al. 2006). The progenitors undergo either proliferative or neurogenic

(differentiative) divisions. Proliferative divisions maintain the size of the progenitor pools, determining the size of the cortex. Conversely, neurogenic divisions generate neurons determining cortical thickness (Pontious et al. 2008). Cell cycle dynamics are regulated by both the total length of the cell cycle and G1 phase (which increases as neurogenesis proceeds) (Caviness and Takahashi 1995) as well as the ratio of cells reentering or exiting the cell cycle. The molecular cues controlling proliferative versus neurogenic progenitor divisions are not well understood. We postulate that

ERK1/2, influenced by global morphogens, maintain a crucial balance between proliferative and neurogenic divisions in the developing cortex through their downstream effectors p27Kip1 and cyclin D1. In support of this hypothesis we show

95 that: 1) ERK1/2 activity mirrors the neurogenic gradient within the proliferative

zones of the developing cortex and that the progressive loss of ERK1/2 results in a

significantly smaller cortex, in a gene dose-dependent manner; 2) Significantly, both

CKO and DKO mice show lamina specific deficits with altered number of mature

cortical neurons; 3) Loss of ERK1/2 activity alters the progenitor pool size and cell

cycle dynamics, resulting in prolonged cell cycle length, premature cell cycle exit and

progenitor pool depletion; 4) The cell cycle defects correlate with dysregulation of

ERK-regulated potent cell cycle modulators: cyclin D1 and p27Kip1; 5) These deficits

may then alter the organization, composition and functional output of the cortical

circuitry changing network dynamics and potentially be involved in behavioral

deficits which phenocopy developmental disorders observed in individuals with copy number variations of the ERKs and NCFC syndromes. It is important to note that loss of ERKs at early stages of neurogenesis has a different spectrum of effects than observed when the genes are inactivated later during this process (Samuels et al.

2008), owing to the newly recognized capacity of the ERKs to regulate progenitor pool size and ultimately the number of neurons populating the cortical lamina.

There is now good evidence supporting the hypothesis that the length of the cell cycle is a critical determinant of cell fate where increasing G1 length allows enough time for neurogenic vs. proliferative division (Calegari and Huttner 2003;

Dehay and Kennedy 2007). Analyses on a population level confirm that neurogenic progenitors exhibit longer G1 then coexisting proliferative ones (Lange et al. 2009).

A reduction in cyclin D1 levels results in lengthened G1 and this alone is necessary and sufficient for a switch to neurogenic fate (Lange et al. 2009). The ERKs

96 transcriptionally regulate the major G1-phase active cyclin D1, which is expressed

specifically in the neural progenitors in the VZ (Glickstein et al. 2007) and its

expression mirrors the neurogenic gradient, just like the activated ERK1/2. We show

that in the absence of ERK1/2, levels of cyclin D1 are decreased, causing precocious

elongation of G1-phase, favoring neurogenic divisions in the developing

telencephalon. Conversely, overexpression of p27Kip1 in neural progenitors in mid-

neurogenesis increases the proportion of exiting cells independently from the cell

cycle (Tarui et al. 2005). Aberrant accumulation of p27Kip1 in Tbr2+ neural

progenitors is likely to contribute to the increased cell cycle exit we observed in the

CKO mice. We argue that in the absence of ERKs, the reduction in cyclin D1 levels, coupled with enhanced accumulation of p27Kip1, act in parallel to force neural

progenitors into premature cell cycle exit where the prolonged cell cycle promotes an

aberrant neurogenic cell fate switch. Thus, ERK1/2 activity confined to the

neurogenic hub, acting through cyclins and their inhibitors modulate the cell cycle

length and progenitor proliferation dynamics, precisely controlling the rate of

neurogenesis in the developing cortex. This balance between neurogenic and

proliferative divisions is crucial for normal brain development and function. Loss of

ERK1/2 causes premature genesis of too many neurons at the expense of progenitors resulting in premature progenitor pools depletion. In mice, changes in progenitor number are associated with abnormal cortical thickness (Sahara and O'Leary 2009;

Quinn et al. 2007). Basal progenitors amplify neuronal output, divide away from the ventricle and produce mostly neurons (Haubensak et al. 2004; Miyama et al. 1997;

Noctor et al. 2007; Kowalczyk et al. 2009). Some evidence suggests that basal

97 progenitors generate neurons destined for upper cortical layers (II-IV) (Tarabykin et

al. 2001), however, they are present throughout all stages of cortical neurogenesis and

contribute to all cortical layers (Kowalczyk et al. 2009; Haubensak et al. 2004;

Miyama et al. 1997; Noctor et al. 2004; Shen et al. 2006; Smart 1973). Our data shows a deficit in progenitor number, as well as frequency of their generation, which we postulate is due to defects in apical and basal progenitor proliferation dynamics.

As a consequence of these developmental perturbations, the CKO and DKO mice

show altered number and frequency of projection neurons in cortical layers II to V.

We attribute the laminar deficit in supragranular neurons in layers II-IV to premature depletion of IPC pool and this view is corroborated by our BrdU birth dating study and similar to results reported by others (Arnold et al. 2008; Kriegstein et al. 2006).

Consistent with this hypothesis, we also report an increase in layer V neurons, presumably a result of premature neurogenesis. It is also worth noting that we did not observe any migration deficits within the mutant cortex (data not shown), a finding which differs from that of Imamura et al., (Imamura et al. 2010). We also conducted a series of neuronal reconstructions in layer II/III somatosensory cortex and found significant changes in axon length and dendritic arborization. A critical outcome of the present study demonstrates the cellular mechanism underlying ERK actions in corticogenesis which centers on the importance of ERK regulation of cyclin D1 and p27Kip1 in governing the choice of neurogenic vs. self renewing divisions in the

developing cortex.

Importantly, this study reveals that intrinsic electrophysiological properties of the projection neurons in layer II-III were altered in the absence of ERK2 activity,

98 exhibiting decreased maximal firing rate and input dynamic range, as well as a longer

response latency to depolarizing stimuli. Together, these results suggest that in the

absence of ERK2 principal layer II/III neurons will have a more restricted input and

output range and will be less responsive to transient inputs. These defects are likely to

impede their proper functioning within the cortical circuit. At the network level,

alterations in cortical cytoarchitecture and neuronal morphology correspond to less

recurrent excitation in layer II/III of the CKO cortex. The combination of altered cell-

intrinsic and network properties is likely to account for profound deficits in global

network activity where the excitability of the network is significantly diminished. It is

possible that these changes may contribute to the behavioral deficits seen in these

mice, specifically the anxiety phenotype which has not been previously observed. In

fact, in patients microdeletion of 22q11.21-22 that encompasses the ERK2 gene,

MAPK1 was found to be associated with developmental abnormalities and profound

anxiety (Verhoeven et al. 2011). Significantly, copy number variations in ERK1 gene,

MAPK3 have been genetically linked with autism and mental retardation (Sanders et al. 2011). It is also interesting to note that deletion to ERK2 at different times during cortical neurogenesis results in significantly different phenotypes. The present study

provides novel insights into why the human brain is so sensitive to even minor

dysregulation of the ERK MAPK pathway which results in pathology associated with

NCFC Syndromes and related disorders.

Author contribution statement:

99 J.P. and J.C.K. generated the CKO and DKO animals and performed western blot analyses. J.P. performed all embryonic and postnatal histology as well as the cell proliferation analyses and imaging. J.P. and G.C. performed the behavioral analyses.

R.F.G and P.A.P. generated the electrophysiology data. G.E. L., J.P., R.F.G and

P.A.P. designed the experiments and wrote the manuscript.

Acknowledgments:

We thank Dr. Lynn Landmesser, Dr. Evan Deneris, Dr. Stephen Maricich and Joseph

Vithayathil for helpful suggestions and Dr. Gemma Casadesus and the Rodent

Behavior Core at Case Western Reserve University. This work was supported by The

Mount Sinai Health Care Foundation and The Alfred P. Sloan Foundation (RFG).

100 References: Arlotta P, Molyneaux BJ, Chen J, Inoue J, Kominami R, and Macklis JD. 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45: 207–221.

Arnold SJ, Huang G-J, Cheung AFP, Era T, Nishikawa S-I, Bikoff EK, Molnár Z, Robertson EJ, and Groszer M. 2008. The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone. Genes & Development 22: 2479–2484.

Bulfone A, Martinez S, Marigo V, Campanella M, Basile A, Quaderi N, Gattuso C, Rubenstein JL, and Ballabio A. 1999. Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development. Mech. Dev. 84: 133–138.

Calegari F, and Huttner WB. 2003. An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis. Journal of Cell Science 116: 4947–4955.

Campbell DB, Li C, Sutcliffe JS, Persico AM, and Levitt P. 2008. Genetic evidence implicating multiple genes in the MET receptor tyrosine kinase pathway in autism spectrum disorder. Autism Res 1: 159–168.

Caviness VS, and Takahashi T. 1995. Proliferative events in the cerebral ventricular zone. Brain Dev. 17: 159–163.

Chen B, Schaevitz LR, and McConnell SK. 2005. Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc. Natl. Acad. Sci. U.S.A. 102: 17184–17189.

Cubelos B, Sebastián-Serrano A, Kim S, Moreno-Ortiz C, Redondo JM, Walsh CA, and Nieto M. 2008. Cux-2 controls the proliferation of neuronal intermediate precursors of the cortical subventricular zone. Cerebral Cortex 18: 1758–1770.

Dehay C, and Kennedy H. 2007. Cell-cycle control and cortical development. Nat Rev Neurosci 8: 438–450. del Rio JA, and Soriano E. 1989. Immunocytochemical detection of 5'- bromodeoxyuridine incorporation in the central nervous system of the mouse. Brain Res. Dev. Brain Res. 49: 311–317.

Eder C, and Heinemann U. 1994. Current density analysis of outward currents in acutely isolated rat entorhinal cortex cells. Neuroscience Letters 174: 58–60.

Englund C, Fink A, Lau C, Pham D, Daza RAM, Bulfone A, Kowalczyk T, and Hevner RF. 2005. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. Journal of Neuroscience 25: 247–251.

101 Feldmeyer D, Lübke J, and Sakmann B. 2006. Efficacy and connectivity of intracolumnar pairs of layer 2/3 pyramidal cells in the barrel cortex of juvenile rats. The Journal of Physiology 575: 583–602.

Fernandez BA, Roberts W, Chung B, Weksberg R, Meyn S, Szatmari P, Joseph- George AM, Mackay S, Whitten K, Noble B, et al. 2010. Phenotypic spectrum associated with de novo and inherited deletions and duplications at 16p11.2 in individuals ascertained for diagnosis of autism spectrum disorder. J. Med. Genet. 47: 195–203.

Gillies K, and Price DJ. 1993. The fates of cells in the developing cerebral cortex of normal and methylazoxymethanol acetate-lesioned mice. Eur. J. Neurosci. 5: 73– 84.

Glickstein SB, Alexander S, and Ross ME. 2007. Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets. Cereb. Cortex 17: 632–642.

Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JLR, and Jones KR. 2002. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. Journal of Neuroscience 22: 6309–6314.

Götz M, and Huttner WB. 2005. The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6: 777–788.

Götz M, Stoykova A, and Gruss P. 1998. Pax6 controls radial glia differentiation in the cerebral cortex. Neuron 21: 1031–1044.

Haubensak W, Attardo A, Denk W, and Huttner WB. 2004. Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc. Natl. Acad. Sci. U.S.A. 101: 3196–3201.

Hille B. 2001. Ion channels of excitable membranes. Sinauer Associates Inc.

Hooks BM, Hires SA, Zhang Y-X, Huber D, Petreanu L, Svoboda K, and Shepherd GMG. 2011. Laminar analysis of excitatory local circuits in vibrissal motor and sensory cortical areas. Plos Biol 9: e1000572.

Iansek R, and Redman SJ. 1973. An analysis of the cable properties of spinal motoneurones using a brief intracellular current pulse. The Journal of Physiology 234: 613–636.

Imamura O, Pagès G, Pouysségur J, Endo S, and Takishima K. 2010. ERK1 and ERK2 are required for radial glial maintenance and cortical lamination. Genes Cells 15: 1072–1088.

Karasewski L, and Ferreira A. 2003. MAPK signal transduction pathway mediates agrin effects on neurite elongation in cultured hippocampal neurons. J. Neurobiol.

102 55: 14–24.

Kowalczyk T, Pontious A, Englund C, Daza RAM, Bedogni F, Hodge R, Attardo A, Bell C, Huttner WB, and Hevner RF. 2009. Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex. Cerebral Cortex 19: 2439–2450.

Kriegstein A, Noctor S, and Martínez-Cerdeño V. 2006. Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion. Nat Rev Neurosci 7: 883–890.

Lange C, Huttner WB, and Calegari F. 2009. Cdk4/CyclinD1 Overexpression in Neural Stem Cells Shortens G1, Delays Neurogenesis, and Promotes the Generation and Expansion of Basal Progenitors. Cell Stem Cell 5: 320–331.

Lukas J, Bartkova J, and Bartek J. 1996. Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint. Mol. Cell. Biol. 16: 6917–6925.

Martynoga B, Morrison H, Price DJ, and Mason JO. 2005. Foxg1 is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis. Developmental Biology 283: 113–127.

Miyama S, Takahashi T, Nowakowski RS, and Caviness VS. 1997. A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium. Cereb. Cortex 7: 678–689.

Newbern J, Zhong J, Wickramasinghe RS, Li X, Wu Y, Samuels I, Cherosky N, Karlo JC, O'Loughlin B, Wikenheiser J, et al. 2008. Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development. Proceedings of the National Academy of Sciences 105: 17115– 17120.

Noctor SC, Martínez-Cerdeño V, and Kriegstein AR. 2007. Contribution of intermediate progenitor cells to cortical histogenesis. Arch. Neurol. 64: 639–642.

Noctor SC, Martínez-Cerdeño V, Ivic L, and Kriegstein AR. 2004. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci 7: 136–144.

Ormerod MG. 1997. Analysis of cell proliferation using the bromodeoxyuridine/Hoechst-ethidium bromide method. Methods Mol. Biol. 75: 357–365.

Perron JC, and Bixby JL. 1999. Distinct neurite outgrowth signaling pathways converge on ERK activation. Mol. Cell. Neurosci. 13: 362–378.

103 Pontious A, Kowalczyk T, Englund C, and Hevner RF. 2008. Role of intermediate progenitor cells in cerebral cortex development. Dev Neurosci 30: 24–32.

Price DJ, Aslam S, Tasker L, and Gillies K. 1997. Fates of the earliest generated cells in the developing murine neocortex. J. Comp. Neurol. 377: 414–422.

Quinn JC, Molinek M, Martynoga BS, Zaki PA, Faedo A, Bulfone A, Hevner RF, West JD, and Price DJ. 2007. Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism. Developmental Biology 302: 50–65.

Rakic P, and Caviness VS. 1995. Cortical development: view from neurological mutants two decades later. Neuron 14: 1101–1104.

Roy K, Kuznicki K, Wu Q, Sun Z, Bock D, Schütz G, Vranich N, and Monaghan AP. 2004. The Tlx gene regulates the timing of neurogenesis in the cortex. Journal of Neuroscience 24: 8333–8345.

Sahara S, and O'Leary DDM. 2009. Fgf10 regulates transition period of cortical stem cell differentiation to radial glia controlling generation of neurons and basal progenitors. Neuron 63: 48–62.

Saitta SC, Harris SE, McDonald-McGinn DM, Emanuel BS, Tonnesen MK, Zackai EH, Seitz SC, and Driscoll DA. 2004. Independent de novo 22q11.2 deletions in first cousins with DiGeorge/velocardiofacial syndrome. Am. J. Med. Genet. A 124A: 313–317.

Samuels IS, Karlo JC, Faruzzi AN, Pickering K, Herrup K, Sweatt JD, Saitta SC, and Landreth GE. 2008. Deletion of ERK2 mitogen-activated protein kinase identifies its key roles in cortical neurogenesis and cognitive function. Journal of Neuroscience 28: 6983–6995.

Samuels IS, Saitta SC, and Landreth GE. 2009. MAP'ing CNS development and cognition: an ERKsome process. Neuron 61: 160–167.

Sanders SJ, Ercan-Sencicek AG, Hus V, Luo R, Murtha MT, Moreno-De-Luca D, Chu SH, Moreau MP, Gupta AR, Thomson SA, et al. 2011. Multiple Recurrent De Novo CNVs, Including Duplications of the 7q11.23 Williams Syndrome Region, Are Strongly Associated with Autism. Neuron 70: 863–885.

Schrader LA. 2005. ERK/MAPK regulates the Kv4.2 potassium channel by direct phosphorylation of the pore-forming subunit. AJP: Cell Physiology 290: C852– C861.

Shen Q, Wang Y, Dimos JT, Fasano CA, Phoenix TN, Lemischka IR, Ivanova NB, Stifani S, Morrisey EE, and Temple S. 2006. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat Neurosci 9: 743– 751.

104 Shepherd GMG, Stepanyants A, Bureau I, Chklovskii D, and Svoboda K. 2005. Geometric and functional organization of cortical circuits. Nat Neurosci 8: 782– 790.

Sherr CJ, and Roberts JM. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & Development 13: 1501–1512.

Smart IH. 1973. Proliferative characteristics of the ependymal layer during the early development of the mouse neocortex: a pilot study based on recording the number, location and plane of cleavage of mitotic figures. Journal of Anatomy 116: 67–91.

Takahashi T, Nowakowski RS, and Caviness VS. 1996. Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall. J. Neurosci. 16: 5762–5776.

Tarabykin V, Stoykova A, Usman N, and Gruss P. 2001. Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression. Development 128: 1983–1993.

Tarui T, Takahashi T, Nowakowski RS, Hayes NL, Bhide PG, and Caviness VS. 2005. Overexpression of p27 Kip 1, probability of cell cycle exit, and laminar destination of neocortical neurons. Cereb. Cortex 15: 1343–1355.

Tidyman WE, and Rauen KA. 2008. Noonan, Costello and cardio-facio-cutaneous syndromes: dysregulation of the Ras-MAPK pathway. Expert Rev Mol Med 10: e37.

Verhoeven W, Egger J, Brunner H, and de Leeuw N. 2011. A patient with a de novo distal 22q11.2 microdeletion and anxiety disorder. Am. J. Med. Genet. A 155A: 392–397.

Wu S-X, Goebbels S, Nakamura Kouichi, Nakamura Kazuhiro, Kometani K, Minato N, Kaneko T, Nave K-A, and Tamamaki N. 2005. Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone. Proc. Natl. Acad. Sci. U.S.A. 102: 17172–17177.

Xiao J, and Liu Y. 2003. Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system. Journal of Neurochemistry 86: 1516–1523.

105 Figure 2-1: ERK activity is abrogated in the dorsal telencephalon of ERK2 CKO and ERK1/2 DKOs.

106 Figure 2-1

ERK activity is abrogated in the dorsal telencephalon of ERK2 CKO and ERK1/2 double knock outs. ERK2 inactivation is driven by the Emx 1-cre within the dorsal telencephalon beginning at E9.5 (Gorski et al., 2002). pERK activity in the developing WT cortex shows low-medial to high-later gradient in the dorsal telencephalon at E14.5 (a). ERK activity is lost in the DKO mice at E14.5 as shown by DAB staining (a-a’), where a’ shows higher magnification of the telencephalon marked in a. Western blot analysis of cortical lysates at E14.5 shows absence of

ERK1 and/or ERK2 protein in CKO and DKO mice, respectively (b). ERK activity was analyzed by immunohistochemical analysis of pERK expression (green) in DKO mice at E14.5 (c-e). During mid-neurogenesis pERK activity was detected within the proliferative zone of the lateral cortex, but not in the postmitotic neurons (c-e). Strong expression of ERK activity was detected in sub-pallial ventricular zone mitotic progenitors in both WT and DKO cortices, but absent from pallial mitotic PH3+(red)

(d) and Nestin+(red) (c) progenitors (marked by asterisk). A clear boundary of cre expression is observed in the DKO mice and marked by an arrow (c). V, ventricle;

GE, ganglionic eminence. The cortical size and body weight is reduced in the ERK2

CKO and DKO mice (f-j). Dorsal view of WT and DKO mutant mice at P21 (f).

Body weight was measured in CKO (n=5); P≤0.001) and DKO (n=5; P≤0.0001) mice and compared to WT (n=11) (h). Schematic representation of cortical parameters measured: area size, A-P length and cortical length (g). Dorsal images of whole brains: WT (left), CKO (middle) and DKO (right) mice at P10 (i). Quantitative comparison of brain weight and cortical size between WT, CKO, and DKO mice at

107 P10 (j). Measurements of cortical parameters (g) were obtained using Image J software. At least 6 brains were analyzed per genotype.

108 Figure 2-2: Loss of ERK1/2 changes morphology and cytoarchitecture of the postnatal cortex.

109 Figure 2-2

Loss of ERK1 and ERK2 leads to layer perturbations in the adult cortex.

Coronal brain sections of WT, CKO and DKO mice at P2 (a). Immunohistochemical analysis using layer specific markers: Brn1, layer II-IV (green) used to quantify

Brn1+ cells per cortical area (P=0.0009); Ctip2+, layer V motor neurons (green),

(P=0.0021) (a-b). Tbr1 in layer V/VI expression (red) was unchanged (P=0.7091) (a- b). SatB2, layer II-V to quantify colossal projections (P=0.0042) (red) and Cux1

(green), marker for layer II-III neurons (P=0.0304) (c-d). Reconstructed layer II/III

PCs from WT and CKO somatosensory cortex (e). Axon length (µm) from reconstructed PCs (WT: n = 6; CKO: n = 7; P < 0.05, bootstrap analysis) (f). Sholl profile of basal dendrites plotted as mean number of dendritic intersections. Note the significant difference in number of dendritic intersections at 25µm and 35µm away from cell soma (WT: n = 6; CKO: n = 7; P < 0.05, bootstrap analysis) (g). At minimum 6 animals analyzed per group per genotype.

110 Figure 2-3:

The frequency of neurons generated during mid-neurogenesis is altered in mutant mice lacking ERK1 and/or ERK2.

111 Figure 2-3

The frequency of neurons generated during mid-neurogenesis is altered in

mutant mice lacking ERK1 and/or ERK2.

The number of NeuN+ neurons (green) across 10 equally spaced cortical bins in WT

and CKO mice (a) quantified at P2 (b). Bin 2-3 corresponds to supragranular neurons

in layer II-IV and bin 7 corresponds to layer V (b). The frequency of neuronal birth

was analyzed at P2 by examining the number of BrdU+/DAPI+ cells that were

labeled with BrdU (green) at E14.5 (a). For layer II-IV analysis mice were labeled

with BrdU (red) at E14.5 and evaluated for Brn1 co-expression (green) at P2 (P≤

0.0001) (c,d). Layer V neurons were BrdU (green) labeled at E12.5 and co-expression with Ctip2 evaluated at P2 (red); (P=0.0032) (c,e). Minimum of 6 mice evaluated per genotype.

112 Figure 2-4: ERK2 CKO mice show reduction in Pax6+ progenitors, mitotic radial glia and transition to intermediate progenitor cells.

113 Figure 2-4

ERK2 CKO mice show reduction in Pax6+ progenitors, mitotic radial glia and transition to intermediate progenitor cells.

We evaluated the number of Pax6+ radial glia (a,c); (P=0.0002). The number of mitotic radial glia was analyzed by co-labeling with a mitotic marker, PH3 (red)) at

E14.5 (P=0.0014); (a,d). In addition, we analyzed the number of transitioning progenitors by co-labeling with the intermediate progenitor marker, Tbr2 (red); (b,e).

At least 5 mice per genotype were evaluated.

114 Figure 2-5: Loss of ERK2 disrupts basal progenitor frequency and generation, resulting in premature progenitor pool depletion.

115 Figure 2-5

Loss of ERK2 disrupts basal progenitor frequency and generation, resulting in premature progenitor pool depletion.

The number of intermediate progenitors was analyzed by immunohistochemical analysis with Tbr2 (red) at E14.5; (P=0.0080) (a-b). A short BrdU (50ng/g) pulse was

IP injected 30 minutes prior to sacrificing the pregnant dam. The number of BrdU+ cells (green) was counted at E14.5 (c,d). To analyze the frequency of cycling SVZ progenitors we used a short-pulse BrdU labeling paradigm (green), to immunolabel

Tbr2+ (red) progenitors in the S phase, Tbr2+/BrdU+ (n = 5; P=0.0022) (e-f). Basal progenitor generation from apical progenitors was assayed by co-labeling of Tbr2+ cells with BrdU 16 hours post BrdU injection, allowing for some apical progenitors to migrate into the SVZ and express Tbr2 (red); (n=5; P=0.0001) (g-h).

116 Figure 2-6: ERK2 CKO neural progenitors exhibit premature cell cycle exit and lengthening of the cell cycle during mid-neurogenesis.

117 Figure 2-6

ERK2 CKO neural progenitors exhibit premature cell cycle exit and lengthening of the cell cycle during mid-neurogenesis.

A single BrdU injection was administered at E13.5 and embryos were fixed 24 h later. Coronal sections were immunolabeled with BrdU (green). Cells that reenter the cell cycle show distinct puncta and are less intensely labeled, whereas cells that exited the cell cycle and become postmitotic are brightly labeled (a). Brightly labeled BrdU+ cells (at least 80% of cell covered) were counted across the cortical anlage (b). The cortical entity was divided into 10 vertical bins of equal thickness. The histogram shows a statistical analysis of the number of brightly labeled cells in each bin in CKO and WT cortex (a-b); (n=3; P≤0.001). Immunostaining with Ki67 (green) and BrdU

(red) antibodies after 24 hours BrdU pulse at E13.5 (c-d). All cells that exited the cell cycle (BrdU+/Ki67-) were counted and their percentage of total BrdU+ cells evaluated 24 hours post injection was analyzed (d). To evaluate the fate of newly born cells we co-label with a postmitotic marker, Tbr1 (red) and BrdU (green) 24 hours after injection; (n=4(WT), n=5(CKO); P=0.0383) (e-f). The total number of

Tbr1+ post mitotic neurons was also evaluated at E12.5 (P=0.0034), E14.5

(P=0.0007) and E16.5 (P=0.0140) (g). Cell cycle during mid-neurogenesis: Diagram adopted from Martynoga et al.(Martynoga et al. 2005) showing a schematic of the double injection (IdU/BrdU) paradigm (h). Pregnant dams were injected with IdU and

BrdU 1.5h apart and sacrificed 0.5h later. Coronal sections were immunolabeled with

IdU (green) and BrdU (red) at E14.5 (i). Cells that are in the S-phase by t = 2h will be

118 labeled with both BrdU and IdU (S cells: yellow)(k); (P<0.05). Cells were counted in

100μm-wide bins and are shown as colored dots (j).

119 Figure 2-7:

Loss of ERK2 alters expression of key cell cycle regulators cyclin D1 and p27Kip1 in the developing cortex.

120 Figure 2-7

Loss of ERK2 alters expression of key cell cycle regulators cyclin D1 and p27Kip1 in the developing cortex.

We evaluated cyclin D1 levels by IHC (red) (a) and Western analysis (WT, 1,000 ±

0.08104, n=6, CKO; 0.6873 ± 0.05215, n = 5) (P< 0.05) (b). Expression of p27Kip1

(green) is aberrantly upregulated, by IHC, in intermediate progenitors marked by

Tbr2 (red) as well as postmitotic neurons, marked by Tbr1 expression (red) (c,c,d).

Quantified in (e,f). Higher magnification confocal images show an increase in

Tbr2+/p27+ cells in CKO mice (c’). The number of Tbr2+/p27+ cells was quantified;

(n=4(WT), n=7(CKO) (f). and confirmed by western analysis (P=0.0202) (b).

121 Figure 2-8:

Cell-intrinsic electrophysiological parameters are altered in ERK2 mutant mice.

122 Figure 2-8

Cell-intrinsic electrophysiological parameters are altered in Erk2 mutant mice.

(a) F-I curve slope distributions from LII/III PCs (WT: n = 21; CKO: n = 18; P >

0.05, bootstrap analysis). Crosses connected by black lines demarcate means of the

distributions. (b) Median firing frequency – input current (F-I) curves with shaded

regions representing the area between the 1% and 99% confidence levels. Examples

of layer II/III PCs firing at maximal rates from (c) WT (n = 21) and (d) Erk2 (n = 18)

CKO mice. (e) Maximal firing frequency distributions (**P < 0.05, bootstrap

analysis). (e) Distributions of input dynamic range, defined as the range of input

current values between firing onset and maximal firing frequency (*P < 0.05,

bootstrap analysis). Examples of voltage responses to ramp current stimulation in

layer II/III PCs from (g) WT (n = 24) and (h) Erk2 (n = 35) CKO mice. (i) Latency to

the first spike after stimulation with a ramp current (**P < 0.05, bootstrap analysis).

(j) Median transient outward K+ current with shaded regions representing the area between the 1% and 99% confidence levels (WT: n = 25; CKO: n = 31). (k) Number of spikes fired per ramp (*P < 0.05, bootstrap analysis). (l) Firing window, defined as time elapsed between first and last spike during ramp current stimulation (**P < 0.05, bootstrap analysis).

123

Figure 2-9:

Network-level electrophysiological parameters are altered in Erk2 mutant mice.

124 Figure 2-9

Network-level electrophysiological parameters are altered in Erk2 mutant mice.

Examples of spontaneous network activity (i.e. high conductance events, HCEs) shown as voltage traces in (a) WT and (b) CKO disinhibited cortical slices. (c)

Number of spontaneous HCEs recorded during a 10-minute interval (WT n = 33,

CKO n = 32; **P < 0.05, bootstrap analysis). (d) Event-triggered average (ETA) taken as the mean shape of the network burst. (e) Mean area under the HCE tail in relation to the resting potential (**P < 0.05. bootstrap analysis). (f) Example of a network HCE recorded in layer II/III PC from a cortical slice of a WT mouse. Note the difference in number of EPSPs 2 seconds before the HCE (pre-HCE) and 2 seconds after HCE onset (post-HCE). Insets show EPSPs detected using a custom detection algorithm. Red lines over the voltage trace represent detected EPSPs during the pre- and post-HCE periods. (g) Box plot of EPSPs detected during the pre- and post-HCE periods in WT and CKO animals. Red lines and crosses on the box plots represent the distribution means and outliers, respectively. (h) Cumulative distribution function of mEPSC amplitude with insets showing sample mEPSC traces

(WT: n = 25, CKO: n = 23; P > 0.05, two-sample Kolmogorov-Smirnov test). (i)

Cumulative distribution function of mIPSC amplitude with insets showing sample mIPSC traces (P < 0.05, two-sample Kolmogorov-Smirnov test). (j) Cumulative distribution function of mEPSC inter-event interval (**P < 0.05, two-sample

Kolmogorov-Smirnov test). (k) Cumulative distribution function of mIPSC inter- event interval (**P < 0.05, two-sample Kolmogorov-Smirnov test).

125 Figure 2-10:

Loss of ERK2 leads to behavioral alterations and cognitive impairment in adult mice.

126 Figure 2-10

Loss of ERK2 leads to behavioral alterations and cognitive impairment in adult

mice.

Elevated plus maze: percentage of time spent in different arms during testing and

number of head dips (*P < 0.05) (a). Dark ↔ light exploration (***P < 0.001) (b).

Open field (*P < 0.05) (c). Continuous alteration and delayed T-maze: percent of

alterations, time spent and number of entrees into the novel arm, (*P < 0.05) (e). No

significant differences observed with rotarod test (f) or pain sensitivity (d). We

evaluated 3 months old male CKO (n=6) and WT (n=7) mice.

Table 1:

PARAMETER Mean+/-SD Mean+/-SD P-value WT ERK CKO

Input Resistance (MΩ) 126 ± 73 125 ± 102 0.61

Membrane Resistance (MΩ) 222 ± 96 192 ± 86 *0.031

Membrane Tau (ms) 9.5 ± 4.5 11.3 ± 3.4 *0.005

Membrane Capacitance (pF) 43 ± 13 42 ± 13 0.38

Spike Threshold (mV) -32 ± 9.1 -30.0 ± 4.8 0.12

Resting Potential -67 ± 14 -70 ± 7.0 0.11 (mV) Area of Mean Up-State 1.3 ± 0.52 1.49 ± 0.49 0.13 (mV· s)

*Statistically significant

127 Supplementary Figure 2-1: The cortical size and body weight is reduced in the ERK2 CKO and DKO mice.

128 Supplementary Figure 2-1

The cortical size and body weight is reduced in the ERK2 CKO and DKO mice.

Dorsal view of WT and DKO mutant mice at P21 (a). Body weight was measured in

CKO (n=5); P≤0.001) and in DKO 67% (n=5; P≤0.0001) mice when compared to

WT (n=11) (c). Schematic representation of cortical parameters measured (e,g,i): area size, A-P length and cortical length (b). Dorsal images of whole brains: WT (left),

CKO (middle) and DKO (right) mice at P2 (d), P10 (f) and P21 (h). Quantitative comparison of brain weight and cortical size between WT, CKO, and DKO mice at

P2, P10 and P21 (e,g,i). DKO mice show a 38% decrease in brain weight (P≤0.0001);

48% decrease in cortical area size (P=0.0009); 42% decrease in A-P length

(P≤0.0001); and 25% decrease in cortical length (P=0.0046) at P21 (i). Measurements of cortical parameters at each age (b) were obtained using Image J software. At least

6 brains were analyzed per genotype and per age group.

129 Supplementary Figure 2-2:

Apical and basal mitosis are reduced in the CKO and DKO mice during mid- neurogenesis.

130

Supplementary Figure 2-2:

Apical and basal mitosis are reduced in the CKO and DKO mice during mid- neurogenesis.

Phosphorylated histone H3 (PH3) immunolabeling of coronal sections from WT,

CKO and DKO dorsal telencephalon at E14.5 (a). The number of apical (in the VZ) and basal, (SVZ and IZ) PH3+ mitotic cells was counted throughout the entire length of the cortical slice and quantified by statistical analysis (*p≤0.05) (b).

131

Supplementary Figure 2-3:

Cell death in not significantly altered in the developing dorsal telencephalon of

CKO mice.

132 Supplementary Figure 2-3

Cell death in not significantly altered in the developing dorsal telencephalon of

CKO mice.

We analyzed the cortices of CKO and WT mice at E14.5 for the cleaved caspase-3 activity (red). Confocal images were taken and double-labeled with DAPI (blue) and

Caspase -3 (red). No significant changes were observed.

133 Chapter 3: Cortical Interneurons in ERK deficient mice.

The effects of ERK1 and ERK2 deletion on the number and migration pattern of

cortical interneurons in NCFC mouse model.

Introduction:

There are two different types of neurons in the rodent cerebral cortex:

glutamatergic projection neurons and GABAergic inhibitory, non-pyramidal

interneurons (DeFelipe and Farinas, 1992). Excitatory pyramidal cells comprise about

80% of the total neuronal population in the cortex whereas the inhibitory interneurons

contribute the remaining 20%. The idea that cortical projection neurons and

interneurons originate from two distinct germinal zones has revolutionized the field

of cortical development. Within the last 15 years it became clear that cortical

interneurons are born within the ventral telencephalon and must migrate along the

telencephalic ventricles to reach their final destination in the dorsal telencephalon

(Marin and Rubenstein, 2003). The newly born interneurons migrate tangentially and

then exit the migratory streams in order to find their appropriate partners, and

establish networks with a precise balance of excitation and inhibition. How the

processes of interneuron sorting, migration, regional and laminar distribution are

achieved is still debated and an area of extensive research.

The cortical inhibitory interneurons are crucial for normal brain function. The proper assembly of microcircuits between projection neurons and GABAergic interneurons is necessary for advanced cortical functions such as sensory perception,

134 motor function and cognition (Hensch, 2005). In the properly functioning brain the

firing activity of cortical projection neurons must be modulated by a precise level of

inhibition. In the murine model, interneurons enter the cortex at E12.5 via two

streams: the marginal zone and the second, much broader stream in the subventricular

zone (McManus et al., 2004; Metin et al., 2006). At a specific time the interneurons

exit the migratory streams and migrate radially into the cortical plate. By E15.5 they

are widely present across the cortex and the hippocampus but they do not achieve

their final laminar position until the first postnatal week. In the rodent, there are many diverse types of interneurons that can be further divided into many classes and

subtypes based on their immunochemical, morphological and physiological properties

(Somogyi et al., 1998; Kawaguchi and Kondo, 2002; Markram et al., 2004; Ascoli et

al., 2008). Based on their chemical properties they can be categorized into

parvalbumin (PV), somatostatin (SOM), calretinin (CR) and cholecystokinin (CCK)

types. Although most cortical interneurons are generated from the ventral

telencephalon, the distinct classes of interneurons originate from distinct spatial and

temporal origins. All cortical interneurons are generated from multiple progenitor

hubs of the lateral, medial and caudal eminences (LGM, MGE and CGE). Initial

patterning of the MGE requires the presence of Nkx2.1 transcription factor, which is

induced or permitted by the FGF and SHH signaling (Chiang et al., 1996; Fuccillo et

al., 2004; Gutin et al., 2006). Nkx2.1 in turn induces the expression of Lhx6 which is

also required for a proper MGE specification (Du et al., 2008; Liodis et al., 2007).

The precise specification, proper differentiation and migration of interneurons can be

regulated by intrinsic as well as extrinsic factors.

135 Transcription factors such as Dlx 1,2,5,6 and Lhx6 homeobox genes

contribute to the proper specification of interneurons and their migration into the

dorsal pallium. Deletion of Dlx1/2 genes in mice results in almost complete block of

migration of all cortical and hippocampal interneurons, where the newly born cells

never leave their germinal zones in the ventral telencephalon (Anderson et al., 1997a;

Pleasure et al., 2000). In addition, mice lacking either Dlx5 or Dlx5/6 exhibit defects

in PV+ interneurons (Wang et al., 2010). Dlx genes are important because along with

Lhx6, they promote the expression of CXCR4 and CXCR7, seven-transmembrane

chemokine receptors, which are important for proper interneuron migration (Stumm

and Hollt, 2007; Lopez-Bendito et al., 2008). These receptors are located on interneurons and bind CXCL12 ligand (also know as stromal cell-derived factor-1,

SDF1) (Balabanian et al., 2005). Mice with mutations in CXCR4 or its ligand,

CXCL12, show positional defects in cortical interneurons in both migratory streams.

In CXCR4 null mice interneurons prematurely exit the migratory stream and invade

the cortical plate resulting in abnormal interneuron distribution (Li et al., 2008;

Lopez-Bendito et al., 2008). Furthermore, CXCL12 chemokine is also necessary for

normal stream migration (Stumm et al., 2003). CXCL12 is expressed in the meninges

where it is crucial for proper organization of Cajal-Retzius cells (Borrell and Marin,

200), as well as the SVZ, where it is important for interneuron migration into the

cortex (Tiveron et al., 2006). In fact, reduced levels of CXCL12 result in excessive

branching, retarded rate of migration and premature exit from the migratory streams

(Lysko et al., 2011). Together, CXCL2 ligand and its receptor, CXCR4 act as an

136 attractant, controlling the initial sorting of interneurons into the migratory streams as

well as the timing of their exit (Li et al., 2007).

Interneurons that express PV and SST markers originate from the medial

ganglionic eminence, whereas CR+ interneurons originate mainly from the caudal

ganglionic eminence (Butt et al., 2005; Wichterle et al., 2001). Furthermore, within

the MGE, the SST+ interneurons come from the dorsal portion of the MGE and the

PV+ cells tend to come from the ventral side (Flames et al., 2007; Wonders et al.,

2008). Interestingly, cortical interneurons tend to assume the laminar positioning of the projection neurons born approximately at the same time (Miller, 1985). This

observation led to the idea that this birth-date matching may be due to projection

neurons and interneurons responding to similar environmental clues within the

developing cortex. On the other hand, MGE transplant experiments showed that the

cortical environment may not play an important role in the birth-date matching

phenomenon and that, in fact, it is the projection neurons themselves that directly

guide and instruct the interneurons to adopt a proper laminar destination (Pla et al.,

2006). Additionally, work from Fishell’s lab (Miyoshi and Fishell, 2010) argues that

is it the positioning and the birthplaces rather then the birth-time that more accurately

predicts the laminar fate of newly born interneurons. In their studies, they show that

MGE derived interneurons preferentially populate deeper cortical layers, whereas

caudally derived interneurons allocate mostly to the superficial cortical layering,

irrespective of their actual birthdates. It is feasible to speculate, that the layer specific

projection neurons can selectively distinguish between different subtypes of

interneurons and communicate proper laminar information to them. Taking advantage

137 of known mutants with altered numbers of layer specific cortical projection neurons, such as Fezf2 mutant, or the ERK1/2 mutants, it is possible to investigate whether the cortical projection neurons confer specific information to the appropriate interneuron sub-types, thus guiding them toward their final laminar destination. Since, the ERK2

CKO and DKO mice show deficits in progenitor proliferation as well as in the upper cortical projection neurons and an increase in the sub-cortical layer V neurons, we hypothesized that the cortical changes in lamina specific neuronal populations may lead to altered distribution of different subtypes of interneurons. To establish whether the projection neurons and their precursors are critical for interneurons to establish proper laminar distribution we crossed the ERK2 CKO mice with mice in which

SST+ interneurons and PV+ interneurons are genetically linked to GFP reporter.

Figure 3-1

Do laminar alterations in the dorsal telencephalon caused by ERK1/2 deletion affect the migration and final positioning of the sub-pallially derived interneurons?

138

Results:

The cellular and molecular cues that navigate interneurons through the complex environment of the developing brain to position within specific cortical layers are poorly understood. Appropriate cell-autonomous development of cortical interneurons is crucial for the correct positioning of GABAergic interneurons, since mutant mice with perturbed neuronal differentiation show defective laminar distribution (Alifragis et al., 2004; Azim et al., 2009; Batista-Brito et al., 2009; Butt et al., 2008; Cobos et al., 2006; Liodis et al., 2007; Wang et al., 2010). However, new research indicates that the projection neurons within the developing cortex may also play an instructive role in regulating distribution of cortical interneuron across the cortical milieu. Several lines of evidence seem to strengthen this hypothesis: 1).

139 GABAergic interneurons and projection neurons generated at the same time preferentially pair with each other, suggesting linked mechanisms of layer distribution (Fairen et al., 1986; Miller, 1985; Peduzzi, 1988; Pla et al., 2006;

Valcanis and Tan, 2003). 2). Newly generated interneurons invade the cortical plate only after their projection neuron partners, possibly suggesting a need for signals from appropriately located projection neurons (Lopez-Bendito et al., 2008). 3). In the reeler mouse with nearly inverted lamination, interneuron distribution in the cortex is abnormal (Hammond et al., 2006; Hevner et al., 2004; Pla et al., 2006; Yabut et al.,

2007).

Our research provides preliminary evidence that projection neurons are important for proper laminar distribution of interneurons and, that this may be neuron subtype specific.

ERK1/2 CKO and DKO mice show severe deficits in colossal projection neurons due to premature depletion of radial glia and intermediate progenitor pools in the developing telencephalon. As previously postulated defects in cortical neurogenesis and cytoarchitecture may affect tangential and radial migration of interneurons and their proper laminar distribution.

Since the migration of interneurons from the ventral pallium is partially controlled through cues that are extrinsically derived from the surrounding cells and the cortical environment we examined the ERK CKO and DKO mice for any developmental migration defects. Extrinsic factors, such as CXCL12, are derived from the neural intermediate progenitor cells, which are Tbr2+ and are generated in the SVZ of the dorsal pallium. We took advantage of our ERK1/2 knock out mice,

140 which show deficits in this population of progenitor cells, and predicted that the

interneuron migration may be altered due to perturbations in extrinsic signaling cues.

To evaluate this hypothesis, we examined the migratory pattern of different

populations of interneurons in the developing telencephalon be measuring the

distance they travelled from the pallial boundary into to dorsal cortex. We found that

in the ERK2 CKO/SST-GFP+ mice the newly generated ventral interneurons did not

migrate as far into the cortical milieu as the WT controls at E13.5 (Figure 3-2A, C).

However, the total number of interneurons was not significantly different between the

two groups (Figure 3-2B). In addition, we also evaluated the pattern of tangential

migration at E16.5, where we divided the developing dorsal cortex into four zones

(Figure 3-2B). We observed a decrease in the migratory distance in zone 2 and 4 in

the ERK2 CKO/SST-GFP+ mice when compared to the WT (Figure 3-3A, D). The

total number of SST+ interneurons was not changed (Figure 3-3C). Therefore, our

preliminary data suggests that tangential migration of at least SST+ interneurons is

altered in the dorsally deficit ERK2 mice. This change in migration pattern may be a

reflection of diminished number of cortical progenitors, which may not provide a sufficient level or gradient of chemo-attractants such as the CXCL12 chemokine.

Figure 3-2

The migration pattern but not the total number of SST+ interneurons is altered in the ERK2 deficient dorsal cortex at E13.5.

141

Figure 3-3

The migration pattern but not the total number of SST+ interneurons is altered in the ERK2 deficient dorsal cortex at E16.5.

142

We are currently in the process of expanding this analysis by examining tangential

migration patterns of PV+, and CR+ interneuron populations in the ERK2 deficient

dorsal telencephalon. We also plan to examine the potential mechanism by which

cortical progenitors and newly generated interneurons communicate and guide each other. We will utilize immunohistochemistry and western analysis to quantify and compare the expression of various receptors and their ligands such as ErbB4/NRG1 and CXCR4&7/CXCL12 in WT and ERK2 CKO dorsal cortex, which may be involved in the tangential and radial migration process, respectfully.

143 In addition, we also set out to explore how deficits in specific cortical layers of ERK

CKO and DKO mice affect distribution of interneurons and their specific subtypes in

the mature brain. There are two phases of interneuron migration: tangential migration

and radial migration. During tangential migration, interneurons migrate medially into

the dorsal pallium and are guided by various intrinsic and extrinsic signaling factors.

Once they reach their proper place along the medio-lateral plane, they migrate radially along the apical-basal axis. This migratory pattern is partly guided by specific cues released by the layer specific excitatory neurons. The work of Miyoshi and

Fishell points to the idea that specific subtypes of pyramidal cortical neurons may in fact attract and communicate with a specific type of interneuron. We propose that layer specific perturbations (deficits in layer II/III and increase in layer V of ERK2

CKO) confer disruptions in the lamination of their corresponding inhibitory interneurons. To study this, we used immunohistochemical analysis of different interneuron populations (SST+, PV+ and CR+) in the postnatal brain across cortical lamina of dorsal, medial and lateral sections of the somatosensory cortex. We observed that ERK2 CKO/SST-GFP+ mice show an increase in the total number of

SST+ interneurons in the somatosensory cortex when compared to WT (Figure 3-4

A-C). This increase was specific to dorsal but not lateral cortex of P10 CKO mice. In addition, this increase was especially pronounced in the area of zone 2 (Figure 3-4 C) which corresponds to cortical layer 3-5.

Figure 3-4

Loss of ERK2 from dorsal telencephalon results in an increase in SST+ interneurons in dorsal but not lateral postnatal cortex.

144

Furthermore, we also analyzed the ERK2 CKO mice for Calretinin+ interneurons. We show that the number of CR+ interneurons is reduced in ERK2 deficient mice when compared to WT controls (Figure 3-5).

Figure 3-5

Loss of ERK2 from dorsal telencephalon results in a decrease in Calretinin+ interneurons in the cortex of P10 mice.

145

Discussion:

Neurodevelopmental disorders such as schizophrenia, autism and intellectual disabilities originate from a broad set of causes but also exhibit overlapping phenotypes and genetics, which may suggest common deficits. Recently, the idea that disruption of inhibitory circuits may be partially responsible for some of the clinical symptoms associated with these disorders is gaining acceptance. Molecular genetics and animal models demonstrate that this disruption of inhibition is linked to defects in the development and function of interneurons. Even subtle changes in specific circuits result in aberrant information processing and cognitive impairment which lead to neuropsychiatric diseases.

146 Global balance between excitation and inhibition is crucial for development

and function of cortical networks (Maffei et al., 2006; Yizhar et al., 2011). Our

preliminary data suggests that the migration patter and laminar distribution of SST+

and CR+ interneurons is altered in the ERK2 deficient mice, where the total number

of interneurons is not significantly changed. There are two hypothesis which may

account for this alteration: 1) Migratory cues necessary for proper tangential

migration and exit from the migratory stream may be altered due to disrupted

progenitor proliferation in the ERK deficient mice; 2) Radial migration and proper

distribution of interneurons is disrupted due to changes in the cytoarchitecture of the

mature cortex. It is also possible that both progenitors and mature neuron contribute to the interneuronal changes observed in the ERK deficient mice. It is interesting to note, that the cortical reduction in superficial projection neurons of upper cortical layers correlates with a deficiency in CR+ interneurons. This suggests that caudally derived interneurons (CR+) allocating mostly to the superficial cortical layers, irrespective of their actual birthdates, may be indeed influenced in layer and sub-type specific fashion by upper cortical neurons. Additionally, the medially derived SST+ interneurons postulated to be destined for deeper cortical layers, may be attracted to the aberrant excess of deeper cortical projection neurons observed in the ERK2 CKO mice. Therefore, our preliminary data supports the idea that lamina-specific project neurons can discern the intrinsic properties of the subtype specific interneurons and

thus confer the proper laminar information to them. Therefore, we argue that

projection neurons choose their partner interneurons mostly by their molecular

footprint rather than their birthdate. Whether PV+ interneurons follow the same patter

147 as the SST+ interneurons remains to be investigated. The possible cues responsible for the specific pairing between interneurons and their projection neurons remain largely unknown.

148 DISCUSSION:

ERK1 and ERK2 are ubiquitously expressed protein kinases that can be activated by a diverse number of stimuli (Cobb et al., 1991). ERKs are central elements of an evolutionarily conserved signaling module involved in proliferation, differentiation and many other metabolic processes. Both isoforms are expressed in all species from yeast through mammals with the exception of ERK1, which is not found in frogs and chicks (Lefloch et al., 2008). ERK activity is required for proper brain development but its expression is also present in the mature brain (Boulton and

Cobb, 1991; Ortiz et al., 1995). In adult mice ERK signaling is also necessary for long term potentiation and learning and memory (Sweatt et al., 2001). In addition,

ERK MAP kinases are also important for memory consolidation in the hippocampus

(Eckel-Mahan et al., 2008). It is very clear that ERKs may have very distinct roles during brain development and in mature neurons or other cells. However, they have been studied the most in the context of cell cycle progression. ERKs were first identified from studies in mesenchymal fibroblasts, where upon mitogenic stimulation

Erk activity was observed throughout G1 phase and therefore required for G1/S-phase cell cycle progression (Meloche and Pouysségur, 2007). Further in vitro studies confirmed that ERK1/2 were able to influence G1/S transition by regulating and stabilization of various components of the cell cycle. These include up-regulation of

Cyclin D1, up-regulation of p21 leading to stabilization of Cyclin D-Cdk4 complexes and downregulation of anti-proliferative genes such as Sox6 and Jund1 (Lavoie et al.,

1996; Cheng et al., 1999; Yamamoto et al., 2006).

149 In addition, roles for ERK signaling in later stages of the cell cycle including

G2/M progression, have been investigated but yielded questionable results which

remain controversial mainly due to off-target effects of MEK inhibitors used in these

studies (Roberts et al., 2002; Liu et al., 2004; Shinohara et al., 2006; Kim et al.,

2008). Moreover, the majority of ERK1/2 cell cycle effects have been studied

predominantly in fibroblast cell lines, and given the highly cell type specific

responses associated with these kinases should not be assumed to function similarly

in other tissues. For example, recent work in epidermal cells in which ERK1/2 were

acutely knocked down showed epidermal hypoplasia and hypoproliferation where

ERK1/2-depleted keratinocytes exhibited G2/M cell cycle arrest but showed no deficit in G1/S phase progression. Importantly, the aberrant progression through

G2/M stage of the cell cycle was due to deregulation of core G2/M transition

controllers, including cyclin B1 but not cyclin D (Dumesic et al., 2009). These

findings contrast with the G1 arrest and cyclin D1 dysregulation observed in Erk1/2-

depleted fibroblasts. Together these findings indicate that control of cell cycle

progression by ERK MAPKs is cell type specific and may require other unknown

mechanisms. It is also possible that ERKs may also act as transcription factors,

modulating cell cycle dynamics by yet unknown mechanism. The ratio of ERK

isoforms varies in different cell types. For example, in the developing brain

expression of ERK2 is 14 times greater than the expression of ERK1, however in

epidermal progenitors the levels of ERK1 are twice the levels of ERK2 (Dumesic et

al., 2009). There is, however, some evidence supporting the idea that ERK2 may be a

more potent mitogen then ERK1, since hyperactivated ERK2 is just as prevalent in

150 epithelial tumors as phospho-ERK1, despite a significant difference in their

expression levels (Pelech et al., 2006). In conclusion, a substantial body of evidence

suggests that ERK1 and ERK2 may compete in respect to MEK activation with

ERK2 being activated more than ERK1 (Vantaggiato et al., 2006). The in vitro

experiments in different cell types point to the obvious roles of ERK kinases in the

control of the cell cycle. Our research and the data presented in this document aim to

investigate the mechanistic role ERKs may play in control of the cell cycle during one of the most complex developmental processes: the development of the brain and specifically, corticogenesis.

Figure 4-1

Cell cycle progression during cortical development.

151

FGF Signaling, ERKs and Cortical Development

Changes in brain size due to proliferation defects as well as aberrant shifts in arealization of the cortical milieu have the capacity to alter animal and human behavior and physiology (Cholfin and Rubenstein, 2008; O’Leary and Sahara, 2008).

There are currently very few animal models which allow for association of aberrant

behavior mimicking human developmental disorders with causative cellular and

molecular mechanism. Mouse models with altered FGF signaling or ERK activity are

just beginning to be explored in the context of known developmental and cognitive

disorders such as NCFC and Autism Spectrum Disorders. These models serve as

152 critical steps in elucidating a direct link between a mechanism and abnormal behavior

which can then be applied to human brain pathologies.

The technological advances in genetics and molecular mapping diagnostic techniques revealed that children with developmental syndromes including autism and 22q11.2 harbor copy number variations of ERK genes which result in altered

ERK activity during CNS development. Additionally, children with mutations in upstream or downstream elements of the MAPK/ERK cascade present with some common phenotypic characteristics observed in children hemizygous for ERK1 or

ERK2. We believe that the aberrant output of the MAPK/ERK pathway may play a central role in pathophysiology associated with these disorders.

ERK kinases are uniquely positioned to influence the magnitude and timing of corticogenesis but their mechanism of action during this stage of brain development is not well understood. We have shown that during neurogenesis ERK activity is confined to the ventricular zone, with high activity present in radial glia as well as in

IPCs, but not in postmitotic neurons. Cortical expansion is one of the most complex biological processes in the mammalian evolution. The developing cortex is exquisitely sensitive to even minor perturbations in the crucial balance between proliferation and differentiation. Despite the fact that the cellular composition of the developing cortex has been elucidated, the molecular cues controlling proliferative versus neurogenic progenitor divisions are not well understood. During a limited embryonic period two mitotically active layers, the VZ and the SVZ, generate the complex cytoarchitecture and determine the ultimate size of the adult cortex across all

153 species (Gotz and Huttner, 2005; Kriegstein et al., 2006). This process is potentiated in mammals due to a significant increase in the overall brain size. The progenitors undergo either proliferative or neurogenic (differentiative) divisions. Proliferative divisions maintain the size of the progenitor pools, ultimately establishing the size of the cortex. Conversely, neurogenic divisions generate neurons thus determining cortical thickness (Pontious et al., 2008). Cell cycle dynamics are regulated by both the total length of the cell cycle and G1 phase (which increases as neurogenesis proceeds) (Takahashi et al., 1995) and the ratio of cells reentering or exiting the cell cycle.

In our studies we postulate that ERK1/2 kinases, influenced by global morphogens, maintain a crucial balance between proliferative and neurogenic divisions in the developing cortex through their downstream effectors: p27Kip1 and cyclin D1. Therefore, we show that targeted dorso-telencephalic loss of ERK activity results in reduction in cyclin D1 expression levels in the VZ and augmented premature accumulation of p27Kip1. Together the disregulation of these potent cell cycle regulators forces neural progenitors into premature cell cycle exit where the elongated cell cycle length promotes an aberrant neurogenic cell fate switch, thus disrupting the balance between proliferation and differentiation. Therefore, ERK1/2 activity confined to the neurogenic hub, acting through cyclins, cdks and their inhibitors, modulates the cell cycle length and progenitor proliferation dynamics, precisely controlling the rate of neurogenesis in the developing cortex. It is also likely that other cell cycle regulators may be affected by the loss of ERK activity during cortical development. Whether or not ERK activity is necessary for proper function

154 and progression of other components and stages of the cell cycle will require further

investigation. For example, it would be of interest to examine the levels of c-fos,

which is a known transcriptional target of the ERKs that controls cyclin B1 levels and

G2/M cell cycle transition in other cell types (Wright et al., 1999; Liu et al., 2004). In

addition, RSK1 and RSK2 (ribosomal S6 kinase), downstream effectors of the ERKs,

have pleiotropic roles in the regulation of cell cycle, protein synthesis, cell growth,

and differentiation (Silverman et al., 2004; Houge et al., 2006). Importantly, male

children with mutations in RSK2 exhibit psychomotor and growth retardation, facial

dysmorphism and moderate mental disability with various skeletal anomalies

commonly observed in other disorders of the MAPK pathway (Temtamy et al., 1975;

Pereira et al., 2010). Behavioral studies in RSK2 knockout mice revealed profound retardation in spatial learning and a deficit in long-term spatial memory, providing evidence that RSK2 has similar roles in mental function in both mice and humans

(Poirier et al., 2007). An increase in ERK phosphorylation was also reported but

potential changes in corticogenesis or hippocampal function were not addressed.

The role of FGF signaling in the development of the cerebral cortex has been well established in the past decade. However, the downstream signaling pathways and the molecular mechanisms that may be involved in the FGF mutants are just beginning to be explored. Corson et al. (2003) was the first to report elevated ERK activation in regions normally associated with FGF signaling. Further in vitro data

utilizing cultured cortical progenitors treated with FGF2, FGF8 and FGF15 showed

transient activation of pERK (phosphorylated ERK) by Western blot analysis.

Furthermore, mice harboring a gain of function FGFR3 allele showed downstream

155 activation of MAP kinases which resulted in increased cell proliferation at E11.5

(Thomson et al., 2007). We believe that the downstream mechanism of the FGF signaling at least partially involves the ERK MAP kinases and their ability to control cell cycle dynamics during neurogenesis. In addition, we evaluated E14.5 cortical lysates from WT, CKO and DKO mice and examined the activation of downstream effectors of other, developmentally important pathways which are known to play a role in early cortical neurogenesis and cell cycle progression. We did not observe any significant changes in downstream elements of the Wnt or Notch pathways by

Western analysis or immunohistochemistry in ERK2 deficient mice but DKO mice showed a significant reduction. Wnt signaling is mediated through many intracellular pathways, including the β-catenin-mediated canonical Wnt pathway. During later stages of neurogenesis, activated β-catenin controls cell proliferation by regulating cell cycle exit in cortical progenitors. However, initial onset of neurogenesis is blocked by aberrant increase in Wnt signaling. It is a possible that changes in progenitor cell cycle dynamics in ERK deficient mice may be also due to altered levels of Wnt signaling observed in DKO mice.

Figure 4-2

The Wnt and Notch developmental pathways that are important in corticogenesis are not altered in ERK deficient mice.

156 NOTCH

C-Notch/Notch C-Notch/Actin 1.5 3

1.0 2

0.5 1

0.0 0

T O O W K WT K C DKO CKO D

WNT

Copy of B-catenin/Actin Axin2/Actin 2.5 4

2.0 3 1.5 2 1.0

1 0.5

0.0 0

T T O W KO W K CKO D CKO D

pGSK/GSK GSK/actin 1.5 4

3 1.0

2

0.5 1

0.0 0

T O WT KO W KO CKO D C DK

ERKs and Cortical Progenitor Dynamics

During cortical development, proliferation and neuronal differentiation can be viewed as two polarizing forces, where proliferation results in expansion or maintenance of progenitor pools and differentiation depletes it. The importance of the proper balance between proliferation and differentiation cannot be underestimated.

157 Many transcription factors and their downstream effectors work in concert to ensure

proper brain development. The bHLH transcription regulator family is implicated in

this delicate balance (Ross et al., 2003). Some of them like Id and Hes families are

important for maintenance of proliferative activity and therefore suppress

differentiation, whereas others, such as Ngn1 and Ngn2, promote a “proneural” fate

commitment inducing cell cycle exit (Sun et al., 2001). Other transcription factors

such as Emx2 and Tlx regulate progenitor proliferation, whereas Pax6 promotes

neurogenesis (Roy et al., 2004; Heins et al., 2001; Scardigli et al., 2003).

Interestingly, other transcription factors such as Lef1/TCF switch from proliferative

to neurogenic as cortical development progresses (Chenn et al., 2002). Upon initiation

of neurogenesis, other transcription factors guide the fate and laminar specification of newly born neurons. These differentiation factors include other members of the bHLH members such as NeuroD, NeuroD2 and Math2 (Ross et al., 2003). In addition, the T-domain transcription factors, Tbr2 and Tbr1, are sequentially expressed in many, if not all, differentiating projection neurons (Hevner et al., 2001;

Englund et al., 2005). Although laminar fate is precisely linked to chronological neuronal birth date, transcription factors controlling “temporal identity” have not been described in the mammalian cortex. However, sequential expression patterns related to neuronal type have been described in Drosophila (Pearson et al., 2004). On the other hand, several transcription factors have been implicated in fate specification in the cortex. For example, Er81 (an Ets transcription factor) is expressed in a small group of progenitors and a subset of layer 5 neurons, thus perhaps specifying layer 5 identity (Hasegawa et al., 2005). Cux1/2 is transiently expressed in mitotic cells of

158 the SVZ and in upper-layer neurons, suggesting its importance in upper-layer fate specification (Nieto et al., 2004; Zimmer at al., 2004). Overall, many different transcriptional regulators and morphogens act in concert, creating unique molecular footprints, which in turn guide many decisions during cortical development. How

ERKs fit into these combinatorial patterns and how they help with executing of their molecular codes is just beginning to be unraveled and will be explored in future research.

The changes in neural progenitor proliferation during cortical development observed in the ERK knockouts further expand and validate in vitro and in vivo experimental data from mutations in other components of the MAPK signaling pathway. For example, mutations in the ERK scaffolding protein FRS2 result in reduced cortical thickness, decreases in intermediate progenitor cells and deficits in mature projection neurons (Yamamoto et al., 2005). Furthermore, the ERK activity in these mice was reduced during critical stages of neurogenesis, but other pathways, such as AKT, were not changed.

In addition, recent studies from the Miller lab showed that mutations in the H-

Ras gene, which cause Costello Syndrome in humans, enhance neural precursor cell proliferation, perturbing neuronal generation, which may contribute to the cortical abnormalities and cognitive dysfunction seen in Costello patients (Paquin et al.,

2009). Importantly, the ERK activity was significantly elevated in these studies, yet again confirming the importance of ERKs in progenitor proliferation dynamics during brain development.

159 ERKs and Cognition

It has become generally accepted that long-term potentiation (LTP) is one of

the cellular mechanisms responsible for learning and memory. In 1997, England et al.

reported that application of the MEK inhibitor PD98059 abolished LTP in mice,

providing the first evidence that ERK signaling is important in synaptic plasticity.

Furthermore, in 2001 Dicristo et al. showed that ERK activity is required for cortical

neurons to participate in the LTP pathway. Independent behavioral experiments

further confirmed the importance of ERK signaling in learning and memory. For

example, Blum et al. reported that ERK activity was significantly elevated after mice

were trained in the water maze test (Blum et al., 1999). Administration of MEK

inhibitors weakened the ability of rats to learn a new odor associated with avoidance

response (Atkins et al., 1998). Furthermore, new studies demonstrated the

involvement of the MAPK/ERK pathway in human cognition. Silva et al. showed that

mutations associated with the NF1gene, which causes neurofibromatosis type I

blastoma, intellectual disability and hyperactivates the ERK pathway, results in

decrease in learning and memory (Silva et al., 1997; Denayer et al., 2008). Our

results, among others, complement the evidence that loss of ERK activity results in

deficits in learning and memory (Satoh et al., 2007; Samuels et al., 2008; Imamura et

al. 2010). In the present study we first examined our mutant mice for learning and

memory deficits, utilizing the classical fear conditioning paradigm which requires cooperative collaboration between the hippocampus, cortex and the amygdala. ERK2

CKO mice were also found to exhibit a strong anxiety phenotype, which had not been

previously reported and was distinctly different from other ERK knockouts (our data).

160 Therefore, we evaluated working and hippocampus-dependent memory using continuous alteration and delayed T-maze tests, respectively. We report that deletion of ERK2 during early neurogenesis resulted in anxiety-like behavior with deficits in working and hippocampal memory components. In contrast, studies by Satoh et al.,

2011, in which ERK2 was inactivated in the CNS with a Nestin-cre, reported decreased anxiety-like behaviors. In addition, these mice also showed abnormalities in multiple aspects of social behaviors related to autism-spectrum disorders including elevated aggression, deficits in maternal nurturing, poor nest building, and lower levels of social familiarity and social interaction (Satoh et al., 2011). This contrasting behavioral phenotype can be attributed to the different onset of cre-mediated deletions of ERK gene (Emx1-cre recombination is initiated 1.5 days before Nestin- cre) as well as the fact that Nestin-cre recombines in almost the entire CNS including the ventral telencephalon. Therefore the more global deletion of ERK2, which encompasses other brain regions including interneurons, can account for different behavioral outcomes reported by Satoh (Satoh et al., 2011).

It is important to interpret these behavioral results in the context of ERK2 deletions. The long arm of chromosome 22 harbors the MAPK1 gene which encodes the ERK2 protein. It is also a “hot spot” of chromosomal microdeletions due to a presence of high homology low copy repeats (LCRs) which cause faulty recombination events (Emanueal et al., 2001). In humans, the 22q11 3 Mb deletion is associated with this putative region and results in 22q11/DiGeorge Syndrome. This more common deletion encompasses the proximal region of chromosome 22 that does not include the ERK2 gene. In fact, previous research in our lab showed that these

161 patients exhibit normal levels of the ERK2 protein, which was examined by Western

analysis in lymphoblasts obtained from these patients (Samuels et al., 2008). On the

other hand, recent studies identified a new cohort of patients with a 1Mb deletion just

distal to the more common proximal deletion (Rauch et al., 1999; Saitta et al., 1999;

Ben-Shachat et al., 2008). Although both deletions shared the same LCR mediated

mechanism, they did not overlap. Importantly, the distal deletion included

MAPK1/ERK2 but shared the phenotypic characteristics typically associated with classic DiGeorge’s Syndrome. The distal deletion exhibited DiGeorge-like symptoms which include microcephaly and neurodevelopmental as well as neuro-cognitive deficits that are not caused by genes deleted in the larger proximal deletion.

Furthermore, other genes within the 1Mb deleted region such as HIC2, YPEL1 and others have not been previously associated with human pathology. Therefore, we argue that loss of ERK2 in children with small distal deletion on chromosome 22 is responsible for their neurodevelopmental and cognitive deficits. More importantly, murine models of ERK deficiency generated in this study recapitulate cognitive and behavioral deficits observed in these human patients. It is intriguing that our behavioral analysis also revealed that ERK2 CKO mice show anxiety-like phenotype,

since patients with microdeletion of 22q11.21-22 that encompasses the ERK2 gene

only exhibit developmental abnormalities as well as profound anxiety (Verhoeven et

al., 2011). In mice, changes in progenitor number are associated with abnormal

cortical thickness (Sahara et al., 2009; Quinn et al., 2007). It has been recently

reported that Emx-1-derived pallial Pax6+ progenitors contribute to the developing

amygdala during early corticogenesis (Cocas et al., 2009; Stoykova et al., 1996).

162 Therefore, the reduced activity in the mature cortex as well as diminished progenitor

pools may disinhibit the amygdala, leading to an anxiety phenotype observed in our

mutant mice. At the same time, lack of cortical drive to the hippocampus may account

for memory deficits seen in the ERK2 CKO mice. It has been previously reported that

the ERKs regulate synaptic remodeling (Wu et al., 2001) and formation and stabilization of dendritic spines during long-term storage of information in the CNS

(Sweatt et al., 2004). Our studies indicate that the ERK2 CKO mice exhibit a

reduction in axonal length and the number of dendritic arborizations in

somatosensory layer II/III pyramidal neurons. These changes may also further

contribute to the behavioral phenotype of the ERK2 deficient mice.

The combination of altered cell-intrinsic and network properties is likely to

account for profound deficits in global network activity where the excitability of the

network is significantly diminished. It is possible that these changes may contribute

to the behavioral deficits seen in these mice, specifically the anxiety phenotype which

had not been previously observed. On the other hand, the question of whether the

behavioral deficits observed in the mutant mice are a consequence of developmental perturbations of cortical cytoarchitecture and global network physiology or intrinsic

postnatal deficits in mature neurons is addressed in ongoing studies. Our preliminary

data shows that the developmental changes in postnatal cortical structure affect the

formation and function of the hippocampus. We report that mice deficient in ERK

signaling have abnormal hippocampi with altered number of mitotic and Tbr2+

intermediate progenitor cells when compared to wild type controls at P2 and P10.

163 This phenotype is also dose dependent since mice with completely abrogated ERK

activity exhibit more drastic hippocampal deficits (Figure 4-3).

Figure 4-3

ERK deficient mice exhibit changes in hippocampal volume and severely

reduced Tbr2+ progenitor population in the dentate gyrus.

Disorders of the Ras/MAPK pathway and Cognitive Deficits

164

Intellectual disability is characterized by a significant decrease in intellectual functions such as mental capacity, learning, reasoning and problem solving as well as adaptive behavior. It affects about 2–3% of the population, where 75–90% of the intellectually disabled individuals are mildly affected (Daily et al., 2000). About a quarter of all cases of intellectual disability are caused by a genetic disorder. In the

US the term mental retardation has been replaced by intellectual disability, which belongs to a larger group of developmental disabilities that also include epilepsy, autism, cerebral palsy and other disorders that develop during the critical period of development (birth to age 18). Intellectual disability is also distinct from mental illnesses such as schizophrenia or depression. At present, there is no "cure" for an established disability, only supportive and behavioral therapy. The most common congenital causes of intellectual disability in the US include Down syndrome,

22q11.2 or DiGeorge deletion syndrome (formerly velo-cardio-facial syndrome), and fetal alcohol syndrome, as well as other genetic mutations (Daily et al., 2000). The mechanisms that cause all of the associated features of these syndromes are largely unknown. However, it is evident that proper brain development is crucial for normal cognition and that structural or functional changes result in disability. A substantial body of work implies that the MAPK pathway plays an important role in cognitive function (Mazzucchelli and Brambilla, 2000). The genetic evidence from numerous mutations in the upstream and downstream elements of the ERK cascade clearly illustrates a link between cognitive function and the MAP kinases. For example, mutations in RSK2, which lead to Coffin Lowery Syndrome, are associated

165 with reduced brain volume and cognitive impairment in mice, most likely due to

aberrant over-activation of ERK and its downstream effectors such as c-fos

(Schneider et al., 2011). Studies in Drosophila also show that RSK plays an inhibitory

role in the ERK pathway by anchoring ERK in the cytoplasm (Kim et al., 2006), and

during mouse embryogenesis by decreasing ERK activation and target gene

expression (Myers et al., 2004). In addition, studies in PC12 cells show that RSK2

negatively regulates ERKs by phosphorylating son of sevenless, which then inhibits

Ras (Douville and Downward, 1997). Other targets of the ERK cascade which are important in memory and cognition include the Ets Like gene1 (ELK1) transcription factor (Yang et al., 1998), the immediate early gene (IEG) c-Fos (Murphy et al.,

2002) and the cAMP response element-binding (CREB) transcription factor. The

induction of c-Fos has also been linked to synaptic plasticity mechanisms and

memory processes (Davis and Laroche, 2006; Messaoudi et al., 2007; Alberini, 2009;

Davis et al., 2010). Therefore, the ERK/MAPK signaling cascade and CREB and

ELK1-mediated gene transcription are important in synaptic plasticity and memory

(Thiels et al., 2002; Davis and Laroche, 2006). In addition, other forms of intellectual

disability have been associated with mutations in RSK4 (Yntema et al., 1999),

MNK1/2 (MAPK-interacting kinase (MNK); Fragile X-Syndrome) and CREB

binding protein CBP which causes Rubenstein Taybi Syndrome (Kim et al., 2008;

Roelfsema and Peters, 2007; Bartholdi et al., 2007).

166 Figure 4-4

Mutations in upstream and downstream elements of the MAPK/ERK pathway

lead to NCFC and other developmental syndromes.

Recent advances in molecular genetic research have led to the definition of a

new group of clinically related genetic syndromes of the Ras/MAPK pathway termed

Neuro-cardio-facial cutaneous (NCFC) syndromes. They comprise Noonan syndrome

and related disorders (cardio-facio-cutaneous (CFC) and Costello syndromes), as well as neurofibromatosis type 1. It has only recently been appreciated that they result

167 from germline mutations in genes encoding kinases and other upstream and downstream elements of one central pathway: the RAS-MAPK pathway (Figure 4-4).

The new insights into the array of gene mutations within the MAPK cascade and associated clinical manifestations allowed for a better refinement of genotype- phenotype correlations. In addition, novel studies revealed early prenatal manifestation and tumor risk which may contribute to early diagnostic criteria. These syndromes commonly present with characteristic facial features, cardiac defects, cutaneous abnormalities, growth deficits and predisposition to malignancies. In addition, each syndrome also exhibits unique features that probably reflect genotype- related specific biological effects. Importantly, these syndromes are associated with cognitive impairment and psychiatric disease, which are now thought to result from early perturbations in CNS development.

In this study, we hypothesized that the pathophysiology associated with these disorders results from aberrant ERK signaling during development in many different neuralcrest-derived tissues. Specifically, we postulated that aberrant changes during cortical development may be responsible for the cognitive and physiological deficits seen in these patients. We tested our hypothesis by generating mice in which ERK expression was inactivated during the early stages of cortical neurogenesis within the dorsal telencephalon. Our data elucidates the potential mechanisms by which ERKs control crucial aspects of progenitor proliferation dynamics and neuronal differentiation. We demonstrate that loss of ERK1 and ERK2 in mice alters the crucial balance between neurogenic and proliferative progenitor divisions, resulting in

168 aberrant layering of the adult cortex. Mechanistically, we show that deletion of

ERK1/2 affects apical and basal progenitor proliferation by altering the length and

dynamics of the cell cycle. Specifically, elongation of G1 phase results in an aberrant

switch where neurogenic divisions are favored at the expense of proliferation. The

precocious neurogenesis causes premature depletion of the progenitor pool, reducing

the number of pyramidal neurons in the mature cortex. As an alternative hypothesis, it

is also possible that neuroepithelial cell (NE) divisions which unsheathe the ventricles before the onset of the neurogenic expansion, may be altered by absence of ERK activity. In fact, proliferative defects in NE cells may contribute to fewer Pax6+ radial glia in the VZ. This remains to be further investigated. These early embryonic deficits affect global cortical circuitry altering normal physiology and behavior in the adult mice. In addition, we observed vascular bleeding in the cortex of DKO mice. This observation was also made by Satoh et al. who reported intracerebral hemorrhages in nestin driven DKO mice. However, these vascular bleeds did not correlate with the impairments in neurogenesis or increased apoptosis (Satoh et al., 2011).

Our mouse models with perturbations in ERK1/2 signaling present a unique opportunity to directly study potential molecular and cellular mechanisms which are important in cortical development and how they link to physiology and animal behavior. We established a mechanistic explanation and furthered our understanding of the link between loss of ERK1/2 and its consequences on development and the pathophysiology and behavioral abnormalities associated with disorders of the

RAS/MAPK pathway, including DiGeorge Syndrome and autism spectrum disorders.

169 Figure 4-5

General schematic illustrating changes in cortical development in ERK deficient mice.

170 Figure 4-6

The cell cycle dependent mechanism of ERK action during cortical development.

The contribution of MAPK/ERK signaling to Autism Spectrum Disorders

associated with copy number variation of 16p11.2.

A number of recent findings have suggested that key processes in early brain

development may significantly contribute to, and perhaps even initiate, the

pathological hallmarks of autism spectrum disorders (ASD). In particular, distinct studies of genetic mutations, gene expression, and the neuropathology of the ASD

171 brain have all suggested that the size of the population of cortical neurons and/or the patterning of the cortex may be altered in the context of ASD patients. For example, macrocephaly is associated with ~20% of all cases of ASD, where recent reports suggest a dramatic overproduction of neurons in the prefrontal cortex in the ASD brains (Amaral et al., 2008). It is interesting that mutations which decrease the expression of FGF8 and FGF17 are associated with this altered growth of the dorsomedial frontal cortex specifically. Therefore, some forms of ASD may implicate the FGF signaling pathway. The exact mechanism which could increase the number of cortical progenitors and thus mature neurons in the frontal/somatosensory cortex has not been identified. Furthermore, the behavioral consequences of such an increase have not been addressed. In addition, copy number variations of genes associated with ASD that strongly predispose to autism also correlate with increased head circumference (Sebat et al., 2007). More importantly, exome sequencing reveals de novo mutations in several genes with potential roles in proliferation of cortical neuronal progenitors. Finally, both genetic and transcriptome analyses of postmortem

ASD brains revealed altered patterning of the developing cortex and implicate genes known to play a role in cortical patterning. Together, this data strongly suggests that cortical development and neurogenesis may play an important role in pathology associated with at least some ASD patients. This furthers the idea that proper excitation/inhibition balance and cortical size ratio in the mature brain are essential for normal brain function and may impact cognition (Hensch et al., 2005; Rubenstein et al., 2010)

172 The single most common genetic linkage to autism spectrum disorders (ASD) is associated with deletions and duplications of a region on chromosome 16p11.2

(Sanders et al., 2011; Levy et al., 2011) and is associated with more than 1% of all

ASD patients (Zoghbi and Schaaf, 2011). The extracellular signal-regulated kinases,

ERK1 and ERK2, are genetically linked to ASDs and cognition. The pivotal 16p11.2 locus contains 27 genes including the ERK1 gene (MAPK3) and Major Vault Protein

(MVP) gene. MVP is reported to regulate signaling through the ERKs (Liang et al.,

2010; Kolli et al., 2004). Furthermore, a smaller deletion of the 16p11.2 locus, also associated with ASD patients, contains only 5 genes and includes the MVP gene. It is likely that altered expression of one or more genes in the putative 16p11.2 region may converge onto and significantly alter the ERK/MAP kinase signaling pathway resulting in pathology and cognitive deficits associated with ASD.

The ERKs play critical roles in corticogenesis through their capacity to regulate proliferation of neural progenitors within the proliferative zones of the developing cortex. They are also genetically linked to ASD patients. Our data concludes that changes in progenitor proliferation dynamics result in altered brain cytoarchitecture, which may contribute to behavioral and physiological deficits observed in ASD patients. A mouse model harboring a deletion of the chromosomal region corresponding to 16p11.2, which is genetically linked to ASD, has recently been generated (Horev et al., 2011). Half of the 16p11.2 deletion neonatal heterozygotes die postnatally, which may have relevance to incidence of autism. The cause of death is currently unknown. The 16p11.2 mice were also evaluated by MRI and show significant changes in eight different brain regions including the forebrain,

173 superior colliculus, fornix, hypothalamus, mammillothalamic tract, medial septum,

midbrain, and periaqueductal gray. In addition, the 16p11.2 deletion mice exhibit

hyperactivity, difficulty adapting to change, sleeping abnormalities, and repetitive or

restricted behaviors (Horev et al., 2011). Given these finding it would be of great

interest to examine the 16p11.2 mice for ERK1 and ERK2 expression as well as

activation levels. Furthermore, if ERK activity is perturbed in these animals, we will

extend our investigation to cortical neurogenesis and postnatal cytoarchitecture and

compare them to the ERK1 KO and ERK2 CKO mice. The results from these

experiments will determine if ERK signaling is altered in the 16p11.2del mouse and

may be responsible for the developmental defects accompanying 16p11.2 deletions in the 16p11.2 patients.

We hypothesize that ERK activity in the developing brain may be altered in

the16p11.2del mice due to compound loss of ERK pathway elements. Recent data

(Indrigo et al., 2010) showed that ERK1 is a partial agonist of ERK2, suggesting that

ERK1 slows ERK signaling by competing with ERK2 for substrates. Our lab and the

present study as well as others (Immamura et al., 2010) have established that the ERK

isoforms can compensate for each other during cortical development and that loss of

either isoform can alter the ERK pathway and its activity. Therefore, it would be of

interest to investigate whether the 16p11.2del/dup mice exhibit altered ERK signaling

due to loss of ERK pathway signaling elements, specifically ERK1 and MVP. To

address whether ERK2 activity is altered in the 16p11.2 del/dup mice, the levels of

ERK2 expression and activity during development needs to be addressed. The ERK

activity can be assayed by immunoblotting to determine protein levels of ERK1/2,

174 phospho-ERK1/2 and Elk-1 (an ERK1/2 target) at mid-neurogenesis. In addition, we will examine global ERK expression pattern and activity (phospho-ERK) within the developing brain by immunohistochemistry. We postulate that loss of ERK1 in the

16p11.2 mouse may lead to hyperactivation of ERK2. Alternatively, due to the presence of other genes that regulate ERK signaling within the critical region, we may observe differences in ERK signaling in the 16p11.2del compared to the ERK1

KO alone. Either result would identify whether the ERK signaling pathway is affected and whether other genes within the 16p11.2 region are regulating ERK signaling. Since the physiological actions of MVP are not well understood the future study will investigate the contribution of MVP to ERK signaling by using in vitro shRNA assays. Cortical neuronal cultures will be transfected with control or MVP shRNA constructs at E14.5. Protein levels of ERK1/2, pERK1/2 and Elk-1 will be assessed in these cultures and compared to cortical cultures of the 16p11.2del mice. If

16p11.2del mice display ERK signaling that differs from ERK1KO, loss of MVP in the ERK1KO may reduce these differences, which would indicate that these two genes are converging on the ERK signaling pathway to enhance the phenotype. Our data suggest that the changes in apical and basal progenitor proliferation in the ERK2

CKO mice are a consequence of deregulation of downstream ERK effectors and potent cell cycle regulators such as cyclin D1 and p27Kip1. Therefore, the next step would be to examine and compare cortical development in the 16p11.2del mouse with ERK1 KO and ERK2 CKO mice. Cortical progenitor dynamics using immunohistochemistry (IHC) and BrdU pulse-chase experiments in the developing cortex would be conducted. Further examination of the cell cycle dynamics using

175 IHC and Western analysis as well as ERK dependent cell cycle regulators: p27Kip1 and cyclin D1 would point to an ERK dependent mechanism. Furthermore, any changes in cortical cytoarchitecture in postnatal brains and somatosensory cortex specifically using layer-specific cortical markers would be noted.

Potential Impact on Autism Research

Developmental deficits in the cortex play a pivotal role in pathology of many human disorders including autism and ASD. It has been recently appreciated that perturbations in proliferation and migration of neurons alters cortical microcircuitry, leading to pathology seen in these disorders. Therefore, it is of great importance to understand basic molecular mechanisms responsible for cortical development, which is necessary for formation of normal cortical circuitry. We have documented that the

ERK MAP kinases are important for cortical development and function. They also provide an avenue for potential therapeutic intervention, since MAP kinase inhibitors are available and have been widely studied and indeed, suggested for treatment of

NCFC Syndromes.

Changes in early neuronal proliferation and brain patterning may play a significant role in the ASD pathology. The early developmental proliferative dynamics are not well studied within the ASD paradigm. Our current research allowed us to be uniquely positioned to evaluate whether changes in the most

176 prominent regulators of proliferation, the ERKs, can provide a direct link to ASD

pathogenesis.

Towards a Treatment for Developmental Disorders of the MAPK Pathway

One of the first findings suggesting that aberrant ERK1/2 activity might

contribute to disease was the finding that the ERK1/2 cascade was a downstream

target of receptor tyrosine kinases, as well as Ras, which is commonly mutated in

human cancers (Vojtek et al., 1993; Kyriakis et al., 1992). Since the Ras/MAPK

signaling is hyper-activated in most human tumors, it warranted an immediate attempt

to identify drugs that would modulate the ERK pathway, providing an effective

treatment for cancers (Sebolt-Leopold, J.S. and Herrera, R. 2004). Recent

technological advances allowed for better understanding of the genetic mutations and

the mechanism of their action in individuals with developmental disorders of the

MAPK pathway. Better understanding of the biochemical and molecular mechanisms

of these disorders also raised the possibility of future therapeutic intervention.

Although treatment of aberrant hyperactivity of the MAPK cascade in cancers

involves selective targeting of malignant cells exclusively, children with developmental disorders carry the insidious mutation in every cell. Since targeting of the drugs has been an issue in cancer treatments, it would appear that modulation of an abnormal activity in patients harboring a germ line mutation is more straightforward than attempting to distinguish ‘bad cells’ from ‘good cells’ in cancer therapy. On the other hand, modulating the activity of the MAPK cascade in all

177 tissues may result in unexpected deleterious effects in some tissues which have

readapted the same signaling networks for other purposes. The question of whether an

inhibitor would benefit affected patients should also be considered.

Craniofacial malformations are involved with approximately three-quarters of

all congenital human defects, affecting neck, head, or face development (Chai et al.,

2006). ERKs may be an important target for therapeutic intervention, since many

inhibitors are currently in development, or in clinical trials, and some are FDA

approved. For example, germline missense mutations in human PTPN11, which

encodes SHP2, are linked to Noonan and LEOPARD syndromes which belong to a

larger group of NCFC developmental syndromes discussed earlier. They are

characterized by short stature, craniofacial defects, cardiac defects, and

developmental disability. In addition, somatic SHP2 mutations are also associated

with several types of human malignancies, including common juvenile and juvenile

myelomonocytic leukemia (Loh et al., 2004a; Tartaglia et al., 2005). Nakamura et al.

reported that SHP2 mutations associated with Noonan syndrome hyperactivate the

ERK1/2 pathway in mice, leading to craniofacial defects that included smaller skull lengths, greater inner canthal distances, and taller frontal bone heights. Importantly, perinatal injection of the MAPK/ERK inhibitor U0126 resulted in inhibition of

ERK1/2 activity and effectively diminished the severity of the craniofacial defects

(Nakamura et al., 2009). In addition, pilot clinical trials with small molecule inhibitors and statins revealed that they may reverse cognitive deficits in children with NF1 mutations by decreasing the enhanced p21Ras activity in the brain

178 (unpublished data). In zebrafish, treatment of CFC embryos with MEK inhibitors can

restore normal early development of the embryo without any other developmental side effects.

One of the most promising multi-kinase inhibitors is Sorafenib which modulates MAPK pathway activity. It is an orally active RAF inhibitor, which is

FDA approved and typically prescribed for renal cell and hepatocellular carcinoma.

Although along with other inhibitors such as MEK inhibitors, combinatorial cancer chemotherapy has shown some success in clinical trials, none of these drugs have

been tested in patients with developmental disorders.

The disorders of the MAPK pathway, although seemingly phenotypically

heterogeneic, exhibit overlapping phenotypes with respect to their neurodevelopment,

cardiac and cognitive symptoms. The overlapping characteristics allow for the possibility of drug development centered on the defects in the elements of the MAPK cascade. It may become possible to alter the course of the pathological hallmarks of these syndromes and perhaps even reverse some of their manifestations.

179 Conclusion:

The present study contributes to our understanding of why the human brain is so sensitive to even minor dysregulation of the MAPK pathway resulting in pathology associated with NSFC Syndromes and other behavioral disorders. The biology of the

ERKs may have broader implications in understanding the dynamics of cortical progenitor populations which are of key importance for stem cell biology and evolutionary expansion of the cerebral cortex. This study is the first, to our knowledge, to provide in vivo evidence for the mechanistic role of ERK1/2 kinases in corticogenesis through the ability to regulate progenitor proliferation dynamics.

180 CHAPTER 5

Citations:

Aboitiz F, López J, Montiel J (2003a) Long distance communication in the human brain: timing constraints for inter-hemispheric synchrony and the origin of brain lateralization. Biol Res 36:89–99.

Aboitiz F, Morales D, Montiel J (2003b) The evolutionary origin of the mammalian isocortex: towards an integrated developmental and functional approach. Behav Brain Sci 26:535–52–discussion552–85.

Ahamed S, Foster JS, Bukovsky A, Wimalasena J (2001) Signal transduction through the Ras/Erk pathway is essential for the mycoestrogen zearalenone-induced cell-cycle progression in MCF-7 cells. Mol Carcinog 30:88–98.

Alberini CM (2009) Transcription factors in long-term memory and synaptic plasticity. Physiol Rev 89:121–145.

Algan O, Rakic P (1997) Radiation-induced, lamina-specific deletion of neurons in the primate visual cortex. J Comp Neurol 381:335–352.

Alifragis P, Liapi A, Parnavelas JG (2004) Lhx6 regulates the migration of cortical interneurons from the ventral telencephalon but does not specify their GABA phenotype. Journal of Neuroscience 24:5643–5648.

Amaral DG, Schumann CM, Nordahl CW (2008) Neuroanatomy of autism. Trends in Neurosciences 31:137–145.

Anderson SA, Marin O, Horn C, Jennings K, Rubenstein JL (2001) Distinct cortical migrations from the medial and lateral ganglionic eminences. Development 128:353– 363.

Anderson SA, Qiu M, Bulfone A, Eisenstat DD, Meneses J, Pedersen R, Rubenstein JL (1997) Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons. Neuron 19:27– 37.

Angevine JB, Sidman RL (1961) Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192:766–768.

Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, Tanaka Y, Filocamo M, Kato K, Suzuki Y, Kure S, Matsubara Y (2005) Germline mutations in HRAS proto- oncogene cause Costello syndrome. Nat Genet 37:1038–1040.

181 Aragona BJ, Liu Y, Curtis JT, Stephan FK, Wang Z (2003) A critical role for nucleus accumbens dopamine in partner-preference formation in male prairie voles. Journal of Neuroscience 23:3483–3490. Arimatsu Y, Ishida M, Kaneko T, Ichinose S, Omori A (2003) Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1. J Comp Neurol 466:180–196.

Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD (1998) The MAPK cascade is required for mammalian associative learning. Nat Neurosci 1:602–609.

Azim E, Jabaudon D, Fame RM, Macklis JD (2009) SOX6 controls dorsal progenitor identity and interneuron diversity during neocortical development. Nature Publishing Group 12:1238–1247.

Bagui TK, Cui D, Roy S, Mohapatra S, Shor AC, Ma L, Pledger WJ (2009) Inhibition of p27Kip1 gene transcription by mitogens. Cell Cycle 8:115–124.

Balabanian K, Lagane B, Infantino S, Chow KYC, Harriague J, Moepps B, Arenzana- Seisdedos F, Thelen M, Bachelerie F (2005) The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem 280:35760–35766.

Balmanno K, Cook SJ (1999) Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells. Oncogene 18:3085–3097.

Bani-Yaghoub M, Tremblay RG, Lei JX, Zhang D, Zurakowski B, Sandhu JK, Smith B, Ribecco-Lutkiewicz M, Kennedy J, Walker PR, Sikorska M (2006) Role of Sox2 in the development of the mouse neocortex. Developmental Biology 295:52–66.

Barkovich AJ (1996) Malformations of neocortical development: magnetic resonance imaging correlates. Curr Opin Neurol 9:118–121.

Barkovich AJ (2000) Morphologic characteristics of subcortical heterotopia: MR imaging study. AJNR Am J Neuroradiol 21:290–295.

Barkovich AJ, Kuzniecky RI (1996) Neuroimaging of focal malformations of cortical development. J Clin Neurophysiol 13:481–494.

182 Barkovich AJ, Kuzniecky RI, Dobyns WB, Jackson GD, Becker LE, Evrard P (1996) A classification scheme for malformations of cortical development. Neuropediatrics 27:59–63. Bartholdi D, Roelfsema JH, Papadia F, Breuning MH, Niedrist D, Hennekam RC, Schinzel A, Peters DJM (2007) Genetic heterogeneity in Rubinstein-Taybi syndrome: delineation of the phenotype of the first patients carrying mutations in EP300. J Med Genet 44:327–333.

Batista-Brito R, Fishell G (2009) The developmental integration of cortical interneurons into a functional network. Curr Top Dev Biol 87:81–118.

Batista-Brito R, Rossignol E, Hjerling-Leffler J, Denaxa M, Wegner M, Lefebvre V, Pachnis V, Fishell G (2009) The cell-intrinsic requirement of Sox6 for cortical interneuron development. Neuron 63:466–481.

Bayer SA, Altman J, Russo RJ, Dai XF, Simmons JA (1991) Cell migration in the rat embryonic neocortex. J Comp Neurol 307:499–516.

Bellus GA, Spector EB, Speiser PW, Weaver CA, Garber AT, Bryke CR, Israel J, Rosengren SS, Webster MK, Donoghue DJ, Francomano CA (2000) Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype. Am J Hum Genet 67:1411–1421.

Bishop KM, Goudreau G, O'Leary DD (2000) Regulation of area identity in the mammalian neocortex by Emx2 and Pax6. Science 288:344–349.

Blum S, Moore AN, Adams F, Dash PK (1999) A mitogen-activated protein kinase cascade in the CA1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory. J Neurosci 19:3535–3544.

Borrell V, Marín O (2006) Meninges control tangential migration of hem-derived Cajal-Retzius cells via CXCL12/CXCR4 signaling. Nat Neurosci 9:1284–1293.

Boulton TG, Cobb MH (1991) Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul 2:357–371.

Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J, Cobb MH (1990) An insulin-stimulated protein kinase similar to yeast kinases involved in

183 cell cycle control. Science 249:64–67.

Böttcher RT, Niehrs C (2005) Fibroblast growth factor signaling during early vertebrate development. Endocr Rev 26:63–77.

Brasil AS, Malaquias AC, Wanderley LT, Kim CA, Krieger JE, Jorge AAL, Pereira AC, Bertola DR (2010) Co-occurring PTPN11 and SOS1 gene mutations in Noonan syndrome: does this predict a more severe phenotype? Arq Bras Endocrinol Metabol 54:717–722.

Breckpot J, Thienpont B, Bauters M, Tranchevent L-C, Gewillig M, Allegaert K, Vermeesch JR, Moreau Y, Devriendt K (2012) Congenital heart defects in a novel recurrent 22q11.2 deletion harboring the genes CRKL and MAPK1. Am J Med Genet A 158A:574–580.

Brondello JM, Pouysségur J, McKenzie FR (1999) Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 286:2514–2517.

Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouysségur J (1999) Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 18:664–674.

Brückner G, Biesold D (1981) Histochemistry of glycogen deposition in perinatal rat brain: importance of radial glial cells. J Neurocytol 10:749–757.

Bucan M et al. (2009) Genome-wide analyses of exonic copy number variants in a family-based study point to novel autism susceptibility genes. PLoS Genet 5:e1000536.

Buday L, Downward J (1993a) Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73:611–620.

Buday L, Downward J (1993b) Epidermal growth factor regulates the exchange rate of guanine nucleotides on p21ras in fibroblasts. Mol Cell Biol 13:1903–1910.

184 Butt SJB, Fuccillo M, Nery S, Noctor S, Kriegstein A, Corbin JG, Fishell G (2005) The temporal and spatial origins of cortical interneurons predict their physiological subtype. Neuron 48:591–604.

Cajal S (1911) Histologie du Systeme Nerveux de l'Homme et des Vertebres Maloine. Paris (1909–1911).

Calegari F, Huttner WB (2003) An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis. Journal of Cell Science 116:4947–4955.

Campbell K, Götz M (2002) Radial glia: multi-purpose cells for vertebrate brain development. Trends in Neurosciences 25:235–238.

Castiel A, Danieli MM, David A, Moshkovitz S, Aplan PD, Kirsch IR, Brandeis M, Krämer A, Izraeli S (2011) The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr. Journal of Cell Science 124:532–539.

Cau E, Gradwohl G, Casarosa S, Kageyama R, Guillemot F (2000) Hes genes regulate sequential stages of neurogenesis in the olfactory epithelium. Development 127:2323–2332.

Caviness VS, Frost DO, Hayes NL (1976) Barrels in somatosensory cortex of normal and reeler mutant mice. Neuroscience Letters 3:7–14.

Caviness VS, Rakic P (1978) Mechanisms of cortical development: a view from mutations in mice. Annu Rev Neurosci 1:297–326.

Caviness VS, Takahashi T (1995) Proliferative events in the cerebral ventricular zone. Brain Dev 17:159–163.

Caviness VS, Takahashi T, Nowakowski RS (1995) Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model. Trends in Neurosciences 18:379–383.

Chandran S, Kato H, Gerreli D, Compston A, Svendsen CN, Allen ND (2003) FGF- dependent generation of oligodendrocytes by a hedgehog-independent pathway.

185 Development 130:6599–6609.

Chen B, Schaevitz LR, McConnell SK (2005) Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc Natl Acad Sci USA 102:17184–17189.

Cheng JD, Werness BA, Babb JS, Meropol NJ (1999a) Paradoxical correlations of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1 in metastatic colorectal carcinoma. Clin Cancer Res 5:1057–1062.

Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM, Sherr CJ (1999b) The p21(Cip1) and p27(Kip1) CDK “inhibitors” are essential activators of cyclin D- dependent kinases in murine fibroblasts. EMBO J 18:1571–1583.

Chenn A (2002) Regulation of Cerebral Cortical Size by Control of Cell Cycle Exit in Neural Precursors. Science 297:365–369.

Chi P, Greengard P, Ryan TA (2003) Synaptic vesicle mobilization is regulated by distinct synapsin I phosphorylation pathways at different frequencies. Neuron 38:69– 78.

Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA (1996) Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383:407–413.

Choi BH, Lapham LW (1978) Radial glia in the human fetal cerebrum: a combined Golgi, immunofluorescent and electron microscopic study. Brain Research 148:295– 311.

Choi BH, Lapham LW, Amin-Zaki L, Saleem T (1978) Abnormal neuronal migration, deranged cerebral cortical organization, and diffuse white matter astrocytosis of human fetal brain: a major effect of methylmercury poisoning in utero. J Neuropathol Exp Neurol 37:719–733.

Cholfin JA, Rubenstein JLR (2007) Genetic regulation of prefrontal cortex development and function. Novartis Found Symp 288:165–73–discussion173–7–276– 81.

186 Cholfin JA, Rubenstein JLR (2008) Frontal cortex subdivision patterning is coordinately regulated by Fgf8, Fgf17, and Emx2. J Comp Neurol 509:144–155.

Cobb MH, Robbins DJ, Boulton TG (1991) ERKs, extracellular signal-regulated MAP-2 kinases. Curr Opin Cell Biol 3:1025–1032.

Cobos I, Long JE, Thwin MT, Rubenstein JL (2006) Cellular patterns of transcription factor expression in developing cortical interneurons. Cereb Cortex 16 Suppl 1:i82– i88.

Cocas LA, Miyoshi G, Carney RSE, Sousa VH, Hirata T, Jones KR, Fishell G, Huntsman MM, Corbin JG (2009) Emx1-lineage progenitors differentially contribute to neural diversity in the striatum and amygdala. Journal of Neuroscience 29:15933– 15946.

Coogan AN, O'Leary DM, O'Connor JJ (1999) P42/44 MAP kinase inhibitor PD98059 attenuates multiple forms of synaptic plasticity in rat dentate gyrus in vitro. J Neurophysiol 81:103–110.

Cooper JA (2008) A mechanism for inside-out lamination in the neocortex. Trends in Neurosciences 31:113–119.

Corbin JG, Nery S, Fishell G (2001) Telencephalic cells take a tangent: non-radial migration in the mammalian forebrain. Nat Neurosci 4 Suppl:1177–1182.

Crossley PH, Martin GR (1995) The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121:439–451.

Daily DK, Ardinger HH, Holmes GE (2000) Identification and evaluation of mental retardation. Am Fam Physician 61:1059–67–1070.

Davis S, Laroche S (2006) Mitogen-activated protein kinase/extracellular regulated kinase signalling and memory stabilization: a review. Genes Brain Behav 5 Suppl 2:61–72.

Davis S, Renaudineau S, Poirier R, Poucet B, Save E, Laroche S (2010) The formation and stability of recognition memory: what happens upon recall? Front

187 Behav Neurosci 4:177.

Davis S, Vanhoutte P, Pages C, Caboche J, Laroche S (2000) The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J Neurosci 20:4563–4572.

De Camilli P, Harris SM, Huttner WB, Greengard P (1983) Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J Cell Biol 96:1355–1373.

DeFelipe J, Fariñas I (1992) The pyramidal neuron of the cerebral cortex: morphological and chemical characteristics of the synaptic inputs. Prog Neurobiol 39:563–607.

Dehay C, Kennedy H (2007) Cell-cycle control and cortical development. Nat Rev Neurosci 8:438–450.

Denayer E, Ahmed T, Brems H, Van Woerden G, Borgesius NZ, Callaerts-Vegh Z, Yoshimura A, Hartmann D, Elgersma Y, D'Hooge R, Legius E, Balschun D (2008a) Spred1 Is Required for Synaptic Plasticity and Hippocampus-Dependent Learning. Journal of Neuroscience 28:14443–14449.

Denayer E, de Ravel T, Legius E (2008b) Clinical and molecular aspects of RAS related disorders. J Med Genet 45:695–703.

Desai AR, McConnell SK (2000) Progressive restriction in fate potential by neural progenitors during cerebral cortical development. Development 127:2863–2872.

Dhillon AS, Kriegsheim von A, Grindlay J, Kolch W (2007) Phosphatase and feedback regulation of Raf-1 signaling. Cell Cycle 6:3–7.

Di Cristo G, Berardi N, Cancedda L, Pizzorusso T, Putignano E, Ratto GM, Maffei L (2001) Requirement of ERK activation for visual cortical plasticity. Science 292:2337–2340.

188 Dichter GS, Felder JN, Green SR, Rittenberg AM, Sasson NJ, Bodfish JW (2012) Reward circuitry function in autism spectrum disorders. Soc Cogn Affect Neurosci 7:160–172.

Dichter GS, Sikich L, Mahorney S, Felder JN, Lam KSL, Turner-Brown L, Bodfish J (2010) fMRI tracks reductions in repetitive behaviors in autism: two case studies. Neurocase 16:307–316.

Dikic I, Schlessinger J, Lax I (1994) PC12 cells overexpressing the insulin receptor undergo insulin-dependent neuronal differentiation. Curr Biol 4:702–708.

Dobrowolski S, Harter M, Stacey DW (1994a) Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells. Mol Cell Biol 14:5441–5449.

Dobrowolski SF, Stacey DW, Harter ML, Stine JT, Hiebert SW (1994b) An E2F dominant negative mutant blocks E1A induced cell cycle progression. Oncogene 9:2605–2612.

Douville E, Downward J (1997) EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2. Oncogene 15:373–383.

Du T, Xu Q, Ocbina PJ, Anderson SA (2008) NKX2.1 specifies cortical interneuron fate by activating Lhx6. Development 135:1559–1567.

Dumesic PA, Scholl FA, Barragan DI, Khavari PA (2009) Erk1/2 MAP kinases are required for epidermal G2/M progression. J Cell Biol 185:409–422.

Durand B, Gao FB, Raff M (1997) Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation. EMBO J 16:306–317.

Eckel-Mahan KL, Phan T, Han S, Wang H, Chan GCK, Scheiner ZS, Storm DR (2008) Circadian oscillation of hippocampal MAPK activity and cAmp: implications for memory persistence. Nature Publishing Group 11:1074–1082.

Edelmann L, Pandita RK, Morrow BE (1999) Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome. Am J Hum Genet

189 64:1076–1086.

Edmondson JC, Hatten ME (1987) Glial-guided granule neuron migration in vitro: a high-resolution time-lapse video microscopic study. J Neurosci 7:1928–1934.

Elledge SJ, Harper JW (1994) Cdk inhibitors: on the threshold of checkpoints and development. Curr Opin Cell Biol 6:847–852.

Emanuel BS, McDonald-McGinn D, Saitta SC, Zackai EH (2001) The 22q11.2 deletion syndrome. Adv Pediatr 48:39–73.

English JD, Sweatt JD (1996) Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271:24329–24332.

English JD, Sweatt JD (1997) A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem 272:19103–19106.

Englund C, Fink A, Lau C, Pham D, Daza RAM, Bulfone A, Kowalczyk T, Hevner RF (2005) Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. Journal of Neuroscience 25:247–251.

Ensenauer RE, Adeyinka A, Flynn HC, Michels VV, Lindor NM, Dawson DB, Thorland EC, Lorentz CP, Goldstein JL, McDonald MT, Smith WE, Simon-Fayard E, Alexander AA, Kulharya AS, Ketterling RP, Clark RD, Jalal SM (2003) Microduplication 22q11.2, an emerging syndrome: clinical, cytogenetic, and molecular analysis of thirteen patients. Am J Hum Genet 73:1027–1040.

Esain V, Postlethwait JH, Charnay P, Ghislain J (2010) FGF-receptor signalling controls neural cell diversity in the zebrafish hindbrain by regulating olig2 and sox9. Development 137:33–42.

Eswarakumar VP, Lax I, Schlessinger J (2005) Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16:139–149.

Evans SJ, Choudary PV, Neal CR, Li JZ, Vawter MP, Tomita H, Lopez JF, Thompson RC, Meng F, Stead JD, Walsh DM, Myers RM, Bunney WE, Watson SJ, Jones EG, Akil H (2004) Dysregulation of the fibroblast growth factor system in

190 major depression. Proc Natl Acad Sci USA 101:15506–15511.

Faedo A, Borello U, Rubenstein JLR (2010) Repression of Fgf Signaling by Sprouty1-2 Regulates Cortical Patterning in Two Distinct Regions and Times. Journal of Neuroscience 30:4015–4023.

Fairén A, Cobas A, Fonseca M (1986) Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex. J Comp Neurol 251:67–83. Ferland RJ, Batiz LF, Neal J, Lian G, Bundock E, Lu J, Hsiao Y-C, Diamond R, Mei D, Banham AH, Brown PJ, Vanderburg CR, Joseph J, Hecht JL, Folkerth R, Guerrini R, Walsh CA, Rodriguez EM, Sheen VL (2009) Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia. Hum Mol Genet 18:497–516.

Ferland RJ, Cherry TJ, Preware PO, Morrisey EE, Walsh CA (2003) Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. J Comp Neurol 460:266–279.

Flames N, Pla R, Gelman DM, Rubenstein JLR, Puelles L, Marín O (2007) Delineation of multiple subpallial progenitor domains by the combinatorial expression of transcriptional codes. Journal of Neuroscience 27:9682–9695.

Frantz GD, McConnell SK (1996) Restriction of late cerebral cortical progenitors to an upper-layer fate. Neuron 17:55–61.

Frantz GD, Weimann JM, Levin ME, McConnell SK (1994) Otx1 and Otx2 define layers and regions in developing cerebral cortex and cerebellum. J Neurosci 14:5725– 5740.

Freitag CM, Luders E, Hulst HE, Narr KL, Thompson PM, Toga AW, Krick C, Konrad C (2009) Total brain volume and corpus callosum size in medication-naïve adolescents and young adults with autism spectrum disorder. Biol Psychiatry 66:316– 319.

Fuccillo M, Rallu M, McMahon AP, Fishell G (2004) Temporal requirement for hedgehog signaling in ventral telencephalic patterning. Development 131:5031–5040.

191 Gabay L, Lowell S, Rubin LL, Anderson DJ (2003) Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 40:485–499.

Gadisseux JF, Evrard P (1985) Glial-neuronal relationship in the developing central nervous system. A histochemical-electron microscope study of radial glial cell particulate glycogen in normal and reeler mice and the human fetus. Dev Neurosci 7:12–32.

Gaiano N, Nye JS, Fishell G (2000) Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26:395–404.

Georgopoulou N, Hurel C, Politis PK, Gaitanou M, Matsas R, Thomaidou D (2006) BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation. J Biol Chem 281:33606–33620.

Giachello CNG, Fiumara F, Giacomini C, Corradi A, Milanese C, Ghirardi M, Benfenati F, Montarolo PG (2010) MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity. Journal of Cell Science 123:881–893.

Glessner JT et al. (2009) Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. Nature 459:569–573.

Glickstein SB, Alexander S, Ross ME (2007) Differences in cyclin D2 and D1 protein expression distinguish forebrain progenitor subsets. Cereb Cortex 17:632– 642.

Glickstein SB, Monaghan JA, Koeller HB, Jones TK, Ross ME (2009) Cyclin D2 is critical for intermediate progenitor cell proliferation in the embryonic cortex. Journal of Neuroscience 29:9614–9624.

Goc B, Walencka Z, Włoch A, Wojciechowska E, Wiecek-Włodarska D, Krzystolik- Ładzińska J, Bober K, Swietliński J (2006) Trisomy 18 in neonates: prenatal diagnosis, clinical features, therapeutic dilemmas and outcome. J Appl Genet 47:165– 170.

192 Gotoh Y, Moriyama K, Matsuda S, Okumura E, Kishimoto T, Kawasaki H, Suzuki K, Yahara I, Sakai H, Nishida E (1991) Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. EMBO J 10:2661–2668.

Gotoh Y, Nishida E, Yamashita T, Hoshi M, Kawakami M, Sakai H (1990) Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem 193:661–669.

Gottschalk W, Pozzo-Miller LD, Figurov A, Lu B (1998) Presynaptic modulation of synaptic transmission and plasticity by brain-derived neurotrophic factor in the developing hippocampus. J Neurosci 18:6830–6839.

Götz M, Huttner WB (2005) The cell biology of neurogenesis. Nat Rev Mol Cell Biol 6:777–788. Gripp KW (2005) Tumor predisposition in Costello syndrome. Am J Med Genet C Semin Med Genet 137C:72–77.

Gripp KW, Scott CI, Nicholson L, McDonald-McGinn DM, Ozeran JD, Jones MC, Lin AE, Zackai EH (2002) Five additional Costello syndrome patients with rhabdomyosarcoma: proposal for a tumor screening protocol. Am J Med Genet 108:80–87.

Gupta P, Prywes R (2002) ATF1 phosphorylation by the ERK MAPK pathway is required for epidermal growth factor-induced c-jun expression. J Biol Chem 277:50550–50556.

Gutin G, Fernandes M, Palazzolo L, Paek H, Yu K, Ornitz DM, McConnell SK, Hébert JM (2006) FGF signalling generates ventral telencephalic cells independently of SHH. Development 133:2937–2946.

Hallberg B, Rayter SI, Downward J (1994) Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation. J Biol Chem 269:3913–3916.

Hamasaki T, Leingärtner A, Ringstedt T, O'Leary DDM (2004) EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors. Neuron 43:359–372.

193 Hammond V, So E, Gunnersen J, Valcanis H, Kalloniatis M, Tan S-S (2006) Layer positioning of late-born cortical interneurons is dependent on Reelin but not p35 signaling. Journal of Neuroscience 26:1646–1655.

Han J, Tsukada Y-I, Hara E, Kitamura N, Tanaka T (2005) Hepatocyte growth factor induces redistribution of p21(CIP1) and p27(KIP1) through ERK-dependent p16(INK4a) up-regulation, leading to cell cycle arrest at G1 in HepG2 hepatoma cells. J Biol Chem 280:31548–31556.

Hanafusa H, Torii S, Yasunaga T, Nishida E (2002) Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nature Cell Biology 4:850–858.

Hanna N, Montagner A, Lee WH, Miteva M, Vidal M, Vidaud M, Parfait B, Raynal P (2006) Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: consequences for PI3K binding on Gab1. FEBS Lett 580:2477–2482. Hasegawa H, Ashigaki S, Takamatsu M, Suzuki-Migishima R, Ohbayashi N, Itoh N, Takada S, Tanabe Y (2004) Laminar patterning in the developing neocortex by temporally coordinated fibroblast growth factor signaling. Journal of Neuroscience 24:8711–8719.

Hatano N, Mori Y, Oh-hora M, Kosugi A, Fujikawa T, Nakai N, Niwa H, Miyazaki J- I, Hamaoka T, Ogata M (2003) Essential role for ERK2 mitogen-activated protein kinase in placental development. Genes Cells 8:847–856.

Haubensak W, Attardo A, Denk W, Huttner WB (2004) Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc Natl Acad Sci USA 101:3196–3201.

Hauge C, Frödin M (2006) RSK and MSK in MAP kinase signalling. Journal of Cell Science 119:3021–3023.

Hawthorne AL, Wylie CJ, Landmesser LT, Deneris ES, Silver J (2010) Serotonergic neurons migrate radially through the neuroepithelium by dynamin-mediated somal translocation. Journal of Neuroscience 30:420–430.

Heins N, Cremisi F, Malatesta P, Gangemi RM, Corte G, Price J, Goudreau G, Gruss P, Götz M (2001) Emx2 promotes symmetric cell divisions and a multipotential fate in precursors from the cerebral cortex. Mol Cell Neurosci 18:485–502.

194 Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA, Chapouton P, Barde Y-A, Götz M (2002) Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci 5:308–315.

Hensch TK (2005) Critical period plasticity in local cortical circuits. Nat Rev Neurosci 6:877–888.

Hensch TK, Fagiolini M (2005) Excitatory-inhibitory balance and critical period plasticity in developing visual cortex. Prog Brain Res 147:115–124.

Hensen U, Lange OF, Grubmüller H (2010) Estimating absolute configurational entropies of macromolecules: the minimally coupled subspace approach. PLoS ONE 5:e9179.

Hevner RF, Daza RAM, Englund C, Kohtz J, Fink A (2004) Postnatal shifts of interneuron position in the neocortex of normal and reeler mice: evidence for inward radial migration. NSC 124:605–618.

Hevner RF, Shi L, Justice N, Hsueh Y, Sheng M, Smiga S, Bulfone A, Goffinet AM, Campagnoni AT, Rubenstein JL (2001) Tbr1 regulates differentiation of the preplate and layer 6. Neuron 29:353–366.

Horev G, Ellegood J, Lerch JP, Son Y-EE, Muthuswamy L, Vogel H, Krieger AM, Buja A, Henkelman RM, Wigler M, Mills AA (2011) Dosage-dependent phenotypes in models of 16p11.2 lesions found in autism. Proceedings of the National Academy of Sciences 108:17076–17081.

Houge G, Boman H, Lybaek H, Ness GO, Juliusson PB (2006) Lack of meiotic crossovers during oogenesis in an apparent 45,X Ullrich-Turner syndrome patient with three children. Am J Med Genet A 140:1092–1097.

Hu T, Yamagishi H, Maeda J, McAnally J, Yamagishi C, Srivastava D (2004) Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors. Development 131:5491– 5502.

Huang YY, Martin KC, Kandel ER (2000) Both protein kinase A and mitogen- activated protein kinase are required in the amygdala for the macromolecular

195 synthesis-dependent late phase of long-term potentiation. J Neurosci 20:6317–6325.

Huffman KJ, Garel S, Rubenstein JLR (2004) Fgf8 regulates the development of intra-neocortical projections. Journal of Neuroscience 24:8917–8923.

Huttner WB, Schiebler W, Greengard P, De Camilli P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol 96:1374–1388.

Iacopetti P, Michelini M, Stuckmann I, Oback B, Aaku-Saraste E, Huttner WB (1999) Expression of the antiproliferative gene TIS21 at the onset of neurogenesis identifies single neuroepithelial cells that switch from proliferative to neuron- generating division. Proc Natl Acad Sci USA 96:4639–4644.

Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, Scherer SW, Lee C (2004) Detection of large-scale variation in the human genome. Nat Genet 36:949– 951. Imamura O, Pagès G, Pouysségur J, Endo S, Takishima K (2010) ERK1 and ERK2 are required for radial glial maintenance and cortical lamination. Genes Cells 15:1072–1088.

Impey S, Obrietan K, Wong ST, Poser S, Yano S, Wayman G, Deloulme JC, Chan G, Storm DR (1998) Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21:869– 883.

Indrigo M, Papale A, Orellana D, Brambilla R (2010) Lentiviral vectors to study the differential function of ERK1 and ERK2 MAP kinases. Methods Mol Biol 661:205– 220.

Inoue K, Terashima T, Nishikawa T, Takumi T (2004) Fez1 is layer-specifically expressed in the adult mouse neocortex. Eur J Neurosci 20:2909–2916.

Itoh N, Ornitz DM (2004) Evolution of the Fgf and Fgfr gene families. Trends Genet 20:563–569.

Jaglin XH, Chelly J (2009) Tubulin-related cortical dysgeneses: microtubule dysfunction underlying neuronal migration defects. Trends Genet 25:555–566.

196 Jovanovic JN, Benfenati F, Siow YL, Sihra TS, Sanghera JS, Pelech SL, Greengard P, Czernik AJ (1996) Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions. Proc Natl Acad Sci USA 93:3679– 3683.

Jovanovic JN, Czernik AJ, Fienberg AA, Greengard P, Sihra TS (2000) Synapsins as mediators of BDNF-enhanced neurotransmitter release. Nat Neurosci 3:323–329.

Kahan C, Seuwen K, Meloche S, Pouysségur J (1992) Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. J Biol Chem 267:13369–13375.

Kalay E et al. (2011) CEP152 is a genome maintenance protein disrupted in Seckel syndrome. Nat Genet 43:23–26.

Kao S, Jaiswal RK, Kolch W, Landreth GE (2001) Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells. J Biol Chem 276:18169–18177.

Karandikar M, Xu S, Cobb MH (2000) MEKK1 binds raf-1 and the ERK2 cascade components. J Biol Chem 275:40120–40127.

Kawaguchi A, Ogawa M, Saito K, Matsuzaki F, Okano H, Miyata T (2004) Differential expression of Pax6 and Ngn2 between pair-generated cortical neurons. J Neurosci Res 78:784–795.

Kawaguchi Y, Kondo S (2002) Parvalbumin, somatostatin and cholecystokinin as chemical markers for specific GABAergic interneuron types in the rat frontal cortex. J Neurocytol 31:277–287.

Kerkhoff E, Rapp UR (2001) The Ras-Raf relationship: an unfinished puzzle. Adv Enzyme Regul 41:261–267.

Kesler SR, Simensen RJ, Voeller K, Abidi F, Stevenson RE, Schwartz CE, Reiss AL (2007) Altered neurodevelopment associated with mutations of RSK2: a morphometric MRI study of Coffin-Lowry syndrome. Neurogenetics 8:143–147.

197 Kessaris N, Jamen F, Rubin LL, Richardson WD (2004) Cooperation between sonic hedgehog and fibroblast growth factor/MAPK signalling pathways in neocortical precursors. Development 131:1289–1298.

Kim D-I, Lee S-J, Lee S-B, Park K, Kim W-J, Moon S-K (2008a) Requirement for Ras/Raf/ERK pathway in naringin-induced G1-cell-cycle arrest via p21WAF1 expression. Carcinogenesis 29:1701–1709.

Kim S-H, Hu Y, Cadman S, Bouloux P (2008b) Diversity in fibroblast growth factor receptor 1 regulation: learning from the investigation of Kallmann syndrome. J Neuroendocrinol 20:141–163.

Kim SW, Ha NY, Kim KI, Park JK, Lee YH (2008c) Memory-improving effect of formulation-MSS by activation of hippocampal MAPK/ERK signaling pathway in rats. BMB Rep 41:242–247.

Kolli S (2004) The Major Vault Protein Is a Novel Substrate for the Tyrosine Phosphatase SHP-2 and Scaffold Protein in Epidermal Growth Factor Signaling. Journal of Biological Chemistry 279:29374–29385.

Kontaridis MI, Swanson KD, David FS, Barford D, Neel BG (2006) PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J Biol Chem 281:6785–6792.

Korada S, Zheng W, Basilico C, Schwartz ML, Vaccarino FM (2002) Fibroblast growth factor 2 is necessary for the growth of glutamate projection neurons in the anterior neocortex. Journal of Neuroscience 22:863–875.

Kowalczyk T, Pontious A, Englund C, Daza RAM, Bedogni F, Hodge R, Attardo A, Bell C, Huttner WB, Hevner RF (2009) Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex. Cerebral Cortex 19:2439–2450.

Kriegstein A, Noctor S, Martínez-Cerdeño V (2006) Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion. Nat Rev Neurosci 7:883–890.

198 Kriegstein AR (2005) Constructing circuits: neurogenesis and migration in the developing neocortex. Epilepsia 46 Suppl 7:15–21.

Kumar RA, Pilz DT, Babatz TD, Cushion TD, Harvey K, Topf M, Yates L, Robb S, Uyanik G, Mancini GMS, Rees MI, Harvey RJ, Dobyns WB (2010) TUBA1A mutations cause wide spectrum lissencephaly (smooth brain) and suggest that multiple neuronal migration pathways converge on alpha tubulins. Hum Mol Genet 19:2817–2827.

Kushner SA, Elgersma Y, Murphy GG, Jaarsma D, van Woerden GM, Hojjati MR, Cui Y, LeBoutillier JC, Marrone DF, Choi ES, De Zeeuw CI, Petit TL, Pozzo-Miller L, Silva AJ (2005) Modulation of presynaptic plasticity and learning by the H- ras/extracellular signal-regulated kinase/synapsin I signaling pathway. Journal of Neuroscience 25:9721–9734.

Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J (1992) Raf-1 activates MAP kinase-kinase. Nature 358:417–421.

Labalette C, Bouchoucha YX, Wassef MA, Gongal PA, Le Men J, Becker T, Gilardi- Hebenstreit P, Charnay P (2011) Hindbrain patterning requires fine-tuning of early krox20 transcription by Sprouty 4. Development 138:317–326.

Lange C, Huttner WB, Calegari F (2009) Cdk4/CyclinD1 Overexpression in Neural Stem Cells Shortens G1, Delays Neurogenesis, and Promotes the Generation and Expansion of Basal Progenitors. Cell Stem Cell 5:320–331.

Larsson K, Eeg-Olofsson O (2006) A population based study of epilepsy in children from a Swedish county. Eur J Paediatr Neurol 10:107–113.

Lavoie JN, Rivard N, L'Allemain G, Pouysségur J (1996) A temporal and biochemical link between growth factor-activated MAP kinases, cyclin D1 induction and cell cycle entry. Prog Cell Cycle Res 2:49–58.

Lee M-L, Chen H-N, Chen M, Tsao L-Y, Wang B-T, Lee M-H, Chiu I-S (2006) Persistent fifth aortic arch associated with 22q11.2 deletion syndrome. J Formos Med Assoc 105:284–289.

Lefloch R, Pouysségur J, Lenormand P (2008) Single and combined silencing of ERK1 and ERK2 reveals their positive contribution to growth signaling depending on

199 their expression levels. Mol Cell Biol 28:511–527.

Leingärtner A, Richards LJ, Dyck RH, Akazawa C, O'Leary DDM (2003) Cloning and cortical expression of rat Emx2 and adenovirus-mediated overexpression to assess its regulation of area-specific targeting of thalamocortical axons. Cereb Cortex 13:648–660.

Levitt P, Cooper ML, Rakic P (1981) Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultrastructural immunoperoxidase analysis. J Neurosci 1:27–39.

Levitt P, Cooper ML, Rakic P (1983) Early divergence and changing proportions of neuronal and glial precursor cells in the primate cerebral ventricular zone. Developmental Biology 96:472–484.

Levy D, Ronemus M, Yamrom B, Lee Y-H, Leotta A, Kendall J, Marks S, Lakshmi B, Pai D, Ye K, Buja A, Krieger A, Yoon S, Troge J, Rodgers L, Iossifov I, Wigler M (2011) Rare de novo and transmitted copy-number variation in autistic spectrum disorders. Neuron 70:886–897.

Li G, Adesnik H, Li J, Long J, Nicoll RA, Rubenstein JLR, Pleasure SJ (2008) Regional distribution of cortical interneurons and development of inhibitory tone are regulated by Cxcl12/Cxcr4 signaling. Journal of Neuroscience 28:1085–1098.

Liang P, Wan Y, Yan Y, Wang Y, Luo N, Deng Y, Fan X, Zhou J, Li Y, Wang Z, Yuan W, Tang M, Mo X, Wu X (2010) MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway. Biochem Cell Biol 88:445– 450.

Liodis P, Denaxa M, Grigoriou M, Akufo-Addo C, Yanagawa Y, Pachnis V (2007) Lhx6 activity is required for the normal migration and specification of cortical interneuron subtypes. Journal of Neuroscience 27:3078–3089.

Liu J, Fukunaga K, Yamamoto H, Nishi K, Miyamoto E (1999) Differential roles of Ca(2+)/calmodulin-dependent protein kinase II and mitogen-activated protein kinase activation in hippocampal long-term potentiation. Journal of Neuroscience 19:8292– 8299.

200 Liu X, Yan S, Zhou T, Terada Y, Erikson RL (2004a) The MAP kinase pathway is required for entry into mitosis and cell survival. Oncogene 23:763–776.

Liu Y, Suzuki YJ, Day RM, Fanburg BL (2004b) Rho kinase-induced nuclear translocation of ERK1/ERK2 in smooth muscle cell mitogenesis caused by serotonin. Circ Res 95:579–586.

Lloyd AC (2006) Distinct functions for ERKs? J Biol 5:13. LOCKE S, Angevine JB, YAKOVLEV PI (1961) Limbic nuclei of thalamus and connections of limbic cortex. II. Thalamocortical projection of the lateral dorsal nucleus in man. Arch Neurol 4:355–364.

Loh ML, Reynolds MG, Vattikuti S, Gerbing RB, Alonzo TA, Carlson E, Cheng JW, Lee CM, Lange BJ, Meshinchi S, Children's Cancer Group (2004) PTPN11 mutations in pediatric patients with acute myeloid leukemia: results from the Children's Cancer Group. Leukemia 18:1831–1834.

López-Bendito G, Sánchez-Alcañiz JA, Pla R, Borrell V, Picó E, Valdeolmillos M, Marín O (2008) Chemokine signaling controls intracortical migration and final distribution of GABAergic interneurons. Journal of Neuroscience 28:1613–1624.

Lukaszewicz A, Savatier P, Cortay V, Giroud P, Huissoud C, Berland M, Kennedy H, Dehay C (2005) G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex. Neuron 47:353–364.

Lukaszewicz A, Savatier P, Cortay V, Kennedy H, Dehay C (2002) Contrasting effects of basic fibroblast growth factor and neurotrophin 3 on cell cycle kinetics of mouse cortical stem cells. Journal of Neuroscience 22:6610–6622.

Maffei A, Nataraj K, Nelson SB & Turrigiano GG (2006) Potentiation of cortical inhibition by visual deprivation. Nature 443, 81–84.

Malatesta P, Hartfuss E, Götz M (2000) Isolation of radial glial cells by fluorescent- activated cell sorting reveals a neuronal lineage. Development 127:5253–5263.

Mallamaci A, Stoykova A (2006) Gene networks controlling early cerebral cortex arealization. Eur J Neurosci 23:847–856.

201 Marchi M, D'Antoni A, Formentini I, Parra R, Brambilla R, Ratto GM, Costa M (2008) The N-terminal domain of ERK1 accounts for the functional differences with ERK2. PLoS ONE 3:e3873.

Marchi M, Parra R, Costa M, Ratto GM (2010) Localization and trafficking of fluorescently tagged ERK1 and ERK2. Methods Mol Biol 661:287–301.

Marin O, Rubenstein JL (2001) A long, remarkable journey: tangential migration in the telencephalon. Nat Rev Neurosci 2:780–790.

Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, Wu C (2004) Interneurons of the neocortical inhibitory system. Nat Rev Neurosci 5:793–807.

Marshall CR et al. (2008) Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet 82:477–488.

Marshall M (1995) Interactions between Ras and Raf: key regulatory proteins in cellular transformation. Mol Reprod Dev 42:493–499.

Martínez-Cerdeño V, Noctor SC, Kriegstein AR (2006) The role of intermediate progenitor cells in the evolutionary expansion of the cerebral cortex. Cereb Cortex 16 Suppl 1:i152–i161.

Mason I (2007) Initiation to end point: the multiple roles of fibroblast growth factors in neural development. Nat Rev Neurosci 8:583–596.

Matheny SA, Chen C, Kortum RL, Razidlo GL, Lewis RE, White MA (2004) Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP. Nature 427:256–260.

Matsubara M, Kusubata M, Ishiguro K, Uchida T, Titani K, Taniguchi H (1996) Site- specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. J Biol Chem 271:21108–21113.

Matsui T-A, Murata H, Sowa Y, Sakabe T, Koto K, Horie N, Tsuji Y, Sakai T, Kubo T (2010) A novel MEK1/2 inhibitor induces G1/S cell cycle arrest in human fibrosarcoma cells. Oncol Rep 24:329–333.

202 Mazzucchelli C et al. (2002) Knockout of ERK1 MAP kinase enhances synaptic plasticity in the striatum and facilitates striatal-mediated learning and memory. Neuron 34:807–820.

Mazzucchelli C, Brambilla R (2000) Ras-related and MAPK signalling in neuronal plasticity and memory formation. Cell Mol Life Sci 57:604–611.

McAllister AK (2007) Dynamic aspects of CNS synapse formation. Annu Rev Neurosci 30:425–450.

McConnell SK, Kaznowski CE (1991) Cell cycle dependence of laminar determination in developing neocortex. Science 254:282–285.

McCubrey JA, Steelman LS, Abrams SL, Lee JT, Chang F, Bertrand FE, Navolanic PM, Terrian DM, Franklin RA, D'Assoro AB, Salisbury JL, Mazzarino MC, Stivala F, Libra M (2006) Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Adv Enzyme Regul 46:249–279.

McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EWT, Chang F, Lehmann B, Terrian DM, Milella M, Tafuri A, Stivala F, Libra M, Basecke J, Evangelisti C,

Martelli AM, Franklin RA (2007) Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta 1773:1263–1284.

McGahon B, Maguire C, Kelly A, Lynch MA (1999) Activation of p42 mitogen- activated protein kinase by arachidonic acid and trans-1-amino-cyclopentyl-1,3- dicarboxylate impacts on long-term potentiation in the dentate gyrus in the rat: analysis of age-related changes. NSC 90:1167–1175.

McManus MF, Nasrallah IM, Pancoast MM, Wynshaw-Boris A, Golden JA (2004) Lis1 is necessary for normal non-radial migration of inhibitory interneurons. Am J Pathol 165:775–784.

Meloche S, Pouysségur J (2007) The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene 26:3227– 3239.

203 Métin C, Baudoin J-P, Rakić S, Parnavelas JG (2006) Cell and molecular mechanisms involved in the migration of cortical interneurons. Eur J Neurosci 23:894–900.

Miller MW (1985) Cogeneration of retrogradely labeled corticocortical projection and GABA-immunoreactive local circuit neurons in cerebral cortex. Brain Research 355:187–192.

Minshew NJ, Williams DL (2007) The new neurobiology of autism: cortex, connectivity, and neuronal organization. Arch Neurol 64:945–950.

Mirza B, Vanek VW, Kupensky DT (2004) Pinch-off syndrome: case report and collective review of the literature. Am Surg 70:635–644.

Mitsuhashi T, Aoki Y, Eksioglu YZ, Takahashi T, Bhide PG, Reeves SA, Caviness VS (2001) Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells. Proc Natl Acad Sci USA 98:6435–6440.

Miyoshi G, Fishell G (2011) GABAergic Interneuron Lineages Selectively Sort into Specific Cortical Layers during Early Postnatal Development. Cerebral Cortex 21:845–852.

Mizukami Y, Yoshida KI (1997) Mitogen-activated protein kinase translocates to the nucleus during ischaemia and is activated during reperfusion. Biochem J 323 ( Pt 3):785–790.

Molnár Z, Métin C, Stoykova A, Tarabykin V, Price DJ, Francis F, Meyer G, Dehay C, Kennedy H (2006) Comparative aspects of cerebral cortical development. Eur J Neurosci 23:921–934.

Molyneaux BJ, Arlotta P, Macklis JD (2007a) Molecular development of corticospinal motor neuron circuitry. Novartis Found Symp 288:3–15–discussion15– 20–96–8.

Molyneaux BJ, Arlotta P, Menezes JRL, Macklis JD (2007b) Neuronal subtype specification in the cerebral cortex. Nat Rev Neurosci 8:427–437.

204 Moon AM, Guris DL, Seo J-H, Li L, Hammond J, Talbot A, Imamoto A (2006) Crkl deficiency disrupts Fgf8 signaling in a mouse model of 22q11 deletion syndromes. Developmental Cell 10:71–80.

Moreno-De-Luca D et al. (2010) Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia. Am J Hum Genet 87:618–630.

Morest DK (1970a) The pattern of neurogenesis in the retina of the rat. Z Anat Entwicklungsgesch 131:45–67.

Morest DK (1970b) A study of neurogenesis in the forebrain of opossum pouch young. Z Anat Entwicklungsgesch 130:265–305.

Morrison DK (2001) KSR: a MAPK scaffold of the Ras pathway? Journal of Cell Science 114:1609–1612.

Morrison DK, Davis RJ (2003) Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu Rev Cell Dev Biol 19:91–118.

Morrow T, Song MR, Ghosh A (2001) Sequential specification of neurons and glia by developmentally regulated extracellular factors. Development 128:3585–3594.

Mossink, M. H., van Zon, A., Wiemer, E. A. (2003) Vaults: a ribonucleoprotein particle involved in drug resistance? Oncogene 22, 7458–7467.

Muller M, Obeyesekere M, Mills GB, Ram PT (2008) Network topology determines dynamics of the mammalian MAPK1,2 signaling network: bifan motif regulation of C-Raf and B-Raf isoforms by FGFR and MC1R. FASEB J 22:1393–1403.

Murphy LO, Blenis J (2006) MAPK signal specificity: the right place at the right time. Trends in Biochemical Sciences 31:268–275.

Murphy LO, Smith S, Chen R-H, Fingar DC, Blenis J (2002) Molecular interpretation of ERK signal duration by immediate early gene products. Nature Cell Biology 4:556–564.

Murphy M, Reid K, Ford M, Furness JB, Bartlett PF (1994) FGF2 regulates proliferation of neural crest cells, with subsequent neuronal differentiation regulated

205 by LIF or related factors. Development 120:3519–3528.

Musante L, Kehl HG, Majewski F, Meinecke P, Schweiger S, Gillessen-Kaesbach G, Wieczorek D, Hinkel GK, Tinschert S, Hoeltzenbein M, Ropers H-H, Kalscheuer VM (2003) Spectrum of mutations in PTPN11 and genotype-phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome. Eur J Hum Genet 11:201–206.

Myers AP, Corson LB, Rossant J, Baker JC (2004) Characterization of mouse Rsk4 as an inhibitor of fibroblast growth factor-RAS-extracellular signal-regulated kinase signaling. Mol Cell Biol 24:4255–4266.

Nakamura T, Gulick J, Pratt R, Robbins J (2009) Noonan syndrome is associated with enhanced pERK activity, the repression of which can prevent craniofacial malformations. Proceedings of the National Academy of Sciences 106:15436–15441.

Naruse M, Nakahira E, Miyata T, Hitoshi S, Ikenaka K, Bansal R (2006) Induction of oligodendrocyte progenitors in dorsal forebrain by intraventricular microinjection of FGF-2. Developmental Biology 297:262–273.

Nekrasova T, Shive C, Gao Y, Kawamura K, Guardia R, Landreth G, Forsthuber TG (2005) ERK1-deficient mice show normal T cell effector function and are highly susceptible to experimental autoimmune encephalomyelitis. J Immunol 175:2374– 2380.

Newbern J, Zhong J, Wickramasinghe RS, Li X, Wu Y, Samuels I, Cherosky N, Karlo JC, O'Loughlin B, Wikenheiser J, Gargesha M, Doughman YQ, Charron J, Ginty DD, Watanabe M, Saitta SC, Snider WD, Landreth GE (2008) Mouse and human phenotypes indicate a critical conserved role for ERK2 signaling in neural crest development. Proceedings of the National Academy of Sciences 105:17115– 17120.

Nguyen A, Burack WR, Stock JL, Kortum R, Chaika OV, Afkarian M, Muller WJ, Murphy KM, Morrison DK, Lewis RE, McNeish J, Shaw AS (2002) Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo. Mol Cell Biol 22:3035–3045.

Nguyen L, Besson A, Heng JI-T, Schuurmans C, Teboul L, Parras C, Philpott A, Roberts JM, Guillemot F (2006) p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex. Genes & Development 20:1511–

206 1524.

Nguyen TT, Scimeca JC, Filloux C, Peraldi P, Carpentier JL, Van Obberghen E (1993) Co-regulation of the mitogen-activated protein kinase, extracellular signal- regulated kinase 1, and the 90-kDa ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotrophic factor, nerve growth factor, and the mitogenic factor, epidermal growth factor. J Biol Chem 268:9803–9810.

Nieto M, Monuki ES, Tang H, Imitola J, Haubst N, Khoury SJ, Cunningham J, Götz M, Walsh CA (2004) Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex. J Comp Neurol 479:168–180.

Niihori T et al. (2006) Germline KRAS and BRAF mutations in cardio-facio- cutaneous syndrome. Nat Genet 38:294–296.

Niihori T, Aoki Y, Ohashi H, Kurosawa K, Kondoh T, Ishikiriyama S, Kawame H, Kamasaki H, Yamanaka T, Takada F, Nishio K, Sakurai M, Tamai H, Nagashima T, Suzuki Y, Kure S, Fujii K, Imaizumi M, Matsubara Y (2005) Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia. J Hum Genet 50:192–202.

Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409:714–720.

Noctor SC, Flint AC, Weissman TA, Wong WS, Clinton BK, Kriegstein AR (2002) Dividing precursor cells of the embryonic cortical ventricular zone have morphological and molecular characteristics of radial glia. Journal of Neuroscience 22:3161–3173.

Noor A et al. (2010) Disruption at the PTCHD1 Locus on Xp22.11 in Autism spectrum disorder and intellectual disability. Sci Transl Med 2:49ra68.

O'Leary DD, Sahara S (2008) Genetic regulation of arealization of the neocortex. Curr Opin Neurobiol 18:90–100.

O'Leary DDM, Chou S-J, Sahara S (2007) Area patterning of the mammalian cortex. Neuron 56:252–269.

207 O'Neill E, Rushworth L, Baccarini M, Kolch W (2004) Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science 306:2267– 2270.

Obermeier A, Bradshaw RA, Seedorf K, Choidas A, Schlessinger J, Ullrich A (1994) Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma. EMBO J 13:1585–1590.

Ohkubo Y, Chiang C, Rubenstein JLR (2002) Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles. NSC 111:1–17.

Ohnuma S, Philpott A, Harris WA (2001) Cell cycle and cell fate in the nervous system. Curr Opin Neurobiol 11:66–73.

Ohnuma S, Philpott A, Wang K, Holt CE, Harris WA (1999) p27Xic1, a Cdk inhibitor, promotes the determination of glial cells in Xenopus retina. Cell 99:499– 510.

Ohnuma S-I, Hopper S, Wang KC, Philpott A, Harris WA (2002) Co-ordinating retinal histogenesis: early cell cycle exit enhances early cell fate determination in the Xenopus retina. Development 129:2435–2446.

Ohtsuka T, Sakamoto M, Guillemot F, Kageyama R (2001) Roles of the basic helix- loop-helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain. J Biol Chem 276:30467–30474.

Ortiz J, Harris HW, Guitart X, Terwilliger RZ, Haycock JW, Nestler EJ (1995) Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine. J Neurosci 15:1285– 1297.

Ozaki K, Miyazaki S, Tanimura S, Kohno M (2005) Efficient suppression of FGF-2- induced ERK activation by the cooperative interaction among mammalian Sprouty isoforms. J Cell Science 118; 5861-5871.

Pagès G, Guérin S, Grall D, Bonino F, Smith A, Anjuere F, Auberger P, Pouysségur J (1999) Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice.

208 Science 286:1374–1377.

Pagès G, Lenormand P, L'Allemain G, Chambard JC, Meloche S, Pouysségur J (1993) Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90:8319–8323.

Paquin A, Hordo C, Kaplan DR, Miller FD (2009) Costello syndrome H-Ras alleles regulate cortical development. Developmental Biology 330:440–451.

Paul LK, Brown WS, Adolphs R, Tyszka JM, Richards LJ, Mukherjee P, Sherr EH (2007) Agenesis of the corpus callosum: genetic, developmental and functional aspects of connectivity. Nat Rev Neurosci 8:287–299.

Pearson BJ, Doe CQ (2004) Specification of temporal identity in the developing nervous system. Annu Rev Cell Dev Biol 20:619–647.

Pearson LL, Castle BE, Kehry MR (2001) CD40-mediated signaling in monocytic cells: up-regulation of tumor necrosis factor receptor-associated factor mRNAs and activation of mitogen-activated protein kinase signaling pathways. Int Immunol 13:273–283.

Peduzzi JD (1988) Genesis of GABA-immunoreactive neurons in the ferret visual cortex. J Neurosci 8:920–931.

Pelech S (2006) Dimerization in protein kinase signaling. J Biol 5:12. Pereira PM, Schneider A, Pannetier S, Heron D, Hanauer A (2010) Coffin-Lowry syndrome. Eur J Hum Genet 18:627–633.

Perez JA, Clinton SM, Turner CA, Watson SJ, Akil H (2009) A new role for FGF2 as an endogenous inhibitor of anxiety. Journal of Neuroscience 29:6379–6387.

Petersen PH, Zou K, Krauss S, Zhong W (2004) Continuing role for mouse Numb and Numbl in maintaining progenitor cells during cortical neurogenesis. Nat Neurosci 7:803–811.

Petilla Interneuron Nomenclature Group et al. (2008) Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nat Rev

209 Neurosci 9:557–568.

Pierce KL, Tohgo A, Ahn S, Field ME, Luttrell LM, Lefkowitz RJ (2001) Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding. J Biol Chem 276:23155–23160.

Pinto D et al. (2010) Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466:368–372.

Pla R, Borrell V, Flames N, Marín O (2006) Layer acquisition by cortical GABAergic interneurons is independent of Reelin signaling. Journal of Neuroscience 26:6924– 6934.

Pleasure SJ, Anderson S, Hevner R, Bagri A, Marin O, Lowenstein DH, Rubenstein JL (2000) Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons. Neuron 28:727–740.

Poirier R, Jacquot S, Vaillend C, Soutthiphong AA, Libbey M, Davis S, Laroche S, Hanauer A, Welzl H, Lipp HP, Wolfer DP (2007) Deletion of the Coffin-Lowry syndrome gene Rsk2 in mice is associated with impaired spatial learning and reduced control of exploratory behavior. Behav Genet 37:31–50.

Pontious A, Kowalczyk T, Englund C, Hevner RF (2008) Role of intermediate progenitor cells in cerebral cortex development. Dev Neurosci 30:24–32.

Posada J, Cooper JA (1992a) Molecular signal integration. Interplay between serine, threonine, and tyrosine phosphorylation. Mol Biol Cell 3:583–592.

Posada J, Cooper JA (1992b) Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science 255:212–215.

Pouysségur J, Volmat V, Lenormand P (2002) Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem Pharmacol 64:755–763.

Pozzo-Miller LD, Gottschalk W, Zhang L, McDermott K, Du J, Gopalakrishnan R, Oho C, Sheng ZH, Lu B (1999) Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF

210 knockout mice. Journal of Neuroscience 19:4972–4983.

Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, Temple S (2000) Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells. Neuron 28:69–80. Quinn JC, Molinek M, Martynoga BS, Zaki PA, Faedo A, Bulfone A, Hevner RF, West JD, Price DJ (2007) Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism. Developmental Biology 302:50–65.

Rakic P (1974) Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183:425–427.

Rakic P (1988) Specification of cerebral cortical areas. Science 241:170–176.

Rakic P, Caviness VS (1995) Cortical development: view from neurological mutants two decades later. Neuron 14:1101–1104.

Rash BG, Grove EA (2006) Area and layer patterning in the developing cerebral cortex. Curr Opin Neurobiol 16:25–34.

Rash BG, Lim HD, Breunig JJ, Vaccarino FM (2011) FGF Signaling Expands Embryonic Cortical Surface Area by Regulating Notch-Dependent Neurogenesis. Journal of Neuroscience 31:15604–15617.

Rauch A, Pfeiffer RA, Leipold G, Singer H, Tigges M, Hofbeck M (1999) A novel 22q11.2 microdeletion in DiGeorge syndrome. Am J Hum Genet 64:659–666.

Repasky GA, Chenette EJ, Der CJ (2004) Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol 14:639–647.

Riva MA, Molteni R, Bedogni F, Racagni G, Fumagalli F (2005) Emerging role of the FGF system in psychiatric disorders. Trends in Pharmacological Sciences 26:228– 231.

Roberson ED, English JD, Sweatt JD (1996) A biochemist's view of long-term potentiation. Learn Mem 3:1–24.

211 Roberts EC, Shapiro PS, Nahreini TS, Pagès G, Pouysségur J, Ahn NG (2002) Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis. Mol Cell Biol 22:7226–7241.

Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, Cruz MS, McCormick F, Rauen KA (2006) Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 311:1287–1290.

Roelfsema JH, Peters DJM (2007) Rubinstein-Taybi syndrome: clinical and molecular overview. Expert Rev Mol Med 9:1–16.

Roovers K, Davey G, Zhu X, Bottazzi ME, Assoian RK (1999) Alpha5beta1 integrin controls cyclin D1 expression by sustaining mitogen-activated protein kinase activity in growth factor-treated cells. Mol Biol Cell 10:3197–3204.

Rosenblum K, Futter M, Jones M, Hulme EC, Bliss TV (2000) ERKI/II regulation by the muscarinic acetylcholine receptors in neurons. Journal of Neuroscience 20:977– 985.

Ross ME, Walsh CA (2001) Human brain malformations and their lessons for neuronal migration. Annu Rev Neurosci 24:1041–1070.

Ross SE, Greenberg ME, Stiles CD (2003) Basic helix-loop-helix factors in cortical development. Neuron 39:13–25.

Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J (2004) Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc Natl Acad Sci USA 101:13489–13494.

Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68:320– 344.

Roy K, Kuznicki K, Wu Q, Sun Z, Bock D, Schütz G, Vranich N, Monaghan AP (2004) The Tlx gene regulates the timing of neurogenesis in the cortex. Journal of Neuroscience 24:8333–8345.

212 Rubenstein JLR (2010) Annual Research Review: Development of the cerebral cortex: implications for neurodevelopmental disorders. Journal of Child Psychology and Psychiatry 52:339–355.

Rubinfeld H, Seger R (2005) The ERK cascade: a prototype of MAPK signaling. Mol Biotechnol 31:151–174.

Ryan AK et al. (1997) Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study. J Med Genet 34:798– 804.

Saba-El-Leil MK, Vella FDJ, Vernay B, Voisin L, Chen L, Labrecque N, Ang S-L, Meloche S (2003) An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep 4:964–968.

Sahara S, O'Leary DDM (2009) Fgf10 regulates transition period of cortical stem cell differentiation to radial glia controlling generation of neurons and basal progenitors. Neuron 63:48–62.

Saitta SC, McGrath JM, Mensch H, Shaikh TH, Zackai EH, Emanuel BS (1999) A 22q11.2 deletion that excludes UFD1L and CDC45L in a patient with conotruncal and craniofacial defects. Am J Hum Genet 65:562–566.

Samuels IS, Karlo JC, Faruzzi AN, Pickering K, Herrup K, Sweatt JD, Saitta SC, Landreth GE (2008) Deletion of ERK2 mitogen-activated protein kinase identifies its key roles in cortical neurogenesis and cognitive function. Journal of Neuroscience 28:6983–6995.

Sanders SJ et al. (2011) Multiple Recurrent De Novo CNVs, Including Duplications of the 7q11.23 Williams Syndrome Region, Are Strongly Associated with Autism. Neuron 70:863–885.

Satoh Y, Endo S, Ikeda T, Yamada K, Ito M, Kuroki M, Hiramoto T, Imamura O, Kobayashi Y, Watanabe Y, Itohara S, Takishima K (2007) Extracellular signal- regulated kinase 2 (ERK2) knockdown mice show deficits in long-term memory; ERK2 has a specific function in learning and memory. Journal of Neuroscience 27:10765–10776.

213 Satoh Y, Endo S, Nakata T, Kobayashi Y, Yamada K, Ikeda T, Takeuchi A, Hiramoto T, Watanabe Y, Kazama T (2011). ERK2 Contributes to the Control of Social Behaviors in Mice. Journal of Neuroscience 31(33):11953–11967.

Scambler PJ (2000) The 22q11 deletion syndromes. Hum Mol Genet 9:2421–2426. Scardigli R, Bäumer N, Gruss P, Guillemot F, Le Roux I (2003) Direct and concentration-dependent regulation of the proneural gene Neurogenin2 by Pax6. Development 130:3269–3281.

Scearce-Levie K, Roberson ED, Gerstein H, Cholfin JA, Mandiyan VS, Shah NM, Rubenstein JLR, Mucke L (2008) Abnormal social behaviors in mice lacking Fgf17. Genes Brain Behav 7:344–354.

Schaaf CP, Sabo A, Sakai Y, Crosby J, Muzny D, Hawes A, Lewis L, Akbar H, Varghese R, Boerwinkle E, Gibbs RA, Zoghbi HY (2011) Oligogenic heterozygosity in individuals with high-functioning autism spectrum disorders. Hum Mol Genet 20:3366–3375.

Schaaf CP, Zoghbi HY (2011) Solving the autism puzzle a few pieces at a time. Neuron 70:806–808.

Schaeffer HJ, Catling AD, Eblen ST, Collier LS, Krauss A, Weber MJ (1998) MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade. Science 281:1668–1671.

Schaeffer HJ, Weber MJ (1999) Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19:2435–2444.

Schneider A, Mehmood T, Pannetier S, Hanauer A (2011) Altered ERK/MAPK signaling in the hippocampus of the mrsk2_KO mouse model of Coffin-Lowry syndrome. Journal of Neurochemistry.

Schuurmans C, Guillemot F (2002) Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr Opin Neurobiol 12:26–34.

Sebat J et al. (2004) Large-scale copy number polymorphism in the human genome. Science 305:525–528.

214 Sebat J et al. (2007) Strong association of de novo copy number mutations with autism. Science 316:445–449.

Sebolt-Leopold JS, Herrera R (2004) Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer 4:937–947.

Selcher JC, Nekrasova T, Paylor R, Landreth GE, Sweatt JD (2001) Mice lacking the ERK1 isoform of MAP kinase are unimpaired in emotional learning. Learn Mem 8:11–19.

Selcher JC, Weeber EJ, Christian J, Nekrasova T, Landreth GE, Sweatt JD (2003) A role for ERK MAP kinase in physiologic temporal integration in hippocampal area CA1. Learn Mem 10:26–39.

Seuntjens E, Nityanandam A, Miquelajauregui A, Debruyn J, Stryjewska A, Goebbels S, Nave K-A, Huylebroeck D, Tarabykin V (2009a) Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors. Nature Publishing Group 12:1373–1380.

Seuntjens E, Umans L, Zwijsen A, Sampaolesi M, Verfaillie CM, Huylebroeck D (2009b) Transforming Growth Factor type beta and Smad family signaling in stem cell function. Cytokine Growth Factor Rev 20:449–458.

Shaikh TH, Kurahashi H, Saitta SC, O'Hare AM, Hu P, Roe BA, Driscoll DA, McDonald-McGinn DM, Zackai EH, Budarf ML, Emanuel BS (2000) Chromosome 22-specific low copy repeats and the 22q11.2 deletion syndrome: genomic organization and deletion endpoint analysis. Hum Mol Genet 9:489–501.

Sharrocks AD (2001) The ETS-domain transcription factor family. Nat Rev Mol Cell Biol 2:827–837.

Shen Q, Wang Y, Dimos JT, Fasano CA, Phoenix TN, Lemischka IR, Ivanova NB, Stifani S, Morrisey EE, Temple S (2006) The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat Neurosci 9:743–751.

Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1- phase progression. Genes & Development 13:1501–1512.

215 Shin DM, Korada S, Raballo R, Shashikant CS, Simeone A, Taylor JR, Vaccarino F (2004a) Loss of glutamatergic pyramidal neurons in frontal and temporal cortex resulting from attenuation of FGFR1 signaling is associated with spontaneous hyperactivity in mice. Journal of Neuroscience 24:2247–2258.

Shin HY, Schwartz EA, Bizios R, Gerritsen ME (2004b) Receptor-mediated basic fibroblast growth factor signaling regulates cyclic pressure-induced human endothelial cell proliferation. Endothelium 11:285–291.

Shinohara M, Mikhailov AV, Aguirre-Ghiso JA, Rieder CL (2006) Extracellular signal-regulated kinase 1/2 activity is not required in mammalian cells during late G2 for timely entry into or exit from mitosis. Mol Biol Cell 17:5227–5240.

Siegenthaler JA, Pleasure SJ (2010) There's no place like home for a neural stem cell. Cell Stem Cell 7:141–143. Silva AJ, Frankland PW, Marowitz Z, Friedman E, Laszlo GS, Cioffi D, Jacks T, Bourtchuladze R, Lazlo G (1997) A mouse model for the learning and memory deficits associated with neurofibromatosis type I. Nat Genet 15:281–284.

Silver J, Lorenz SE, Wahlsten D, Coughlin J (1982) Axonal guidance during development of the great cerebral commissures: descriptive and experimental studies, in vivo, on the role of preformed glial pathways. J Comp Neurol 210:10–29.

Silverman E, Frödin M, Gammeltoft S, Maller JL (2004) Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells. Mol Cell Biol 24:10573–10583.

Smart IH (1973) Proliferative characteristics of the ependymal layer during the early development of the mouse neocortex: a pilot study based on recording the number, location and plane of cleavage of mitotic figures. Journal of Anatomy 116:67–91.

Smith KM, Ohkubo Y, Maragnoli ME, Rasin M-R, Schwartz ML, Sestan N, Vaccarino FM (2006a) Midline radial glia translocation and corpus callosum formation require FGF signaling. Nat Neurosci 9:787–797.

Smith TG, Karlsson M, Lunn JS, Eblaghie MC, Keenan ID, Farrell ER, Tickle C, Storey KG, Keyse SM (2006b) Negative feedback predominates over cross-regulation to control ERK MAPK activity in response to FGF signalling in embryos. FEBS Lett 580:4242–4245.

216 Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, Ye Q, Lobo JM, She Y, Osman I, Golub TR, Sebolt-Leopold J, Sellers WR, Rosen N (2006) BRAF mutation predicts sensitivity to MEK inhibition. Nature 439:358–362.

Solit DB, She Y, Lobo J, Kris MG, Scher HI, Rosen N, Sirotnak FM (2005) Pulsatile administration of the epidermal growth factor receptor inhibitor gefitinib is significantly more effective than continuous dosing for sensitizing tumors to paclitaxel. Clin Cancer Res 11:1983–1989.

Somogyi P, Tamás G, Lujan R, Buhl EH (1998) Salient features of synaptic organisation in the cerebral cortex. Brain Res Brain Res Rev 26:113–135.

Song M-R, Ghosh A (2004) FGF2-induced chromatin remodeling regulates CNTF- mediated gene expression and astrocyte differentiation. Nat Neurosci 7:229–235.

Stankiewicz P, Lupski JR (2002) Molecular-evolutionary mechanisms for genomic disorders. Curr Opin Genet Dev 12:312–319.

Storm EE, Garel S, Borello U, Hébert JM, Martinez S, McConnell SK, Martin GR, Rubenstein JLR (2006) Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers. Development 133:1831–1844.

Stoykova A, Fritsch R, Walther C, Gruss P (1996) Forebrain patterning defects in Small eye mutant mice. Development 122:3453–3465.

Stumm R, Höllt V (2007) CXC chemokine receptor 4 regulates neuronal migration and axonal pathfinding in the developing nervous system: implications for neuronal regeneration in the adult brain. J Mol Endocrinol 38:377–382.

Stumm RK, Zhou C, Ara T, Lazarini F, Dubois-Dalcq M, Nagasawa T, Höllt V, Schulz S (2003) CXCR4 regulates interneuron migration in the developing neocortex. Journal of Neuroscience 23:5123–5130.

Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, Fan G, Greenberg ME (2001) Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104:365–376.

217 Sur M, Rubenstein JLR (2005) Patterning and plasticity of the cerebral cortex. Science 310:805–810.

Sweatt JD (2001) The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. Journal of Neurochemistry 76:1– 10. Takahashi T, Nowakowski RS, Caviness VS (1995a) The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J Neurosci 15:6046–6057. Takahashi T, Nowakowski RS, Caviness VS (1995b) Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall. J Neurosci 15:6058–6068.

Tan PB, Kim SK (1999) Signaling specificity: the RTK/RAS/MAP kinase pathway in metazoans. Trends Genet 15:145–149.

Tarabykin V, Stoykova A, Usman N, Gruss P (2001) Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression. Development 128:1983–1993.

Tartaglia M et al. (2007) Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet 39:75–79.

Tartaglia M, Martinelli S, Iavarone I, Cazzaniga G, Spinelli M, Giarin E, Petrangeli V, Carta C, Masetti R, Aricò M, Locatelli F, Basso G, Sorcini M, Pession A, Biondi A (2005) Somatic PTPN11 mutations in childhood acute myeloid leukaemia. Br J Haematol 129:333–339.

Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H, van der Burgt I, Crosby AH, Ion A, Jeffery S, Kalidas K, Patton MA, Kucherlapati RS, Gelb BD (2001) Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 29:465–468.

Tarui T, Takahashi T, Nowakowski RS, Hayes NL, Bhide PG, Caviness VS (2005) Overexpression of p27 Kip 1, probability of cell cycle exit, and laminar destination of neocortical neurons. Cereb Cortex 15:1343–1355.

Tavormina PL, Bellus GA, Webster MK, Bamshad MJ, Fraley AE, McIntosh I, Szabo J, Jiang W, Jabs EW, Wilcox WR, Wasmuth JJ, Donoghue DJ, Thompson LM, Francomano CA (1999) A novel skeletal dysplasia with developmental delay and

218 acanthosis nigricans is caused by a Lys650Met mutation in the fibroblast growth factor receptor 3 gene. Am J Hum Genet 64:722–731.

Teis D, Wunderlich W, Huber LA (2002) Localization of the MP1-MAPK scaffold complex to endosomes is mediated by p14 and required for signal transduction. Developmental Cell 3:803–814.

Temtamy SA, Miller JD, Dorst JP, Hussels-Maumenee I, Salinas C, Lacassie Y, Kenyon KR (1975) The Coffin-Lowry syndrome: a simply inherited trait comprising mental retardation, faciodigital anomalies and skeletal involvement. Birth Defects Orig Artic Ser 11:133–152.

Terauchi A, Johnson-Venkatesh EM, Toth AB, Javed D, Sutton MA, Umemori H (2010) Distinct FGFs promote differentiation of excitatory and inhibitory synapses. Nature 465:783–787.

Therrien M, Michaud NR, Rubin GM, Morrison DK (1996) KSR modulates signal propagation within the MAPK cascade. Genes & Development 10:2684–2695.

Thiels E, Kanterewicz BI, Norman ED, Trzaskos JM, Klann E (2002) Long-term depression in the adult hippocampus in vivo involves activation of extracellular signal-regulated kinase and phosphorylation of Elk-1. Journal of Neuroscience 22:2054–2062.

Thomson RE, Pellicano F, Iwata T (2007) Fibroblast growth factor receptor 3 kinase domain mutation increases cortical progenitor proliferation via mitogen-activated protein kinase activation. Journal of Neurochemistry 100:1565–1578.

Thornton GK, Woods CG (2009) Primary microcephaly: do all roads lead to Rome? Trends Genet 25:501–510.

Tidyman WE, Rauen KA (2009) The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev 19:230–236.

Tirone F (2001) The gene PC3(TIS21/BTG2), prototype member of the PC3/BTG/TOB family: regulator in control of cell growth, differentiation, and DNA repair? J Cell Physiol 187:155–165.

219 Torii S, Yamamoto T, Tsuchiya Y, Nishida E (2006) ERK MAP kinase in G cell cycle progression and cancer. Cancer Sci 97:697–702.

Traverse S, Gomez N, Paterson H, Marshall C, Cohen P (1992) Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J 288 ( Pt 2):351–355.

Traverse S, Seedorf K, Paterson H, Marshall CJ, Cohen P, Ullrich A (1994) EGF triggers neuronal differentiation of PC12 cells that overexpress the EGF receptor. Curr Biol 4:694–701.

Trommsdorff M, Borg JP, Margolis B, Herz J (1998) Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein. J Biol Chem 273:33556–33560.

Turner CA, Calvo N, Frost DO, Akil H, Watson SJ (2008a) The fibroblast growth factor system is downregulated following social defeat. Neuroscience Letters 430:147–150.

Turner CA, Gula EL, Taylor LP, Watson SJ, Akil H (2008b) Antidepressant-like effects of intracerebroventricular FGF2 in rats. Brain Research 1224:63–68.

Umemori H, Linhoff MW, Ornitz DM, Sanes JR (2004) FGF22 and its close relatives are presynaptic organizing molecules in the mammalian brain. Cell 118:257–270.

Vaccarino FM, Grigorenko EL, Smith KM, Stevens HE (2009) Regulation of cerebral cortical size and neuron number by fibroblast growth factors: implications for autism. J Autism Dev Disord 39:511–520.

Valcanis H, Tan S-S (2003) Layer specification of transplanted interneurons in developing mouse neocortex. Journal of Neuroscience 23:5113–5122.

Vantaggiato C, Formentini I, Bondanza A, Bonini C, Naldini L, Brambilla R (2006) ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially. J Biol 5:14.

220 Vara H, Onofri F, Benfenati F, Sassoè-Pognetto M, Giustetto M (2009) ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I. Proceedings of the National Academy of Sciences 106:9872–9877.

Verhoeven W, Egger J, Brunner H, de Leeuw N (2011) A patient with a de novo distal 22q11.2 microdeletion and anxiety disorder. Am J Med Genet A 155A:392– 397.

Vernon AE, Devine C, Philpott A (2003) The cdk inhibitor p27Xic1 is required for differentiation of primary neurones in Xenopus. Development 130:85–92.

Vernon AE, Philpott A (2003a) The developmental expression of cell cycle regulators in Xenopus laevis. Gene Expr Patterns 3:179–192.

Vernon AE, Philpott A (2003b) A single cdk inhibitor, p27Xic1, functions beyond cell cycle regulation to promote muscle differentiation in Xenopus. Development 130:71–83.

Vitelli F, Morishima M, Taddei I, Lindsay EA, Baldini A (2002a) Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways. Hum Mol Genet 11:915–922.

Vitelli F, Taddei I, Morishima M, Meyers EN, Lindsay EA, Baldini A (2002b) A genetic link between Tbx1 and fibroblast growth factor signaling. Development 129:4605–4611.

Vojtek AB, Hollenberg SM, Cooper JA (1993) Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74:205–214.

Waites CL, Craig AM, Garner CC (2005) Mechanisms of vertebrate synaptogenesis. Annu Rev Neurosci 28:251–274.

Wang Y, Dye CA, Sohal V, Long JE, Estrada RC, Roztocil T, Lufkin T, Deisseroth K, Baraban SC, Rubenstein JLR (2010) Dlx5 and Dlx6 regulate the development of parvalbumin-expressing cortical interneurons. Journal of Neuroscience 30:5334– 5345.

221 Weber JD, Hu W, Jefcoat SC, Raben DM, Baldassare JJ (1997) Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27. J Biol Chem 272:32966–32971.

Weiss LA et al. (2008) Association between microdeletion and microduplication at 16p11.2 and autism. N Engl J Med 358:667–675.

Wichterle H, Turnbull DH, Nery S, Fishell G, Alvarez-Buylla A (2001) In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain. Development 128:3759–3771.

Williams VC, Lucas J, Babcock MA, Gutmann DH, Korf B, Maria BL (2009) Neurofibromatosis type 1 revisited. Pediatrics 123:124–133.

Winder DG, Martin KC, Muzzio IA, Rohrer D, Chruscinski A, Kobilka B, Kandel ER (1999) ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors. Neuron 24:715–726.

Wonders CP, Taylor L, Welagen J, Mbata IC, Xiang JZ, Anderson SA (2008) A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence. Developmental Biology 314:127–136.

Wright JH, Munar E, Jameson DR, Andreassen PR, Margolis RL, Seger R, Krebs EG (1999) Mitogen-activated protein kinase kinase activity is required for the G(2)/M transition of the cell cycle in mammalian fibroblasts. Proc Natl Acad Sci USA 96:11335–11340.

Wu CJ, Qian X, O'Rourke DM (1999) Sustained mitogen-activated protein kinase activation is induced by transforming erbB receptor complexes. DNA Cell Biol 18:731–741.

Yabut O, Renfro A, Niu S, Swann JW, Marín O, D'Arcangelo G (2007) Abnormal laminar position and dendrite development of interneurons in the reeler forebrain. Brain Research 1140:75–83.

Yamamoto T, Ebisuya M, Ashida F, Okamoto K, Yonehara S, Nishida E (2006) Continuous ERK activation downregulates antiproliferative genes throughout G1

222 phase to allow cell-cycle progression. Curr Biol 16:1171–1182.

Yang SH, Whitmarsh AJ, Davis RJ, Sharrocks AD (1998a) Differential targeting of MAP kinases to the ETS-domain transcription factor Elk-1. EMBO J 17:1740–1749.

Yang SH, Yates PR, Whitmarsh AJ, Davis RJ, Sharrocks AD (1998b) The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif. Mol Cell Biol 18:710–720.

Yao Y, Li W, Wu J, Germann UA, Su MSS, Kuida K, Boucher DM (2003) Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation. Proc Natl Acad Sci USA 100:12759–12764.

Yizhar, O (2011) Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477, 171–178.

Yntema HG, van den Helm B, Kissing J, van Duijnhoven G, Poppelaars F, Chelly J, Moraine C, Fryns JP, Hamel BC, Heilbronner H, Pander HJ, Brunner HG, Ropers HH, Cremers FP, van Bokhoven H (1999) A novel ribosomal S6-kinase (RSK4; RPS6KA6) is commonly deleted in patients with complex X-linked mental retardation. Genomics 62:332–343.

Yoon S, Seger R (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24:21–44.

Yordy JS, Muise-Helmericks RC (2000) Signal transduction and the Ets family of transcription factors. Oncogene 19:6503–6513.

York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ (1998) Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392:622–626.

Yun K, Mantani A, Garel S, Rubenstein J, Israel MA (2004) Id4 regulates neural progenitor proliferation and differentiation in vivo. Development 131:5441–5448.

223 Zecevic N, Chen Y, Filipovic R (2005) Contributions of cortical subventricular zone to the development of the human cerebral cortex. J Comp Neurol 491:109–122.

Zezula J, Casaccia-Bonnefil P, Ezhevsky SA, Osterhout DJ, Levine JM, Dowdy SF, Chao MV, Koff A (2001) p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal. EMBO Rep 2:27–34.

Zimmer C, Tiveron M-C, Bodmer R, Cremer H (2004) Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons. Cereb Cortex 14:1408–1420.

Zindy F, Cunningham JJ, Sherr CJ, Jogal S, Smeyne RJ, Roussel MF (1999) Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin- dependent kinases. Proc Natl Acad Sci USA 96:13462–13467.

224