Cortical Development: Genes and Genetic Abnormalities

Novartis Foundation Symposium 288

CORTICAL DEVELOPMENT: GENES AND GENETIC ABNORMALITIES

CORTICAL DEVELOPMENT: GENES AND GENETIC ABNORMALITIES The Novartis Foundation is an international scientiﬁ c and educational charity (UK Registered Charity No. 313574). Known until September 1997 as the Ciba Foundation, it was established in 1947 by the CIBA company of Basle, which merged with Sandoz in 1996, to form Novartis. The Foundation operates independently in London under English trust law. It was formally opened on 22 June 1949. The Foundation promotes the study and general knowledge of science and in particular encourages international co-operation in scientiﬁ c research. To this end, it organizes internationally acclaimed meetings (typically eight symposia and allied open meetings and 15–20 discussion meetings each year) and publishes eight books per year featuring the presented papers and discussions from the symposia. Although primarily an operational rather than a grant-making foundation, it awards bursaries to young scientists to attend the symposia and afterwards work with one of the other participants. The Foundation’s headquarters at 41 Portland Place, London W1B 1BN, provide library facilities, open to graduates in science and allied disciplines. Media relations are fostered by regular press conferences and by articles prepared by the Foundation’s Science Writer in Residence. The Foundation offers accommodation and meeting facilities to visiting scientists and their societies. Information on all Foundation activities can be found at http://www.novartisfound.org.uk Novartis Foundation Symposium 288

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Symposium on Cortical development: genes and genetic abnormalities, held at the Novartis Foundation, London, 6–8 February 2007

Editors: Gregory Bock (Organizer) and Jamie Goode

This symposium is based on a proposal made by Zoltán Molnár

John G. Parnavelas Chair’s introduction 1

Bradley J. Molyneaux, Paola Arlotta and Jeffrey D. Macklis Molecular development of corticospinal motor neuron circuitry 3 Discussion 15

Gordon Fishell Perspectives on the developmental origins of cortical interneuron diversity 21 Discussion 35

Pasko Rakic, Kazue Hashimoto-Torii and Matthew R. Sarkisian Genetic determinants of neuronal migration in the cerebral cortex 45 Discussion 53

Stephen C. Noctor, Veronica Martinez-Cerdeño and Arnold R. Kriegstein Neural stem and progenitor cells in cortical development 59 Discussion 73

Teresa H. Chae and Christopher A. Walsh Genes that control the size of the cerebral cortex 79 Discussion 91

General discussion I 96

Vicki Hammond, Joanne Britto, Eva So, Holly Cate and Seong-Seng Tan Control of cortical neuron layering: lessons from mouse chimeras 99 Discussion 108 v vi CONTENTS Fujio Murakami, Daisuke Tanaka, Mitsutoshi Yanagida and Emi Yamazaki Intracortical multidirectional migration of cortical interneurons 116 Discussion 125

Libing Zhou, Fadel Tissir and André M. Gofﬁ net The atypical cadherin Celsr3 regulates the development of the axonal blueprint 130 Discussion 134

Dennis D. M. O’Leary , Shen-Ju Chou, Tadashi Hamasaki, Setsuko Sahara, Akihide Takeuchi, Sandrine Thuret and Axel Leingärtner Regulation of laminar and area patterning of mammalian neocortex and behavioural implications 141 Discussion 159

Jeremy A. Cholﬁ n and John L. R. Rubenstein Genetic regulation of prefrontal cortex development and function 165 Discussion 173

Henry Kennedy, Rodney Douglas, Kenneth Knoblauch and Colette Dehay Self-organization and pattern formation in primate cortical networks 178 Discussion 195

Nobuhiko Yamamoto, Takuro Maruyama, Naofumi Uesaka, Yasufumi Hayano, Makoto Takemoto and Akito Yamada Molecular mechanisms of thalamocortical axon targeting 199 Discussion 208

Zoltán Molnár, Anna Hoerder-Suabedissen, Wei Zhi Wang, Jamin DeProto, Kay Davies, Sheena Lee, Erin C. Jacobs, Anthony T. Campagnoni, Ole Paulsen, Maria Carmen Piñon and Amanda F. P. Cheung Genes involved in the formation of the earliest cortical circuits 212 Discussion 224

Michael Piper, Amber-Lee S. Dawson, Charlotta Lindwall, Guy Barry, Céline Plachez and Linda J. Richards Emx and Nﬁ genes regulate cortical development and axon guidance in the telencephalon 230 Discussion 242 CONTENTS vii Paul J. Harrison Schizophrenia susceptibility genes and their neurodevelopmental implications: focus on neuregulin 1 246 Discussion 255

Peter B. Crino Focal brain malformations: a spectrum of disorders along the mTOR cascade 260 Discussion 272

Final discussion 276

Index of contributors 282

Subject index 284

Participants

Colin Blakemore Medical Research Council, 20 Park Crescent, London W1B 1AL, UK

Jamel Chelly Institut Cochin (IC), Département de Génétique et Pathologie Moléculaire, Equipe de Génétique et Physiopathologie des Retards Mentaux, 24, rue du Faubourg St Jacques, F-75014 Paris, France

Peter B. Crino Hospital of the University of Pennsylvania, Department of Neu- rology, 3 West Gates Building, 3400 Spruce Street, Philadelphia, PA 19104, USA

Kay Davies MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK

Gordon Fishell NYU School of Medicine, Smilow Neuroscience Program and the Department of Cell Biology, 5th Smilow Bldg, 522 First Avenue, New York, NY 10016, USA

Gaëlle Friocourt-Masse Laboratoire de Genetique Moléculaire et d’Histocom- patibilité, INSERM U613, 46 rue Félix Le Dantec, 29200 Brest, France

André M. Gofﬁ net University of Louvain Medical School, Developmental Neu- robiology Unit, 73 avenue E. Mounier, Box DENE 7382, B-1200 Brussels, Belgium

François Guillemot Division of Molecular Neurobiology, NIMR, Mill Hill, London NW7 1AA, UK

Paul J. Harrison Neurosciences Building, Department of Psychiatry, Warneford Hospital, University of Oxford, Oxford OX3 7JX, UK

Robert Hevner Department of Pathology, University of Washington, Harbor- view, Box 359791, Seattle, WA 98104-9791, USA

David Keays St Anne’s College, University of Oxford, Oxford OX2 6HS, UK ix x PARTICIPANTS Henry Kennedy Stem Cell and Brain Research Institute, INSERM U846, 18 avenue Doyen Lépine, F-69675 Bron Cedex, France

Arnold R. Kriegstein Institute for Regeneration Medicine, UCSF School of Medicine, 513 Parnassus Avenue, HSW 1201, Campus Box 0525, San Francisco, CA 94143-0525, USA

Jeffrey D. Macklis Harvard Medical School, Massachusetts General Hospital, MGH-HMS Center for Nervous System Repair, 50 Blossom Street, EDR 410, Boston, MA 02114, USA

Antonello Mallamaci Laboratory of Cerebral Cortex Development, SISSA— Neurobiology Sector, Research Area of Basovizza, Box Q1, Floor 1, Basovizza S. S. 14, Km 163.5, I-34012 Trieste, Italy

Zoltán Molnár Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX, UK

Fujio Murakami Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan

Dennis D. M. O’Leary Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

John G. Parnavelas (Chair) Department of Anatomy and Developmental Biology, Anatomy Building, University College London, Gower Street, London WC1E 6BT, UK

David Price Biomedical Sciences, Hugh Robson Building, University of Edinburgh, 1 George Square, Edinburgh EH8 9XD, UK

Pasko Rakic Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, SHM C303, PO Box 208001, New Haven, CT 06520-8001, USA

Linda J. Richards The University of Queensland, School of Biomedical Sciences and The Queensland Brain Institute, Department of Anatomy and Developmental Biology, Otto Hirschfeld Building (81), Brisbane, QLD 4072, Australia

John L. R. Rubenstein University of California at San Francisco, Department of Psychiatry, 1550 4th Street, 2nd Floor South, Room RH 284C, Box 2611, San Francisco, CA 94143-2611, USA PARTICIPANTS xi Anastassia Stoykova Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany

Seong-Seng Tan Howard Florey Institute, Cnr. Royal Parade and Grattan Street, University of Melbourne, Parkville VIC 3010, Australia

Christopher A. Walsh Division of Genetics, Children’s Hospital Boston, Howard Hughes Medical Institute, BIDMC, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA

Adam Wilkins BioEssays, 10/11 Tredgold Lane, Napier Street, Cambridge CB1 1HN, UK

Michael Wilson University of New Mexico, Department of Neurosciences, Basic Medical Sciences Building, Albuquerque, NM 87131-5223, USA

Nobuhiko Yamamoto Neuroscience Laboratories, Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan

Chair’s introduction

John G. Parnavelas

Department of Anatomy and Developmental Biolog y, University College London, London WC1E 6BT, UK

This is the third symposium on cortical development to be held at the Novartis Foundation in recent years. The fi rst, entitled Development of the cerebral cortex, was held in the winter of 1994. The second meeting, which focused on Evolutionary development of the cerebral cortex, took place in April 1999. The present symposium, proposed by Dr Zoltán Molnár, will concentrate on genes and genetic abnormalities of the developing cortex. The fi rst meeting in 1994 took place only a couple of years after the publication of the seminal papers by Antonio Simeone, Edoardo Boncinelli and colleagues in Naples on the nested expression domains of four homeobox genes in the developing forebrain (Simeone et al 1992a, b). At that meeting, there was a solitary paper by Edoardo Boncinelli on the expression of these Emx and Otx genes in the developing forebrain that included some faint suggestions about their roles in the developing cortex (Boncinelli et al 1995). In the second meeting, fi ve years later, we heard a bit more about genes with papers by Edoardo Boncinelli and John Rubenstein focusing on the genetic control of regional identity in the developing cortex (Boncinelli et al 2000, Rubenstein et al 2000). Interest in genetic and molecular mechanisms involved in the different phases of cortical formation and in genes associated with human cortical malformations has never been greater. The availability of state-of-the-art molecular techniques, transgenic mouse models and much improved techniques for imaging the human brain have been behind the explosion in the amount and complexity of new information to emerge in the past 10 years, and especially since the turn of the century. The purpose of this symposium is to bring together people with different backgrounds, both from the basic and clinical sciences, to review and discuss the present state of knowledge about the development of the normal cerebral cortex and of cortical developmental abnormalities associated with human disorders such as mental retardation, epilepsy and schizophrenia. As the title of the symposium suggests, the emphasis will be on genes and genetic abnormalities. This volume shows how much progress has been made in the fi eld since the last Novartis Foundation symposium on cortical development in 1999. 1 2 PARNAVELAS References Boncinelli E, Gulisano M, Spada F, Broccoli V 1995 Emx and Otx gene expression in the developing mouse brain. In: Development of the cerebral cortex (Ciba Found Symp 193). Wiley, Chichester, p 100–126 Boncinelli E, Mallamaci A, Muzio L 2000 Genetic control of regional identity in the developing vertebrate forebrain. In: Evolutionary developmental biology of the cerebral cortex (Novartis Found Symp 228). Wiley, Chichester, p 53–66 Rubenstein JLR 2000 Intrinsic and extrinsic control of cortical development. In: Evolutionary developmental biology of the cerebral cortex (Novartis Found Symp 228). Wiley, Chichester, p 67–82 Simeone A, Acampora D, Gulisano M, Stornaiuolo A, Boncinelli E 1992a Nested expression domains of four homeobox genes in developing rostral brain. Nature 358:687–690 Simeone A, Gulisano M, Acampora D, Stornaiuolo A, Rambaldi M, Boncinelli E 1992b Two vertebrate homeobox genes related to Drosophila empty spiracles gene are expressed in the embryonic cerebral cortex. EMBO J 11:2541–2550 Molecular development of corticospinal motor neuron circuitry1

Bradley J. Molyneaux, Paola Arlotta2 and Jeffrey D. Macklis3

MGH-HMS Center for Nervous System Repair, Departments of Neurosurgery and Neurolog y, and Program in Neuroscience, Harvard Medical School; Nayef Al-Rodhan Laboratories, Massachusetts General Hospital, and Department of Stem Cell and Regenerative Biolog y and Harvard Stem Cell Institute, Harvard University, Boston, MA 02114, USA

Abstract. The organization, function and evolution of the brain depends centrally on the precise development of a wide diversity of distinct neuronal subtypes. Furthermore, given the heterogeneity of neuronal subtypes within the CNS and the complexity of their connections, attempts to functionally repair circuitry will require a detailed understanding of the molecular controls over differentiation, connectivity and survival of specifi c lineages. Toward these goals, we recently identifi ed developmentally regulated transcrip- tional programmes for specifi c lineages of long-distance neocortical projection neurons as they develop in vivo (in particular, for corticospinal motor neurons; CSMN). We purifi ed CSMN, a clinically important population of neocortical projection neurons, at distinct stages of development in vivo, and compared their gene expression to that of two other pure populations of neocortical projection neurons. We identifi ed novel and largely uncharacterized genes that are instructive for CSMN development and implicated in key developmental processes. These include Fez f2 (also known as Fezl), a regulator of subcerebral projection neuron identity, and Ctip2 (also known as Bcl11b), a regulator of the fasciculation, outgrowth and pathfi nding of CSMN axonal projections to the spinal cord. Loss-of-function and gain-of-function analysis for multiple identifi ed genes reveal programmes of combinatorial molecular controls over the precise development of key neocortical and other forebrain projection neuron populations that elucidate organization and function of the forebrain, and that might be manipulated toward functional cellular repair of complex brain circuitry. 2007 Cortical development: genes and genetic abnormalities. Wiley, Chichester (Novartis Foundation Symposium 288) p 3–20

1 The text of this review is modifi ed and expanded from one section of a broader review pub- lished in Nature Reviews Neuroscience (Molyneaux et al 2007). 2 Current address: Center for Regenerative Medicine, Department of Neurosurgery, Massachu- setts General Hospital, Harvard Medical School, Boston, MA 02114 USA. 3 This paper was presented at the symposium by Jeffrey Macklis, to whom correspondence should be addressed. 3 4 MOLYNEAUX ET AL The neuronal diversity and precise connectivity of the neocortex underlies high- level cognition, integration and motor control. In addition, previous data from our laboratory demonstrate that new neurons can be added to adult neocortical circuitry from transplanted neural precursors or via manipulation of endogenous precursors in situ (Sheen & Macklis 1995, Magavi et al 2000, Shin et al 2000, Fricker-Gates et al 2002, Chen et al 2004). These data indicate that cellular repair of damaged neocortical and neocortical output circuitry (e.g. corticospinal circuitry) might be possible, if controls over specifi c lineage differentiation are under- stood. Given the heterogeneity of neuronal subtypes in the CNS and the complexity of their connections, a deeper understanding of CNS organization, function and evolution, as well as attempts to functionally repair circuitry will require detailed knowledge of the molecular controls over the differentiation, connectivity and survival of specifi c lineages. Within the neocortex, some of the basic mechanisms that control general neuronal specifi cation, migration and connectivity during development have been identifi ed (Bertrand et al 2002, Marin & Rubenstein 2003, Guillemot 2005). More recently, the discovery of genes that have layer and neuronal subtype specifi city within the neocortex has made it possible to investigate the mechanisms underly- ing the specifi cation of individual projection neuron subtypes (Molyneaux et al 2007). Corticospinal motor neurons (CSMN), also known as upper motor neurons, form the basis of voluntary motor control in humans, and are therefore a subtype of particular interest for investigation. Located in the cerebral cortex, CSMN extend extremely long axons that synapse on lower motor neurons and interneurons in the spinal cord. Clinically, they are an important population, as they degenerate in motor neuron degenerative diseases such as amyotrophic lateral sclerosis (ALS), and their injury contributes to the loss of motor function following spinal cord injury. The anatomical and morphological development of CSMN has been extensively characterized (Jones et al 1982, Terashima 1995, Bareyre et al 2005), but strategies to repair or prevent degeneration of CSMN are limited by a lack of understanding of the molecular controls over CSMN development, including neuron type-specifi c differentiation, survival, and connectivity (Arlotta et al 2005). CSMN are one subtype within the broader class of subcerebral projection neurons (O’Leary & Koester 1993, Arlotta et al 2005), which are defi ned by pos- sessing axons that project below the cerebrum to targets in the spinal cord or brainstem, including the tectum, red nucleus and pons (Wise & Jones 1977, Killackey et al 1989, Legg et al 1989, O’Leary & Koester 1993, Arlotta et al 2005, Molnar & Cheung 2006). Among the different types of cortical projection neurons, subcerebral projection neurons are an ideal model population for studying the mechanisms of subtype specifi cation within the neocortex. They are a discrete, CSMN SPECIFICATION AND DIFFERENTIATION 5 readily identifi able, prototypical projection neuron population, located within layer Vb of neocortex (Fig. 1). After being born in the germinal zone, all subcerebral projection neurons migrate to layer Vb and extend a primary axon through the internal capsule, cerebral peduncle, and pyramidal tract toward the spinal cord. Secondary collaterals sprout from the primary axon only after it has passed other targets such as the superior colliculus and pons (O’Leary & Terashima 1988). Inappropriate connections are later eliminated, leaving different populations of subcerebral projection neurons with specifi c patterns of connectivity. For example, CSMN in sensorimotor cortex project axons to the pons and spinal cord, while corticotectal neurons in the visual cortex project axons to the rostral pons and superior colliculus (O’Leary & Terashima 1988, Schreyer & Jones 1988, O’Leary & Koester 1993). Given this common pattern of initial development, many of the genes controlling early specifi cation and differentiation are likely to be shared among the

FIG. 1. Subcerebral projection neurons are located in layer V of neocortex. CSMN are one subpopulation of projection neurons within the broader class of neurons termed subcerebral projection neurons. All subcerebral projection neurons can be retrogradely labelled via an injection of DiI into the cerebral peduncle. This coronal section of a P14 mouse brain shows the distribution of DiI-labelled subcerebral projection neurons within the neocortex. Scale bar: 250 µm. Reproduced from Arlotta et al (2005). 6 MOLYNEAUX ET AL different types of subcerebral projection neurons (Arlotta et al 2005). Over the last several years, substantial progress has been made towards understanding the molecular controls over the specifi cation and development of CSMN and other subcerebral projection neurons, including the identifi cation of a large number of genes expressed with varying degrees of specifi city in CSMN (Arlotta et al 2005), the identifi cation of Fez f2 as a critical regulator of subcerebral projection neuron identity (Chen et al 2005a, b, Molyneaux et al 2005), and the identifi cation of Ctip2 as a regulator of the fasciculation, outgrowth, and pathfi nding of CSMN axonal projections to the spinal cord (Arlotta et al 2005).

Progressive specifi cation of projection neurons The recent identifi cation of a large number of subcerebral and CSMN specifi c genes has enabled an expanding effort to decipher the programmes controlling CSMN development (Arlotta et al 2005). This was achieved through the compari- son of purifi ed neuronal subtypes by microarray analysis (Arlotta et al 2005). The expression patterns of the identifi ed genes indicate that the fate specifi cation and differentiation of subcerebral projection neurons in general, and CSMN in particular, is likely directed by a combinatorial code of transcription factors and other molecules. These molecules are expressed in a pattern that uniquely identifi es CSMN. For example, a small number of CSMN genes appear restricted to sensorimotor cortex (e.g. Diap3, Igfbp4 and Crim1), suggesting that they distinguish CSMN from other subcerebral projection neurons of layer V (Arlotta et al 2005). Other genes are expressed across the full extent of layer V (e.g. Ctip2, Encephalopsin, Fez f2, Clim1, Pcp4 and S100a10) suggestive of restriction to most subcerebral projection neurons (Arlotta et al 2005). Thus far, the functions of only a few of these genes have been reported, but these studies are already revealing key roles for these genes in subcerebral specifi cation and differentiation (Weimann et al 1999, Arlotta et al 2005, Chen et al 2005a, b, Molyneaux et al 2005). The expression pattern for only a small number of genes has been examined in detail via a combination of retrograde labelling and immunocytochemistry. These include: Ctip2, which is expressed at high levels in subcerebral neurons of layer V and at much lower levels in corticothalamic neurons of layer VI (Arlotta et al 2005); Scip, which is primarily expressed in subcerebral projection neurons of layer V, in addition to lower levels of expression in neurons of layers II–III (Frantz et al 1994b); Otx1, which is expressed in 40–50% of subcerebral neurons as well as a number of cells in layer VI (Weimann et al 1999); Er81, which is expressed in cortico–cortical as well as subcerebral projection neurons of layer V (Hevner et al 2003); and Nfh, which is expressed in subcerebral projection neurons of layer V (Voelker et al 2004). It will be important to perform similar careful investigation of neuronal subtype expression by immunocytochemistry for each of the other CSMN SPECIFICATION AND DIFFERENTIATION 7 genes that have been identifi ed as preferentially expressed in CSMN and other subcerebral projection neurons by microarray or in situ hybridization. While signifi cant progress has been made in identifying markers of post-mitotic subcerebral projection neurons once they have reached the cortical plate, it is unclear whether the same markers can be used to identify progenitors that will give rise to subcerebral projection neurons, or whether such lineage committed progenitors even exist. A number of neuronal subtype specifi c genes are expressed in what appears to be subpopulations of cells within the VZ and SVZ, where they might label progenitors or early post-mitotic neurons of that same neuronal subtype. For example, both Fez f2 and Otx1 are expressed in the VZ prior to and during the generation of subcerebral projection neurons, and are later expressed in post-mitotic subcerebral projection neurons. However, it is important to be extremely cautious about inferring that a gene plays a role in the specifi cation of subcerebral projection neurons at the progenitor level based on restricted expression that is later observed in a particular neuronal subtype; it is entirely possible that the gene has two independent functions during development (Alvarez-Bolado et al 1995). This is best illustrated by considering Lhx2, which is expressed in the VZ and SVZ prior to and during the generation of upper layers and is also expressed in post-mitotic neurons of the upper layers. The fi nding that the loss of Lhx2 results in the absence of neurons of all layers (Bulchand et al 2001, Monuki et al 2001) suggests that Lhx2 likely has two functions during development: in the ventricular zone, it is required to establish the neocortical identity of progenitors of all layers, while later in development it might control more specifi c aspects of upper layer differentiation. Therefore, further study is required for each of the subcerebral projection neuron specifi c genes to defi ne the relationship between progenitors and post-mitotic neurons expressing the same genes.

CSMN and subcerebral projection neuron specifi cation Investigations into the function of some of the subcerebral projection neuron- specifi c layer and subtype-restricted genes are starting to provide insight into how CSMN and subcerebral projection neurons are specifi ed in the neocortex. Brn1 and Brn2 are two genes that appear to play a role early in subcerebral projection neuron specifi cation and migration. They are expressed primarily in neurons of layers II–V and are involved in directing the differentiation and migration of neurons within these layers (McEvilly et al 2002, Sugitani et al 2002, Hevner et al 2003). The Brn1/Brn2 double knockouts possess decreased numbers of neurons of layers II–V, and those that are born exhibit abnormalities in migration, arresting in the VZ/SVZ (McEvilly et al 2002, Sugitani et al 2002). Additionally, some markers of upper layer neurons are expressed in these mutants, while others (e.g. 8 MOLYNEAUX ET AL mSorLa) are absent, suggesting abnormalities in differentiation. In contrast, Tle4 and Tbr1 expressing neurons of layer VI appear to form and migrate normally into the cortical plate in the absence of Brn1 and Brn2 (McEvilly et al 2002, Sugitani et al 2002). Further analysis of Brn1/Brn2 mutants with recently identifi ed markers is needed to illuminate precisely which subtypes of neurons are affected in the absence of Brn1 and Brn2. Fez f2 (Fezl/Zfp312), a putative transcription factor that is expressed in all subcerebral projection neurons from the early stages of development through to adulthood (Inoue et al 2004, Arlotta et al 2005), was recently found to be required for the specifi cation of all subcerebral projection neurons (Chen et al 2005a, b, Molyneaux et al 2005). In the absence of Fez f2 function in null mutant mice, the entire population of subcerebral projection neurons is absent, and there are no projections from the cerebral cortex to either the spinal cord or the brainstem (Fig. 2; Chen et al 2005a, Molyneaux et al 2005). Layer VI neurons and subplate

FIG. 2. Fezl −/− cortex lacks all CSMN and subcerebral projection neurons, indicated by CTIP2 expression. (A) Wild-type neocortex in coronal section, showing subcerebral projection neurons in layer V (arrows) intensely expressing CTIP2. (B) Boxed area in (A). (E) Coronal section matching that in (A) from a Fezl −/− brain showing absence of all CTIP2-positive neurons from layer V (arrows in F). (F) Boxed area in E. (C) Coronal section of wild-type brain showing normal CTIP2 staining across the entire mediolateral extent of layer V, in piriform cortex (Pir), and in striatum (Str). (D) Boxed area in C. (G) Section matching that in (C) from a Fezl −/− brain showing lack of subcerebral projection neurons from layer V, while CTIP2- expressing neurons in piriform cortex and striatum are not affected. (H) Boxed area in (G). Scale bars: (A, C, E, G) 200 µm; (B, F) 20 µm; (D, H) 50 µm. Reproduced from Molyneaux et al (2005). CSMN SPECIFICATION AND DIFFERENTIATION 9 neurons, which express Fez f2 at lower levels than subcerebral projection neurons, exhibit disorganization and abnormalities in gene expression, but are less affected (Hirata et al 2004, Chen et al 2005a, Molyneaux et al 2005). In contrast, upper layer pyramidal neurons are born correctly and appear normal (Chen et al 2005a, Molyneaux et al 2005). Importantly, without Fez f2, neocortical progenitors still produce similar numbers of layer V neurons (Chen et al 2005a, Molyneaux et al 2005), but morphologically they appear to be an expansion of layer VI, instead of exhibiting the distinctive appearance of layer V subcerebral projection neurons. Additionally, alkaline phosphatase expression from the Fez f2 locus in null mutants labels an enlarged anterior commissure, further suggesting that a different type of projection neuron is generated in place of subcerebral projection neurons (Chen et al 2005a). Thus, Fez f2 does not affect the ability of progenitors to generate glutamatergic neurons that position themselves in layer V; it likely acts to direct the next step in the programme of specifi cation, defi ning the characteristics of a subcerebral projection neuron. As additional support for Fez f2 in directing subcerebral projection neuron specifi cation, the overexpression of Fez f2 is suffi cient to induce the birth of entirely new deep layer projection neurons that express Ctip2 and Tbr1 and extend axons through the internal capsule (Chen et al 2005b, Moly- neaux et al 2005). A second set of genes has been identifi ed that control later aspects of subcerebral projection neuron development, likely acting downstream of factors such as Fez f2. These include the transcription factors Ctip2 and Otx1. Ctip2 is a transcription factor that is expressed at high levels in all subcerebral projection neurons while it is not expressed within callosal projection neurons of layer V (Arlotta et al 2005). However, prior loss of function experiments in vivo highlight an important role for CTIP2 in cell-type specifi cation in the immune system (Wakabayashi et al 2003) and suggested that it might play a similar role in the development of CSMN and other subcerebral projection neurons. In the absence of Ctip2, subcerebral projection neuron axons exhibit defects in fasciculation, outgrowth, and pathfi nding, with decreased numbers of axons reaching the brainstem (Fig. 3; Arlotta et al 2005). In addition, reduced Ctip2 expression in Ctip2 heterozygous mice results in abnormal developmental pruning of corticospinal axons (Arlotta et al 2005). These experiments identifi ed Ctip2 as a critical regulator of subcerebral axon extension and the refi nement of collaterals as these neurons mature. Another key transcription factor known to play a role in the target choice of subcerebral projection neurons is Otx1. This protein is expressed in putative deep layer progenitors in the VZ, exhibiting decreasing levels of expression in the VZ during the generation of upper layer neurons (Weimann et al 1999, Inoue et al 2004). As deep layer projection neurons mature, localization of OTX1 shifts from the cytoplasm to the nucleus, indicating a fi ne regulation of the activity of this protein (Frantz et al 1994a, Weimann et al 1999). Postnatally, within layer V, Otx1 10 MOLYNEAUX ET AL

FIG. 3. CSMN in Ctip2 −/− mice display pathfi nding defects and fail to extend to the spinal cord. (A, E) Schematic representations of sagittal views of the brain and proximal spinal cord in wild type and Ctip2 −/− mice, respectively, showing the location of CSMN somas in the cortex (triangles) and their axonal projections toward the spinal cord (lines). (B–D and F–H) Photo- micrographs of boxed areas in (A) and (E) respectively. (B, F) Axonal projections by subcerebral projection neurons showing that (B) P0 wild-type axons are organized in typical axon fascicles (arrows), but (F) matched P0 Ctip2 −/− null mutant axons are very disorganized, non- fasciculated (arrow), and display axonal projections that deviate from the normal pathway and extend to ectopic targets (arrowhead). (C, G) The same axonal fi bres as (B) and (F), at a more caudal location. (C) Wild type axons are highly organized in tight bundles of fi bres progressing unidirectionally toward the pons (arrow), while (G) Ctip2 −/− axons are strikingly reduced in number with many individual fi bres extending to ectopic sites (arrowheads). (D, H) Photo- micrographic montages demonstrating (D) that P0 wild-type axons are abundant through the pons (arrows), and have already reached the pyramidal decussation entering the spinal cord (arrowhead). (H) A much smaller number of axons in Ctip2−/− mice enter the pons (arrows) and no axons extend into the medulla or reach the pyramidal decussation. Scale bars, 100 µm. Reproduced from Arlotta et al (2005). is expressed in 40–50% of subcerebral neurons, primarily those within the visual cortex, while it is not expressed in callosal neurons (Weimann et al 1999). Mice lacking the gene for Otx1 have defects in the development of corticotectal projection neurons. Without Otx1, corticotectal projection neurons maintain an axon to the spinal cord and caudal pontine nuclei, collaterals that are only appropriate for CSMN and are normally eliminated by corticotectal projection neurons CSMN SPECIFICATION AND DIFFERENTIATION 11 (Weimann et al 1999), indicating that Otx1 might play a later role in subcerebral projection neuron development than Fez f2 and Ctip2, controlling the refi nement and pruning of axonal collaterals. Additional axon outgrowth and guidance molecules, such as IGF1 and RYK, have been described that play a role in the extension and guidance of subcerebral projection neuron axons to targets in the brainstem and spinal cord (Liu et al 2005, Harel & Strittmatter 2006, Ozdinler & Macklis 2006). While a comprehensive understanding of the role played by additional subcerebral projection neuron-specifi c genes still awaits substantial experimental work in vivo, based on the data available thus far, a possible model for the generation of subcerebral projection neurons can be put forward that requires sequential steps of progressive differentiation. We propose that the concerted function of FoxG1, Lhx2, Pax6 and Emx2 fi rst gives progenitors neocortical potential, setting the stage for the generation of multiple classes of glutamatergic projection neurons. It is conceivable that radial glia progenitors might then express a sequential series of transcription factors that are maintained in intermediate progenitors and post- mitotic neurons, imparting subtype identity. Thus, during the generation of subcerebral projection neurons, genes such as Brn1 and Brn2 might act on partially specifi ed progenitors to determine aspects of laminar identity as individual subtypes of pyramidal neurons are generated. Fez f2 then specifi es the subcerebral projection neuron lineage within a layer (i.e. layer V), enabling the development of the molecular, morphological, and anatomical projection properties of subcerebral projection neurons. Finally, once this cascade is initiated, the expression of genes such as Ctip2 and Otx1, which govern subcerebral axonal outgrowth and target selection, would act to establish the precise connectivity and later morphological features of subcerebral projection neurons. The direct relationships between these transcription factors and the many, yet functionally uncharacterized, genes that act in the cascade of subcerebral projection neuron development remain to be determined. Together, these molecules comprise the fi rst elements of the molecular programme that drives the anatomical model of subcerebral projection neuron development described more than a decade ago (O’Leary & Koester 1993).

CSMN repopulation and repair of corticospinal circuitry Knowledge of the molecular controls over CSMN development and survival might provide new approaches for the treatment of traumatic spinal cord injury and CSMN degenerative diseases such as ALS, primary lateral sclerosis (PLS) and hereditary spastic paraplegia (HSP). These signals could be manipulated to enhance the survival of degenerating CSMN or induce the re-growth of axons after injury. Alternatively, manipulation of the genes that control the progressive 12 MOLYNEAUX ET AL differentiation of progenitors along the CSMN lineage could potentially be used to induce the formation of CSMN from neural precursors in vitro and ultimately in vivo. Previous experiments from our laboratory have shown that distinct subtypes of neurons can be induced to undergo neurogenesis from immature precursors, even in the normally inhibitory environment of the adult mammalian neocortex (Magavi et al 2000, Scharff et al 2000). More recent data indicate that endogenous neural precursors can also be induced to differentiate into CSMN in vivo (Chen et al 2004). However, the number of newborn CSMN is quite low, and only a small percentage of the newborn neurons survive long enough to establish permanent connections to distal targets in the spinal cord (Chen et al 2004). While these experiments demonstrate that new CSMN can be added to the normally inhibitory environment of the adult cortex and extend long-distance projections to the spinal cord, it is likely that the number of functional newborn CSMN could be substantially increased, and functional recovery might be effected by improved understanding of controls over CSMN specifi cation, survival, and connectivity at the molecular level. Such information might enhance survival of developing CSMN and improve the ability of CSMN to connect to proper targets, which in turn might enhance functional connectivity and circuit repair. One gene that might be a candidate for manipulation to generate CSMN from progenitors is Fez f2. Fez f2 is expressed in the ventricular zone during the generation of deep layer neurons, and its expression is maintained in postmitotic neurons of layers V and VI. As development progresses, the expression of Fez f2 in progenitors decreases and disappears by the time upper layer neurons are generated (Hirata et al 2004, Inoue et al 2004, Molyneaux et al 2005). As described above, it is required for the specifi cation of CSMN and all other subcerebral projection neurons. Overexpression of Fez f2 in progenitors soon after the generation of layer V and VI is completed (i.e. in progenitors that give rise to layer IV neurons) is suffi cient to at least partially override this restriction and induce later-stage progenitors to produce neurons with some molecular and anatomical features of earlier-born neurons (Molyneaux et al 2005). Further analysis of Fez f2 transfected neurons with additional positive and negative markers of subcerebral projection neurons is needed to determine the extent of Fez f2’s effect on neuronal phenotype. Interestingly, Fez f2 appears to, at least in part, affect progenitor plasticity late in development, as suggested by the fact that forced expression of Fez f2 in E17 progenitors results in the generation of upper layer neurons that inappropriately express Tbr1 at a higher frequency than is normally observed in upper layer neurons and extend axonal projections to the pons (a feature of deep layer neurons) (Chen et al 2005b). However, Fez f2-overexpressing neurons migrate to the layer appropriate for this late birth date instead of layer V, suggesting some limitations to the ability of Fez f2 alone to alter the fate of late-stage progenitors. While CSMN SPECIFICATION AND DIFFERENTIATION 13 it remains to be elucidated to what extent these late born neurons change their identity in response to Fez f2 overexpression, together, these experiments indicate that cortical progenitors might be more plastic than previously suspected, even late in neurogenesis, if manipulated by the appropriate control molecules. In the future, such neocortical progenitor plasticity might be manipulated much later in development, or even during adulthood, in order to repair damaged cortical circuitry.

Acknowledgements This work was partially supported by grants from the NIH (NS45523, NS49553, NS41590), the Harvard Stem Cell Institute, the Spastic Paraplegia Foundation, and the ALS Association to JDM. PA was partially supported by a Claﬂ in Distinguished Scholar Award and a grant from the ALS Association. BJM was supported by the Harvard M.S.T.P. and the United Sydney Association.

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DISCUSSION O’Leary: What is the source of IGF1 that is infl uencing the axon outgrowth in vivo? Macklis: We don’t know this for sure by direct experiments. From other work we know that it is highly expressed in the forebrain itself during these periods. We have done experiments placing the beads at different distances and angles from the growing axons. It doesn’t seem to be an axon growth guidance molecule. It is active at the cell body as an activator, saying ‘grow out’. IGF1 is present, but we think it takes more than simply the presence of IGF for axon outgrowth. There are a number of molecules that are specifi cally expressed on CSMN that are IGF binders and amassing proteins. It is combinatorial. The growth factor can be present, but if a neuron population pulls it in or excludes it or doesn’t listen to it, this can lead to differential effects. Tan: It is wonderful that your techniques are discriminative enough to give you such specifi c genes in the layer V neurons. Is this corroborated by the Paul Allen brain atlas? Are there any other parts of the brain that express these genes? Macklis: The cerebral cortex is as complex as the rest of the body. Ctip2 can be expressed in T cell progenitors; this doesn’t worry me. It is different in the forebrain than in T cells. Similarly, Ctip2 is expressed in cerebral cortex only in subcerebral neurons. It is expressed in striatum, but in different combinatorial interactions with areal specifi c genes. If you look at the Allen brain atlas, genes that in the cortex are specifi c can be expressed in different populations. You might say that this means that they are non-specifi c. I disagree: within that area they are highly specifi c. This is just the complexity of the forebrain. The same transcrip- tional cassette is being used for different populations. In work we hope to be publishing soon we have shown that the same Ctip2 that is by our reckoning 16 MOLYNEAUX ET AL relatively downstream at the cortical plate projection outgrowth control for subcerebral neurons, is high upstream for medium spiny neurons. Without its function, medium spiny neurons don’t develop correctly, patch matrix organization is eliminated, and repulsive cues that allow the striatum to sit at that complex place and exclude migratory populations, and allow piercing corticospinal neurons is totally eliminated. We have found repeatedly that the same gene can be in a couple of places, but in combination with another it is specifi c. Also, some of these genes are on at X dose in one population and half X dose in another population. This seems to be quite important as well. Molnár: At the beginning of your talk you mentioned that you were working backwards. You identifi ed a specifi c projection neuron population by exploiting the fact that you can access them through their specifi c target sites. Then you mentioned that you do this at P8, P6, P3 and E18.5. So how far did you go back? Surely your ultimate goal must be to understand what cocktail of transcription factors has to be present when these cells are born. Macklis: The earliest that we could get at their projections was E17 injection, E18. Then we take those ‘identity’ genes that are on from E18 and not in any other populations, and then work back by in situ or immunofl ourescence to fi nd, for example, that Fez f2 was on at E11 in ventricular zone progenitors. Then we can march forwards. We use the strategy of marching back to get a foothold. Molnár: Are these steps of target selection, somatodendritic morphology or physiological signatures specifi ed at the very early stages when the cells leave the ventricular zone, or are they imposed slightly later? Macklis: There will be lots of inputs and tweaks. We think that certain elements are there right from the progenitors. We will fi nd this from our fate mapping and some of our other work. People talk about the neural stem cells (what we prefer to term ‘precursors’) as though they are all one population, but I don’t think this is true. There are some which are already partially fate restricted. We think it goes way back, but within the population there may be precursors that really don’t want to cross certain lines. There are precursors that want to be a subcortical neuron. They really want to be a corticospinal neuron, but we can modify them more easily to be a corticothalamic neuron. Our working model is that there are dendritic trees of close evolutionary association. Mallamaci: I have a question about Ctip2. In Ctip2 knockouts, are only corticospinal projections impaired, or does the defect also apply to corticotectal projections? Macklis: We came up with this term ‘subcerebral’ projection neurons. These were genes that were on in at least the two populations we looked at as our starting point, in both the corticotectal and corticospinal neurons. Then when we looked more closely they were on in all subcerebral neurons: all the neurons to brainstem