Specification and integration of interneuron subtypes in the neocortex

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Thesis

Reference

Specification and integration of interneuron subtypes in the neocortex

LIMONI, Greta

Abstract

Cortical microcircuit function relies on the coordinated activity of a large diversity of interneuron (IN) subtypes. These cells originate from discrete areas of the subpallium, namely the medial and caudal ganglionic eminences (MGE and CGE) and the preoptic area (POA).

From these sites of origin, INs migrate long distances to reach the developing neocortex and integrate into the cortical microcircuits. Understanding the emergence of their molecular diversity and how they develop in the neocortex will provide important insights on the architecture of the mature circuit in health and disease. In my PhD project, I studied molecular mechanisms controlling the specification, migration and maturation of IN subclasses. In the first study, I aimed at identifying the developmental origin of a specific subclass of cortical IN called neurogliaform cells (NGCs). These cells are recruited by long-range connections, such as interhemispheric and thalamic projections, and are thought to be the effectors of a powerful inhibitory circuit by activating metabotropic GABAB receptors. Using in vivo lineage-tracing in mice, I found that NGCs [...]

LIMONI, Greta. Specification and integration of interneuron subtypes in the neocortex. Thèse de doctorat : Univ. Genève et Lausanne, 2018, no. Neur. 226

DOI : 10.13097/archive-ouverte/unige:107042 URN : urn:nbn:ch:unige-1070423

Available at:

http://archive-ouverte.unige.ch/unige:107042

Disclaimer: layout of this document may differ from the published version.

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Faculté de Psychologie

DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DE PSYCHOLOGIE Professeur Laszlo VUTSKITS, directeur de thèse

Professeur Alexandre DAYER, co-directeur de thèse

TITRE DE LA THESE

SPECIFICATION AND INTEGRATION OF INTERNEURON SUBTYPES IN THE NEOCORTEX

THESE Présentée à la Faculté de Médecine de l’Université de Genève

pour obtenir le grade de Docteure en Neurosciences

par

Greta LIMONI d’Italie

Thèse N° 226 Genève

Editeur ou imprimeur : Université de Genève 2018

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ACKNOWLEDGEMENTS

Once someone said that the biggest achievement in a journey is the people you meet along. I could not agree more with this, and I am proud to say that during these years I have been lucky enough to be surrounded by lovely people, from whom I learnt a lot and who largely contributed to my professional and personal growth.

First and foremost, I would like to thank my supervisors, Prof. Laszlo Vutskits and Prof.

Alexandre Dayer, who gave me the opportunity to pursue my PhD in their labs, for the scientific discussions, overcoming difficulties by my side and always challenging my thinking and capacities. Thanks for the confidence they accorded to me all along these years. I have largely appreciated working with them.

Thanks to Profs. Valérie Castellani, Camilla Bellone and Denis Jabaudon for kindly accepting to be part of this jury, revising the manuscript and sharing their advices.

Thanks to present and former members of Dayer and Vutskits labs, for the fruitful discussions we had and for the everyday work we shared.

In particular, I would love to thank Mathieu, for holding my hand since I have started my work in Geneva. Mathieu walked by my side in my professional growth, sharing his knowledge and beyond, doing his best in teaching me how to be a good scientist (well, I hope he managed it at least in part!). He’s been more than a colleague and mentor, being a wonderful listener, always there to go through my doubts and worries, helping me to find solutions and confidence in myself. A good guide is the one who manages to take out the student’s best, and he definitely did. I would never be able to word my endless gratitude in his regards and this PhD is for sure his achievement as well. I also thank him for sharing breaks, lunches and beers, for laughing at my ridiculous French and thoroughly teaching me war strategies to re-build the Roman Empire.

Thanks Michèle for her great technical support. Thanks for taking care of my psychological wellbeing by feeding me with chocolate and delicious cakes, for sharing morning coffees and always having a good and reassuring word to say. She has been my “lab-mum” and, I am sure, these years would have not felt the same without her.

Thanks Wafae and Christiane for their invaluable technical and managerial help.

Thanks Julien for the efficient bioinformatics support.

Thanks to Foivos from Holtmaat lab for his acknowledgeable help with electrophysiology and nicely collaborating with us on the POA story.

Thanks to past and present members of Kiss and Jabaudon labs, for sharing lab equipment, space and suggestions.

Thanks Esther, for the endless scientific (and not only) discussions we had, for the brainstorming, for sharing knowledge on circuits and genetics and for always giving me

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constructive and thoughtful advices. Thanks also for the countless restoring beers we had after work and for being such a good friend!

Thanks to François, Olivier, Nicolas and Sergei from the Bioimaging Platform for their invaluable support.

Thanks to Sébastien, René and all the technical staff for all their help.

Thanks to Rachel, Claire and Marie-Ange for their never-ending patience with bureaucratic issues.

Thanks to all the people in the department, for the chats in the corridors and always having smiles, jokes and advices to share.

Also, I would like to thank many other people that, although outside the institute, largely shared this journey, kindly going through my worries and breakdowns.

So, thanks to my Friends, Esther, Ugo, Céline, Gaël, Nicolas, Laura, Seble, Julie, Guillaume, Suba, Raphaël, the “Icelandic crew”, the “Dream Team” and all those I am not mentioning here, but to whom I owe a lot. Thanks for the endless worthy and amusing time we spent together and all the (dis)adventure we shared and survived to.

Thanks to Yoko, Catherine, Ulinka and all the participants of Dansehabile, for sharing this incredible human experience and always fulfilling my heart.

Thanks to Rolf, Vladimir, Aurélien, Rudi, Daniela, Paul, Géraldine, Emilio, Marcela, Ramón, Paolo, Aleksey, the Cie Greffe and all the amazing dancers I have the chance to work with. Besides the countless hours of hard training, I thank them for making me part of their artistic world, boosting my creativity and always knowing how to cheer me up without saying a word.

Last but not least, thanks to my Family, for their boundless care, for supporting my life choices and my new challenging adventures. I thank them for teaching me to dare and be brave enough to follow my dreams, even when they cannot ensure a future. Thanks for letting me go and find myself, thanks for always walking by my side when I got lost. Thanks for blindly believing in me, event when I don’t. Thanks for making me the gipsy person I am.

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ABSTRACT

Cortical microcircuit function relies on the coordinated activity of a large diversity of interneuron (IN) subtypes. These cells originate from discrete areas of the subpallium, namely the medial and caudal ganglionic eminences (MGE and CGE) and the preoptic area (POA).

From these sites of origin, INs migrate long distances to reach the developing neocortex and integrate into the cortical microcircuits. Understanding the emergence of their molecular diversity and how they develop in the neocortex will provide important insights on the architecture of the mature circuit in health and disease. In my PhD project, I studied molecular mechanisms controlling the specification, migration and maturation of IN subclasses.

In the first study, I aimed at identifying the developmental origin of a specific subclass of cortical IN called neurogliaform cells (NGCs). These cells are recruited by long-range connections, such as interhemispheric and thalamic projections, and are thought to be the effectors of a powerful inhibitory circuit by activating metabotropic GABAB receptors. Using in vivo lineage-tracing in mice, I found that NGCs originate from a pool of cells located in the POA, co-expressing the transcription factor Hmx3 and the serotonin receptor 3A (HTR3A).

Through a combination of methods, I found that Hmx3-derived HTR3A⁺ cortical IN exhibited the molecular, morphological and electrophysiological profile of NGCs. Overall, these results indicate that NGCs are a distinct class of INs with a unique developmental trajectory.

In the second study, I focused on mechanisms regulating laminar allocation of superficial neocortical INs. In a previous study, we found that HTR3A controls the migration and laminar positioning of superficial cortical INs, but molecular mechanisms remain unknown.

Using a microarray screen on wild-type and Htr3a-KO INs, I identified PlexinA4 (PlxnA4) as a candidate gene possibly acting downstream the Htr3a and specifically upregulated during the phase of cortical plate invasion. Using in vitro and in vivo strategies, I found that PLXNA4 ligand SEMA3A has chemorepulsive effect on PLXNA4⁺/HTR3A⁺ superficial INs and that these effects are mediated by the PLXNA4/NRP1 receptor complex. Interestingly, SEMA3A was found to be secreted by deep layer INs, which do not express the HTR3A. Overall, these results suggest a new guidance mechanism for migrating HTR3A⁺ INs, involving a HTR3A- dependent upregulation of PLXNA4 in superficial cortical INs. These PLXNA4⁺/HTR3A⁺ cells will become gradually sensitive to the repulsive ligand SEMA3A, secreted by deep layer INs, and will preferentially settle into superficial ones.

In the third study, I aimed at characterizing the role of the potassium/chloride cotransporter KCC2 in INs at early developmental stages. KCC2 plays a main role in driving GABAAR-mediated inhibition in the mature cortex, by tuning intracellular chloride concentration. Interestingly, it is upregulated in INs earlier than in excitatory cells. To determine the role of KCC2 in IN development, I aimed to selectively knock-out this cotransporter in cortical INs using cre-lox approach. Analyses of the somatosensory cortices indicated that IN-specific deletion of KCC2 significantly decreased the density of parvalbumin (PV)-expressing cells in recombined mice and non cell-autonomously affected the morphological maturation of pyramidal neurons. These results suggest an important role for KCC2 in the maturation of PV-expressing INs.

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Taken together, studies performed in this PhD thesis provide new insight on molecular mechanisms regulating the specification, migration and laminar allocation of cortical INs.

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RÉSUMÉ

Le bon fonctionnement des microcircuits corticaux est intimement lié à l’activité coordonnée d’un grand nombre de sous-types d’interneurones (INs). Très diverses, ces cellules proviennent de régions précises du subpallium, notamment des éminences ganglionnaires médiane, caudale (MGE, CGE) et de l’aire préoptique (POA). Après leur genèse, les INs parcourent de longues distances avant de rejoindre le néocortex en développement et d’y intégrer les microcircuits corticaux. Pour mieux étudier l’architecture finale de ces microcircuits dans des cerveaux sains ou malades, il est important de comprendre de quelle manière la diversité émerge au sein des interneurones et comment ils parviennent à maturité.

Mon projet de PhD s’est donc axé sur l’étude des mécanismes moléculaires qui contrôlent la spécification, la migration ainsi que la maturation des sous-classes d’INs.

La première étude visait à découvrir l’origine d’une sous-classe d’INs appelés neurogliaformes (NGCs). On pense que ces cellules, en agissant sur les récepteurs métabotropiques GABAB, sont la principale source d’une inhibition corticale « lente ». Sous l’influence d’afférences distantes comme les projections interhémisphériques et thalamiques, ces cellules seraient ainsi les médiatrices d’un circuit inhibiteur puissant. A l’aide de souris transgéniques permettant de suivre des lignées cellulaires, j’ai découvert qu’un groupe de cellules se situant dans la POA est à l’origine des NGCs et qu’elles expriment le facteur de transcription Hmx3 ainsi que le récepteur 3A à la sérotonine (HTR3A). En utilisant des techniques d’histologie et d’enregistrement, j’ai démontré que les INs corticaux HTR3A⁺ qui dérivent de la lignée Hmx3 ont un profil moléculaire, morphologique et electrophysiologique correspondant aux NGCs. Ces résultats indiquent que les NGCs constituent une classe distincte d’INs dont la trajectoire développementale est unique.

Dans la deuxième étude, je me suis intéressée aux mécanismes qui régissent le positionnement des INs des couches superficielles du cortex. Nous avions montré précédemment que le HTR3A y était impliqué, cependant, l’analyse plus approfondie de la mécanistique moléculaire restait inconnue. Une analyse génétique sur des INs de type sauvage ou dépourvus de HTR3A a permis d’identifier le gène PlxnA4 comme cible potentiel du signal induit par le HTR3A. J’ai montré, par son inactivation, que la PLXNA4 régule à la fois les processus de migration et de positionnement des INs HTR3A⁺ dans les couches superficielles du néocortex. L’utilisation de techniques in vitro et in vivo m’ont permis de montrer qu’un des ligands de la PLXNA4, la Semaphorin 3A (SEMA3A), a un effet chémorepulsif sur les INs HTR3A⁺ et que cet effet est médié par le corécepteur Neuropillin1 (NRP1). Fait intéressant, la SEMA3A est produite par des interneurones des couches profondes. Ces résultats m’ont amené à proposer un mécanisme dans lequel les INs des couches superficielles expriment la PLXNA4 en réponse à l’activation du HTR3A. De cette manière, les INs HTR3A⁺/PLNXA4⁺ deviendraient progressivement sensible à la SEMA3A sécrétée par les cellules des couches profondes et seraient ainsi, par répulsion, piégés dans les couches superficielles.

Dans la dernière étude, j’ai analysé le rôle du cotransporteur potassium/chlore KCC2 dans des stades précoces du développement des INs. KCC2 est exprimé plus fortement et plus précocement dans les INs que dans les cellules excitatrices. J’ai donc cherché à annuler l’expression de ce gène, spécifiquement dans les INs, par le biais du système cre-lox. Ceci m’a

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permis de montrer que la délétion de KCC2 dans les INs réduit la densité des cellules exprimant la parvalbumin (PV) dans le cortex somatosensoriel entrainant par la suite des défauts de maturation des neurones de projection.

Ces études m’ont donc permis d’éclaircir des mécanismes de spécification, de migration et de positionnement des INs néocorticaux.

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ABBREVIATIONS

5-HT Serotonin

AC Anterior commissure

ADP Afterdepolarization

AP Action potential

ApoER2 Apolipoprotein E receptor 2

Ascl1 Achaete-scute homolog 1

BC Basket cell

BMP Bone morphogenic protein

BP Basal progenitor

BPC Bipolar cell

BTC Bitufted cell

Ca²⁺ Calcium

Car4 Carbonic anhydrase 4

CCC Cation-chloride cotransporter

CCK Cholecystokinin

Cdk5 Cyclic-dependent kinease 5

CGE Caudal ganglionic eminence

ChAT Choline acetyltransferase

ChC Chandelier cell

cIN CGE-derived interneuron

Cl^- Chloride

CMS Caudal migratory stream

CNS Central nervous system

CP Cortical plate

CPN Callosa projecting neuron

CR Calretinin

CThPN Cortico-thalamic projecting neuron

Ctip2 Chicken ovalbumin upstream promoter TF-interacting protein 2 Cux (1) Cut-like homeobox (1)

Cx(26) Connexin (26)

Dab1 Disabled homolog1

DBC Double-bouquet cell

Dbx1 Developing brain homeobox 1

DIV (3) Days in vitro (3)

Dkk3 Dickkopf 3

Dlx (1) Distal-less homeobox gene (1)

dMGE Dorsal MGE

DRG Dorsal root ganglion

E (6) Embryonic day (6)

E-I Excitatory/inhibitory (balance)

Emx (1) Empty spiracle homeobox (1)

eNGC Elongated neurogliaform cell

Eph(A4) Ephrin receptor (A4)

Er81 (Etv1) ETS variant 1

F-actin Actin filament

Fezf2 (Fezl) Fez family zinc finger protein 2

FGF Fibroblast growth factor

Foxg1 Forkhead box G1

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GABA Gamma-aminobutyric acid Gad (1) Glutamic acid decarboxylase (1)

GC Growth cone

GFP Green fluorescent protein

GLAST Glutamate aspartate transporter

Gli3 Glioma-associated oncogene family zinc finger 3

Glu Glutamate

Gsx2 (Gsh2) GS homeobox 2

Hmx3 Homeobox coding protein NKX5.1

HTR3A Serotonin receptor 3A

IN Interneuron

IZ Intermediate zone

K⁺ Potassium

KCC2 K⁺/Cl^- cotransporter 2

KO Knock-out

L (1) Layer (1)

LGE Lateral ganglionic eminence

Lhx (2) LIM homeodomain (2)

MGE Medial ganglionic eminence

mIN MGE-derived interneuron

MT Microtuble

MTC Martinotti cell

mTsh1 Homeotic gene teashirt 1

MZ Marginal zone

Na⁺ Sodium

NDUFV2 NADH dehydrogenase ubiquinone flavoprotein 2

NE Neuroepithelial cell

NeuroD2 Neurogenic differentiation factor 2

NGC Neurogliaform cell

Ngn2 Neurogenin 2

NKCC1 Na⁺/K⁺/Cl^- cotransporter 1 Nkx2.1 Thyroid transcription factor 1

NMDA N-methyl-D-aspartate

nNOS1 Neural nitric oxide synthase 1 Npas1 Neuronal PAS domain protein 1

NPY Neuropeptide Y

Nr2f2 (Coup-TFII) Chicken ovalbumin upstream promoter II

Nrg3 Neuregulin 3

NRP (1) Neuropilin (1)

P (3) Postnatal day (3)

Par-3 Protease activated receptor 3

Pax6 Paired box-6

pIN POA-derived interneuron

PLXN (A4) Plexin A4

PN Pyramidal neuron

PNN Perineuronal net

POA Preoptic area

PP Preplate

Prox1 Prospero homeobox 1

PSB Pallial-subpallial boundary

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PV Parvalbumin

RG Radial glia cell

Rnd2 RhoA-like GTP-binding protein 2

Rorβ RAR-related orphan receptor beta S100β S100 calcium binding protein B

Satb(2) Special AT-rich sequence-binding protein (2)

SBC Single bouquet cell

SCPN Subcerebral projecting neuron

SEMA(3A) Semaphorin (3A)

Sfrp2 Secreted frizzled-related protein 2

SHH Sonic hedgehog

Slc12a5 Solute carrier family 12 member 5

Sox (3) SRY-box (3)

SP Subplate

Sp8 Specificity-protein 8

SSC Somatosensory cortex

SST Somatostatin

SVZ Subventricular zone

Tbr (1) T-box brain (1)

tdTOM Tandem dimeric Tomato

TF Transcription factor

TGF-β Transforming growth factor-beta

VIP Vasointestinal peptide

VLDLR Very low density lipoprotein receptor

vMGE Ventral MGE

VZ Ventricular zone

WNT Wingless-Int

WT Wild type

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LIST OF FIGURES

Introduction

Figure 1 Neural tube formation ………... 18

Figure 2 Genes responsible for early telencephalic patterning ……… 20

Figure 3 Subpallial origins of cortical interneurons ……… 22

Figure 4 Proliferation and neurogenesis ………... 25

Figure 5 Generation of excitatory neurons ………... 27

Figure 6 Models for interenuron specification ……… 30

Figure 7 Transcriptional cascade for interneuron specification ……….. 34

Figure 8 Neocortical assembly ………... 36

Figure 9 Modes of radial migration ………. 39

Figure 10 Interneuronal migration dynamics ……… 44

Figure 11 Semaphorins and Plexins diversity ………... 46

Figure 12 Downstream pathway of Semaphorin3A-Neuropilins-PlexinAs ………... 48

Figure 13 Steps of cortical interneuron migration ………. 50

Figure 14 Pyramidal neuron diversity in the neocortex ………. 52

Figure 15 Cortical interneuron molecular diversity ……….. 53

Figure 16 Interneuron morphological diversty in the neocortex ………... 56

Figure 17 Critical period features ………... 59

Figure 18 GABAA-mediated transmission in immature and adult neurons ……….. 61

Figure 19 Interneurons controlling pyramidal neurons firing directly and indirectly ……… 63

Figure 20 KCC2 structure ………. 64

Figure 21 Expression of the cation chloride co-transporters NKCC1 and KCC2 in development ………. 65

Results Figure 22 Microarray and genetic screening results ………... 83

Figure 23 PlexinA4 expression during cortical development ………. 84

Figure 24 Migratory dynamics of Htr3a-GFP⁺ interneurons downregulated for PlexinA4 … 86 Figure 25 Characterization of PlexinA4^-/- cortices ………. 88

Figure 26 Co-receptors expression in CGE-derived interneurons ………... 89

Figure 27 Semaphorin3A induces growth cones collapse in Htr3a-GFP⁺ interneurons in vitro ………... 91

Figure 28 Ex vivo analyses of Htr3a-GFP⁺ cells migration in presence of Semaphorin3A ... 93

Figure 29 Semaphorin3A expression in the developing neocortex ……… 95

Figure 30 Semaphorin3A overexpression in vivo affects Htr3a-GFP⁺ interneurons allocation ………... 96

Figure 31 Developmental expression of KCC2 mRNA in the brain ……….. 99

Figure 32 KCC2 expression in the neocortex at E18.5 and P0 ……….. 100

Figure 33 A tool to downregulate KCC2 in interneurons ……….. 102

Figure 34 KCC2 downregulation preserves cell density and lamination ……… 103

Figure 35 Interneuronal marker expression ………... 104

Figure 36 KCC2 downregulation in interneurons impairs parvalbumin expression ……….. 106

Figure 37 Pyramidal neurons maturation ………... 108

Discussion and conclusion Figure 38 Proposed role for PlexinA4-Semaphorin3A in CGE-derived interneurons migration ………... 119

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LIST OF TABLES

Table 1 Primary antibodies used in the studies ……….. 72 Table 2 In situ hybridization probes used in the studies ……….. 74

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I. INTRODUCTION

Men ought to know that from the brain, and from the brain only, arise our pleasures, joy, laughter and jests, as well as our sorrows, pains, grief and tears. Through it, in particular, we think, see, hear and distinguish the ugly from the beautiful, the bad from the good, the pleasant from the unpleasant […]. It is the same thing that makes us mad or delirious […].

These things that we suffer all come from the brain, when it is not healthy, but becomes abnormally hot, cold, moist, or dry, or suffers any other unnatural affection to which it was not accustomed. Madness comes from its moistness.

Hippocrates – The Sacred Disease (IV sec. B.C.)

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The mammalian cerebral cortex is the largest structure of the nervous system. Its complex cellular and molecular composition underlies our higher cognitive functions, such as attention and language, as well as our emotions and behaviors. Although evolution made the human cortex unique in its complexity and functions, a certain level of similarity is somehow appreciable among different mammalian species. Indeed, homologies are consistently found in cortical cytoarchitecture and genes expression (Molnar 2011, Molnar and Clowry 2012). Of outmost importance to this work, developmental processes underlying brain assembly and maturation are very similar between mammals, making rodents good model to study these biological processes (Molnar 2011, Molnar and Clowry 2012, Aboitiz and Zamorano 2013, Montiel, Vasistha et al. 2016).

My thesis work was aimed at deciphering mechanisms involved in the specification and integration of interneuron subclasses into the neocortex. Particularly, I focused on the generation of interneurons and processes implicated in their cortical settlement. All the experiments were performed on mice models. If not otherwise specified, researches cited in this work were performed on murine models.

1. E

ARLY DEVELOPMENT OF THE TELENCEPHALON

1.1. Neural Tube formation and development of the forebrain

In mice, as early as embryonic day 6 (E6), three layers of germ cells with distinct gene expression programs structure the embryo. Between E8 and 10, part of the outermost cell layer, the ectoderm, thickens into neuroepithelium and is converted into a tubular structure through the process of neurulation (Fig. 1A). Concomitantly, the rest of the ectoderm and germinal layers undergo convergent extensions, in which they tighten and grow along their axis (Arnold and Robertson 2009, Greene and Copp 2009). During the phase of neurulation, the neuroepithelium invaginates along the midline and folds to allow the two extremities to face each other. The stalk of neuroepithelium – between the epithelium and the neural tube – forms the neural crest, a transient structure that will further migrate and give rise to the peripheral nervous system (Kandel, Schwartz et al. 2000, Darnell and Gilbert 2017). During this phase, the notochord has a crucial role in secreting sonic hedgehog (SHH) along the midline to induce and maintain neuronal fate, while the non-neuronal roof plate secretes bone morphogenic proteins (BMPs) and transforming growth factor-beta (TGFβ), which, in turn, inhibit neural fate of ectodermal cells (Kandel, Schwartz et al. 2000, Yamanaka, Tamplin et al. 2007,

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Andoniadou and Martinez-Barbera 2013, Darnell and Gilbert 2017). Finally, the neural tube closes in three discontinuous steps: at the hindbrain/cervical boundary, at the forebrain/midbrain boundary and at its rostralmost part (Greene and Copp 2009, Yamaguchi and Miura 2013, Massarwa, Ray et al. 2014). With its closure, the rostralmost part of the neural tube forms three primary vesicles that will later develop into structures of the central nervous system (CNS), such as the proencephalon/forebrain (telencephalon and diencephalon), the mesencephalon/midbrain and the rhombencephalon/hindbrain (cerebellum, pons and medulla) (Fig. 1B) (Kandel, Schwartz et al. 2000, Darnell and Gilbert 2017).

Figure 1 ½Neural tube formation. A) The future central nervous system (CNS) is induced by the process of neurulation. The neural plate (beige), part of the ectoderm (blue), invaginates in the neural groove and forms the floor plate (green), while the neural folds (violet) converge and form the roof plate. The dorso-ventral axis is conferred by bone morphogenic proteins (BMPs), secreted by the roof plate, and sonic hedgehog (SHH), secreted by the floor plate (Liu and Niswander 2005). B) The anterior part of the neural tube forms three primary vesicles, i.e. proencephalon (forebrain), mesencephalon (midbrain) and rhombencephalon (hindbrain). These will further form secondary vesicles, from which structures of the CNS develop. The neocortex arises from the proencephalic vesicle of the forebrain. C) During development, the neural tube flexes twice (Kandel, Schwartz et al. 2000).

As pioneered by a study in chickens and later confirmed with graft experiments in mice, the forebrain is formed thanks to the anterior visceral ectoderm and the primitive streak, which prevents the caudalization of the proencephalon itself (Eyal-Giladi and Wolk 1970, Beddington 1994, Tam and Steiner 1999, Joubin and Stern 2001). While all these morphogenic structures are necessary to the formation process, they are not sufficient to induce and specify the forebrain. A much more complex scenario has been proposed, in which the role of the aforementioned organizer-areas work as supporters of signaling steps in the forebrain

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formation. Although these steps are yet to be fully unveiled, researchers in the field agree that transient genes, such the SRY-Box3 (Sox3), “activate” future cells of the forebrain, whose fate is further stabilized by the antagonizing action of the caudal fibroblast growth factors (FGFs), retinoic acids and Wingless-Int (WNT)/β-Catenin pathway (Ruiz i Altaba and Jessell 1991, Stern 2001, Houart, Caneparo et al. 2002, Wilson and Houart 2004, Andoniadou and Martinez- Barbera 2013, Tortelote, Hernandez-Hernandez et al. 2013, Yoon, Huang et al. 2015).

1.2. Dorso-ventral patterning and arealization of the telencephalon

The telencephalon arises from the proencephalic vesicle (Kandel, Schwartz et al. 2000).

Around E8.5, the telencephalic primordium is shaped by the induction of the Forkhead box G1 (Foxg1) (Paek, Gutin et al. 2009). Specification of dorsal and ventral domains is initiated by the pan expression of GLI family zinc finger 3 (Gli3), which is rapidly downregulated in the ventral part by the action of SHH and Fgf8 (Kuschel, Ruther et al. 2003, Hebert and Fishell 2008). These latter are concomitantly responsible for the establishment of the dorso-ventral boundary (Rallu, Machold et al. 2002). The differentiation between dorsal (i.e., pallial) and ventral (i.e., subpallial) domains is regulated by sequential expression of different genes, which influence progenitors in a cell-autonomous manner and, thereby, generate discrete cell type to populate the cortex and structures of the telencephalon (Fig. 2) (Dehay and Kennedy 2007). A crucial role in shaping and maintaining the telencephalic anlage is conferred by combinations of extrasignaling factors of the FGF family. Indeed, studies in mutant mice have revealed that loss of Fgfr1, Fgfr2 and Fgfr3 results in cell death and in the absence of both the whole ventral area and cortex (Gutin, Fernandes et al. 2006, Paek, Gutin et al. 2009).

As early as E9.5, the competitive interaction between dorsalizing BMPs and ventralizing SHH starts to shape distinct domains of the telencephalon (Campbell 2003), which becomes more evident with the establishment of the pallial-subpallial boundary (PSB) (Yun, Potter et al. 2001).

The dorsal domain is composed by two main regions: the cerebral cortex (neocortex and hippocampus) and the dorsal midline (cortical hem and choroid plexus). As the neural tube closes, expression of the Empty Spiracles Homeobox 1/2 (Emx1/2) interacts with fgf8 and makes the dorsal telencephalic primordium a distinct area from the roofplate (Fig. 2) (Shinozaki, Yoshida et al. 2004). As studies in chickens and knock-out (KO) mice have demonstrated, lack of Emx1/2 impairs, in a non cell-autonomous manner, the establishment and development of the choroid plexus, which demarcates the brain dorsal midline and, together with the cortical hem, is considered as the regulatory center for neocortex formation (Shinozaki,

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Yoshida et al. 2004, von Frowein, Wizenmann et al. 2006, Subramanian, Klattenhoff et al.

2009). Cross-regulatory interaction between Emx1/2 and the paired box 6 (Pax6) forms rostro- caudal regions within the dorsal pallium (Yoshida, Suda et al. 1997, Bishop, Rubenstein et al.

2002, Subramanian, Klattenhoff et al. 2009). Their complementary gradients of expression in the caudo-medial and rostro-lateral parts, respectively, influence cell proliferation and are likely contributing to the organization of discrete cortical areas (Bishop, Rubenstein et al. 2002, Mallamaci and Stoykova 2006, O'Leary, Chou et al. 2007, Godbole, Roy et al. 2017).

Neocortical induction is critically regulated by the LIM homeodomain 2 (Lhx2), which is secreted by the roofplate and expressed evenly throughout the neocortical anlage except for the dorsal midline (Fig. 2) (Mallamaci and Stoykova 2006, Hebert and Fishell 2008, Godbole, Roy et al. 2017). Loss-of-function studies have demonstrated that this transcription factor (TF) is crucial for the neuronal fate commitment as well as for shaping the neocortex through inhibiting hippocampal fate (Bulchand, Grove et al. 2001, Monuki, Porter et al. 2001, Vyas, Saha et al. 2003, Mangale, Hirokawa et al. 2008, Ypsilanti and Rubenstein 2016, Godbole, Roy et al. 2017). During development, combinations and gradients of TFs contribute in shaping distinct functional areas of the pallium (Mallamaci and Stoykova 2006), with progenitors that will give rise to pyramidal neurons and glia populating the neocortex (Fig. 2).

Figure 2 ½Schematics of genes responsible for early telencephalic patterning. In blue are shown morphogens factors responsible for the main dorso-ventral subdivision, in green downstream targets that shape different structures. Briefly, ventral areas are induced by Sonic hedgehog (SHH), which contrasts the dorsalizing action of GLI3. Concomitantly, SHH and the forkhead box G1 (FOXG1) activate fibroblast growth factor (FGF). FOXG1 and FGF are finally responsible for inducing GSX2 (formerly called GSH2) and NKX2.1 which shape the lateral (LGE) and the medial (MGE) ganglionic eminences, respectively. The dorsal domain is promoted by the expression of bone morphogenic proteins (BMPs) and Wingless-Int proteins (WNTs) by GLI3. In turn, BMPs and

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WNTs allow the expression of EMX proteins, which produce PAX6 and LHX2 to shape neocortex and hippocampus (Hebert and Fishell 2008).

The cortical hem, located along the dorsal midline, is enriched in WNT and BMP genes (Hebert and Fishell 2008). In mice where the hem is ablated, the hippocampus is completely absent and the dorsal cortex is reduced in size, with consequent expansion of the ventro-lateral telencephalon. For these reasons, the cortical hem is considered to be the main regulator of the dorso-ventral patterning and cortex formation (Shimogori, Banuchi et al. 2004, Subramanian, Klattenhoff et al. 2009, Harrison-Uy and Pleasure 2012, Caronia-Brown, Yoshida et al. 2014, Gupta and Sen 2016).

The PSB arises from the antihem, by the competitive action of the dorsalizing Pax6 and ventralizing GS homeobox 2 (Gsx2, formerly called Gsh2) (Yun, Potter et al. 2001, Campbell 2003, Guillemot, Molnar et al. 2006). Cross-repression between Pax6 and Gsx2 influences a heterogeneous cascade of TFs in progenitors, that will later give rise to a group Cajal-Retzius cells (Bielle, Griveau et al. 2005), oligodendrocytes (Naruse, Ishizaki et al. 2017) as well as cells populating the limbic system, the olfactory bulb and the white matter (Hirata, Nomura et al. 2002, Cocas, Georgala et al. 2011, Frazer, Prados et al. 2017). Studies on Pax6-null (also called Sey mice), Gsx2-KO and double mutant mice have shown the requirement of these two TFs for the proper positioning of the PSB (Yun, Potter et al. 2001, Carney, Cocas et al. 2009, Georgala, Carr et al. 2011). Furthermore, both Pax6 and Gsx2 are crucial for the heterogeneous gene expression of developing brain homeobox1 (Dbx1), ETS Variant 1 (Er81), specificity protein-8 (Sp8), homeotic gene teashirt 1 (mTsh1), secreted frizzled related protein 2 (Sfrp2) and Tgfα, necessary for progenitor cells to the limbic system and olfactory bulb here generated (Carney, Cocas et al. 2009).

The ventral telencephalon is primarily shaped by Foxg1, which cell-autonomously induces subpallial-specific TFs (Fig. 2) (Hebert and Fishell 2008, Manuel, Martynoga et al.

2010). The ventral telencephalon is a heterogeneous structure, composed by morphologically and genetically discrete microdomains, anlage for γ-aminobutyric acidergic (GABAergic) cells (here also referred as interneurons), some cholinergic and dopaminergic neurons and some oligodendrocytes and astrocytes populating the neocortex, striatum and other basal nuclei of the CNS (Fig. 3) (Marin, Anderson et al. 2000, Fishell 2007, Kessaris, Pringle et al. 2008, Hu, Vogt et al. 2017). Morphological analyses first and marker studies in the 90s allowed a broad subdivision of the ventral telencephalon in the telencephalic stalk and three ganglionic eminences, expressing specific spatio-temporal cascade of genes (Fishell 2007, Flames, Pla et al. 2007, Hebert and Fishell 2008, Kessaris, Magno et al. 2014, Hu, Vogt et al. 2017). As of

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E12, progenitor cells lying the ventricular and subventricular zones (VZ and SVZ, respectively) of the subpallium are induced by the pan-expression of distal-less homeobox genes 1 and 2 (Dlx1/2) and then by Dlx5/6, to promote the expression of glutamic acid decaboxylase 1 and 2 (Gad1/2, also called GAD67 and 65, respectively) (Zerucha, Stuhmer et al. 2000, Panganiban and Rubenstein 2002, Flames, Pla et al. 2007, Le, Zhou et al. 2017).

The lateral ganglionic eminence (LGE) is shaped by Pax6, in its dorsal most domain, and Gsx2, a TF upstream the Achaete-scute homolog 1 (Ascl1) and the gluticocorticoid-induced transcript 1 (Glcci1) (Wang, Waclaw et al. 2009, Kohli, Nardini et al. 2018). Progenitors in the LGE give rise to oligodendrocytes and neurons in the olfactory bulb and striatum (Wang, Waclaw et al. 2009, Chapman, Waclaw et al. 2013, Diaz-Guerra, Pignatelli et al. 2013). Its contribution in generating a proportion of cortical interneurons has been largely debated (Anderson, Eisenstat et al. 1997, Wonders and Anderson 2006, Ma, Zhang et al. 2012).

However, the recent development of a cre-recombination Gsx2 mouse line, which between E12.5 and E15.5 labels selectively the LGE and the dorsal border of the medial ganglionic eminence (MGE), has confirmed that neurons from this proliferative zone populate the olfactory bulb, the amygdala and the striatum, but not the neocortex (Qin, Madhavan et al.

2016).

Figure 3 ½Subpallial origins of cortical interneurons. Cortical interneurons (INs) originate from discrete areas of the subpallium, i.e. the medial ganglionic eminence (MGE, blue), the preoptica area (POA, red) and the caudal ganglionic eminence (CGE, green). Each of them express peculiar transcriptional codes (Drawn by M. Niquille).

MGE assembly requires the expression of the thyroid transcription factor 1 (Nkx2.1), which is induced and maintained by Shh (Xu, Wonders et al. 2005, Gulacsi and Anderson 2006). Nkx2.1 is expressed in the VZ of the MGE and in post-mitotic cells within this region.

Nkx2.1-null mice develop an LGE-like structure instead of the MGE and display about 50%

reduction of cortical interneurons and striatal cholinergic neurons, indicating the crucial role of

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this TF in specifying this structure and producing specific cell subtypes (Sussel, Marin et al.

1999, Butt, Sousa et al. 2008).

Due to the lack of morphological boundaries, the caudal ganglionic eminence (CGE) has historically been considered as an appendix of the LGE/MGE (Anderson, Marin et al. 2001, Wichterle, Turnbull et al. 2001). Thanks to loss-of-function studies, it has been possible to assess that the development of the CGE occurs independently from genes responsible for LGE or MGE induction, making it a separate eminence that gives rise to a distinct subpopulation of GABAergic neurons (Nery, Fishell et al. 2002, Miyoshi, Hjerling-Leffler et al. 2010).

Nevertheless, is still unknown whether there is a specific gene upstream Dlx2 able to induce the formation of the CGE. So far, the CGE is characterized by the combinatorial motif of TFs that are preferentially, but not exclusively, expressed in this area, such as the chicken ovalbumin upstream promoter II (Coup-TFII or Nr2f2), Sp8 and the prospero homeobox 1 (Prox1) (Kanatani, Yozu et al. 2008, Ma, Zhang et al. 2012, Rubin and Kessaris 2013, Miyoshi, Young et al. 2015).

In addition to the ganglionic eminences, the ventral domain gives also origin to the telencephalic stalk, which includes the preoptic area (POA) and the septum. The POA is a region located around the third ventricle and shares a mix of genes belonging to both MGE and CGE (Puelles, Kuwana et al. 2000, Flames, Pla et al. 2007). Histological studies have shown that the VZ of the whole POA is characterized by the combinatorial expression of Shh, Nkx2.1 and Nkx2.2 (Flames, Pla et al. 2007). In addition, the VZ of the ventro-anterior POA co- expresses TFs such as Nkx6.2, Dbx1 and Lhx2 (Flames, Pla et al. 2007, Hirata, Li et al. 2009, Lischinsky, Sokolowski et al. 2017). Whether these genes are the only ones contributing to the induction of proliferative cells in this region has yet to be fully elucidated. The POA gives mainly origin to cells populating the amygdala, but lineage studies have also revealed a small contribution to cortical GABAergic cells (Gelman, Martini et al. 2009, Hirata, Li et al. 2009, Gelman, Griveau et al. 2011, Lischinsky, Sokolowski et al. 2017). Finally, the septum is the most anterior region of the ventral telencephalon. It shares molecular identities with the adjacent PSB, LGE and MGE, by expressing Nkx2.1 as well as different gradients of Pax6 and Gsx2 along the VZ (Flames, Pla et al. 2007). Although the contribution of the septal region to cortical interneurons is still debated, it seems to only give rise to cells in the olfactory bulb (Taglialatela, Soria et al. 2004, Rubin, Alfonsi et al. 2010, Qin, Ware et al. 2017).

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1.3. Neurogenesis and proliferation

Cells populating the cortex are generated in region- and time-specific manners from a single layer of cells along the ventricular wall. These neuroepithelial cells (NEs) are highly polarized along the apico-basal axis and their cytoplasm surrounded by adherence junctions (Laguesse, Peyre et al. 2015, Arai and Taverna 2017). Although these cells move from the apical surface to the basal lamina during different phases of the cell cycle (Fig. 4B), mitotic division always happens at the ventricular wall, where primary cilia polarize the mitotic spindle through the centrosome and promote communication of extracellular cues in the cerebrospinal fluid (Taverna and Huttner 2010, Lepanto, Badano et al. 2016). In the earliest phase of embryonic development, NEs undergo massive symmetric division to generate two identical daughter cells and increase their number (Gotz and Huttner 2005).

Around E9.5-10.5, NEs upregulate cell adhesion molecules and start divide asymmetrically. During this phase of early neurogenic division, NEs divide into a neuron expressing the neurogenic differentiation factor 2 (NeuroD2; i.e., direct neurogenesis) or a basal progenitor (BP; i.e., indirect neurogenesis) and a radial glia cell (RG) (Fig. 4A) (Gotz and Huttner 2005, Govindan and Jabaudon 2017). BPs are specialized intermediate progenitors that undergo mitosis in the upper VZ or in the SVZ. These cells are distinguishable for the expression of T-box brain1 and 2 (Tbr1, Tbr2/Eomes) and cut-like homeobox 1 and 2 (Cux1, Cux2). BPs normally undergo symmetric division to generate two neurons (Fig. 4A) and, for this reason, they are thought to serve as increasing the neuronal number (Gotz and Huttner 2005, Laguesse, Peyre et al. 2015, Telley, Govindan et al. 2016, Govindan and Jabaudon 2017).

RGs are fate-restricted progenitors that exhibit astoglial markers (such as GLAST and S100β) together with Nestin, Pax6, Sox2 and Vimentin (Gotz and Huttner 2005, Telley, Govindan et al.

2016). These cells have a long basal process to the pia and a short apical one anchored to the VZ by adherence junctions, which recruit cytoplasmatic proteins like β-Catenin (Otero, Fu et al. 2004, Gotz and Huttner 2005, Taverna and Huttner 2010). RGs undergo self-renewing asymmetric division to generate a BP or a neuron and another RG or symmetric division to generate two newborn neurons (Fig. 4A). As demonstrated by time-lapse microscopy experiments, about 15% of RGs give rise to pyramidal neurons in the cortex, while the remaining work as scaffold for radially migrating cells and, by E18.5, specify as cortical astrocytes or oligodendrocytes (Noctor, Flint et al. 2002, Gotz and Huttner 2005, Lepanto, Badano et al. 2016, Govindan and Jabaudon 2017). The fate of RG division is to be attributed to the primary cilia, which are attached to RGs during interphase (but not mitosis) and still

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confer extracellular signals (such as Shh), and is influenced by spindle orientation. Regarding this latter, recent studies on cortical culture from mice and rats have shown that vertical plane of division and oblique/horizontal plane correlate with the rate of direct vs. indirect neurogenesis, respectively. However, self-renewing division does not seem to be dependent on spindle orientation, and it is more likely to be due to the action of Notch signaling and levels of the protease activated receptor 3 (Par-3), that confer daughter cells a RG identity (Postiglione, Juschke et al. 2011, Peyre and Morin 2012, Laguesse, Peyre et al. 2015, Lepanto, Badano et al.

2016, Arai and Taverna 2017).

Figure 4 ½ Proliferation and neurogenesis. A) Schematic showing neurogenesis. Neuroepitelial cells (NE) divide symmetrically to generate two identical daughter cells or asymmetrically to generate a basal progenitor (BP) and a radial glia cell (RG). While normally BPs undergo symmetric, neurogenic division, RGs generate a BP and another RG to increase their number. B) In NEs, the nucleus migrates to the basal surface and back to the apical wall, where it undergoes mitosis. In RGs, nuclear interkinesis is confined to the ventricular zone. Finally, BPs retract from the apical surface and cells divide at the basal wall of the ventricular/subventricular zone (Gotz and Huttner 2005).

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2. G

ENERATION OF CORTICAL NEURON DIVERSITY

Following cell proliferation, timely regulated cascades of TFs are responsible for differentiation of neuronal types populating the cerebral cortex. In the neocortex, about 80% of neurons are excitatory neurons. Two types of excitatory neurons populate the neocortex:

pyramidal neurons (PNs) in layers 2-3 and 5-6 and spiny stellate in L4, all working by glutamatergic (Glu) transmission. PNs project intracortically and long-range to the controlateral hemisphere, in subcortical and subcerebral regions, while spiny stellates make local connections. The remaining 20% is constituted by interneurons (INs), which mainly form local inhibitory connections to control PNs activity via GABAergic neurotransmission.

2.1. Excitatory Neurons

Excitatory neurons are born from PAX6⁺/EMX2⁺ progenitor cells in the dorsal pallium in a time-dependent manner. The first waves of neuroblasts, generated from E12.5, will populate deep layers, while late-born neurons will end up in the outermost ones, building the neocortex in an inside-out fashion (Fig. 5A). Except for spiny stellate neurons in layer (L) 4 of the barrel cortex, PNs have a typical triangular-shaped soma, an apical dendrite towards the pial surface, rich lateral dendrite arborizations and a long axon which departs from the basal midline of the soma to reach the final target (Molyneaux, Arlotta et al. 2007, Leone, Srinivasan et al.

2008, Standring 2016).

It is yet unknown whether RGs belong to different lineages and are fate-determined to produce neurons that eventually settle in a specific layer. The most embraced hypothesis suggests that PNs arise from a unique lineage and time of generation determine an intrinsic molecular cascade that cell-autonomously differentiates sequential waves (Custo Greig, Woodworth et al. 2013, Marin and Muller 2014). In support of this model, heterochronic transplantation studies in mice and ferrets have shown that progenitors aged to give rise to deep or upper layers PNs do not change their final commitment (McConnell and Kaznowski 1991, Frantz and McConnell 1996). However, a study on human tissue found heterogeneity among neurogenic cells across different brain regions. This suggests a degree of diversity that underlie specification of different subtypes of PNs that, in a given area of the brain, establish determined patterns of connectivity (Nowakowski, Bhaduri et al. 2017).

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Figure 5 ½ Generation of excitatory neurons. A) Migrating pyramidal neurons settle to their final allocation in an inside-out manner, i.e., firstly born neurons populate lower layers, while late-born upper ones. B) Peak of generation of different excitatory neurons subtypes (Custo Greig, Woodworth et al. 2013).

On the other hand, post-mitotic control over mature cortical subtypes has been better elucidated. Several TFs in post-mitotic cells control different subtypes of mature PNs in either callosal (CPN), subcerebral (SCPN) or cortico-thalamic (CThPN). SCPNs are generated from Fezf2-expressing cells. Forced induction of this TF has been shown to be sufficient to induce corticofugal features in L4 excitatory neurons (De la Rossa, Bellone et al. 2013). In Fezf2-null cortices, SCPNs and projections to the spinal cord and brain stem are lost and cells are fate- converted into CThPNs or CPNs. Furthermore, TFs downstream Fezf2, such as Ctip2, are no more expressed (Chen, Schaevitz et al. 2005, Molyneaux, Arlotta et al. 2007, Chen, Wang et al. 2008, Canovas, Berndt et al. 2015, Tantirigama, Oswald et al. 2016). Fezf2 is also present at lower levels in CThPNs, but its absence does not prevent their specification (Molyneaux, Arlotta et al. 2007). CThPN fate is regulated by the expression of Tbr1, which represses Fezf2.

In Tbr1^-/- mice, putative CThPNs in L6 overexpress Fezf2 and Ctip2 and extend their axonal projections towards subcerebral regions (Bedogni, Hodge et al. 2010, McKenna, Betancourt et

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al. 2011). Satb2 has a fundamental, but not sufficient, role in establishing CPN identity by repressing Ctip2 (Srinivasan, Leone et al. 2012). CNPs in Satb2^-/- cortices fail to upregulate specific markers such as Dkk3 and Cux1, but interemispherical projections are not fully ablated.

This suggests the requirement of other TFs, like Cux2 and/or Ctip1, to properly develop callosal and associative projections (Alcamo, Chirivella et al. 2008, Britanova, de Juan Romero et al.

2008, Leone, Srinivasan et al. 2008, Franco, Gil-Sanz et al. 2012, Srinivasan, Leone et al. 2012, Greig, Woodworth et al. 2016, Woodworth, Greig et al. 2016).

Spiny stellate neurons in L4 of the somatosensory cortex (SSC) selectively express Rorβ. This TF is sufficient to induce morphological and connectivity-related features of spiny stellate cells (Klingler et al., March 28, 2018; bioRxiv, doi: https://10.1101/290395). Evidences suggest that this identity is acquired in a post-migratory phase, with the establishment of thalamic inputs and/or mutual repression of other TFs (Pouchelon, Gambino et al. 2014, Frangeul, Pouchelon et al. 2016, Oishi, Nakagawa et al. 2016). Whether an earlier post-mitotic transcriptional program exists, it has yet to be discovered.

Integration within the cortex and projections of these PN subtypes are discussed later in this work (cf. chapter 4 par. 4.1).

2.2. Inhibitory Neurons

GABAergic INs are a heterogeneous group of cells that, in the cortex, generally establish local inhibitory connections. They are usually classified according to their morphological, electrophysiological, synaptic and molecular properties and, more recently, for their transcriptomic profiling (Petilla Interneuron Nomenclature, Ascoli et al. 2008, DeFelipe, Lopez-Cruz et al. 2013, Mihaljevic, Benavides-Piccione et al. 2015, Fuzik, Zeisel et al. 2016, Tasic, Menon et al. 2016).

In rodents, all cortical INs are born within the subpallium, mainly from the MGE and, to a lesser extent, from the CGE and the POA (Fishell 2007, Sultan, Shi et al. 2014, Hu, Vogt et al. 2017). In humans, origin of INs is still debated. Although proliferating precursors are found in the dorsal pallial SVZ, these cells present typical features of intermediate progenitors and evidences suggest that, even in higher mammals, INs exclusively originate from the ganglionic eminences. It is more likely, thus, that these precursors only pause in the dorsal SVZ to undergo a further symmetrical division and increase their number (Jakovcevski, Mayer et al.

2011, Zecevic, Hu et al. 2011, Hansen, Lui et al. 2013, Arshad, Vose et al. 2016). In addition, studies have highlighted that groups of INs are generated postnatally, both in rodents and primates. A pool of late-born IN precursors, that will lately populate the anterior cingulated

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cortex, has been identified in the mouse postnatal pallium (Riccio, Murthy et al. 2012). In adult rodents, stem cells for GABAergic granule neurons have been found around the lateral ventricle. These cells migrate along the rostral migratory stream to finally settle in the olfactory bulb (Ming and Song 2011). In humans, neuronal stem cells along the lateral ventricle generate interneurons to the striatum all life long, but only up to 18 months after birth, to the olfactory bulb and the prefrontal cortex (Sanai, Nguyen et al. 2011, Yang, Ming et al. 2011, Ernst, Alkass et al. 2014).

Whether IN progenitors are committed to a specific subtype or their diversity arises from contextual cues is highly debated (Wamsley and Fishell 2017, Telley and Jabaudon 2018).

The “progenitor specification model” believes that IN progenitor cells already have an encoded blueprint that will drive their maturation in the neocortex. The “cardinal-definitive specification hypothesis” states that IN diversity arises from unspecified progenitors and extrinsic activity during a perinatal sensitive time window will determine mature phenotype (Fig. 6) (Wamsley and Fishell 2017, Telley and Jabaudon 2018). Experimental evidences so far accumulated are in support of both hypotheses. However, these two are not mutually exclusive: IN progenitors may be committed to a spatio-temporally determined TF cascade activation, which confers different sensitivity to extrinsic molecules and activity to ultimately determine their mature phenotype. This hypothesis is in accordance with the most recent works implying genetic analyses at single-cell resolution. By analyzing the transcriptomic profile of cells in the ganglionic eminences, these studies failed to find progenitor differences mirroring mature phenotype, but underlined that genetic characteristics are already discernable at post-mitotic level (Mayer, Hafemeister et al. 2018, Mi, Li et al. 2018). In this perspective, a third “mixed model” has been proposed, in which is said that, although differences in mitotic gene expression are negligible, they may be sufficient to drive and specify subtype peculiarities of the mature neocortex (Telley and Jabaudon 2018).

Specification and integration of interneuron subtypes in the neocortex

Thesis

Reference