Neural Ridge  Arc -Activity-Regulated Cytoskeleton-Associated

Acknowledgments

This work would have not been possible without the help and support of Michèle and Crhsitian.

Since my arrival in the lab they gave me responsibility and the opportunity of working on interesting and engaging projects. I must thank both of you for all the time spent in constructive discussions and straightforward teaching of topics and techniques I can now rely on. I would not trade any moment, as they have been in these years the main source of what I learned.

I wish to thank Eloisa Herrera, Xavier Leinekugel and Jacques Noel for accepting to take part in the jury for my discussion, providing helpful insight on how to improve this work. I also loved spending my time in the lab thanks to the people, collegues and friends I have found there. Especially Kawssar Harb, I admired your strength while working back to back with you. No less I enjoyed the brief time spent with Anna Lisa Romano, Audrey Touzot, Nadia Elganfoud, you left the lab, but you never left our tohughts. I am also happy of having shared my daily time with Michele, Bertacchi, Olivier Deschaux, Maria Anna Di Bonito, Josephine Parisot, Mykyta Lukianets, Eya Setti, great people and fantastic colleagues. I wish all the best to Alessandro Simi and Torsten Felske, you will most surely enjoy your time here and I wish we had more time to spend together. I wish to thank all the collaborators that got interested into my work and helped substantially to increase its quality and allowing me to access new experiences. Denis Jabaudon and Laura Frangeul allowed me to learn performing stereotaxic injections. Massimo Mantegazza, Jennifer Lavigne and Maryon Arioult followed granted me to perform thalamo-cortical field recording and followed me in every step of it. Andrea Marcantoni performed MEA recordings showing a great interest and helpful will. I also thank Marjolijn Mertz for helping learn the Matlab language from scratch, and finally Novitch Benett Lab, California, for the Bhlhb5 Antibody. This work was also made possibile thanks to the funding from the AXA research fund, which funded my first three years of Ph.D., and the FRM fin de thése funding for the fourth year.

I am extremely happy of having found many motivated researcher over at the AIRIcerca venture.

We almost never met, in person, but the enthusiasm for science divulgation and its relevance in society tells me how much in common we do share. I owe to thank my family and friends in Italy, who did not let me miss to feel their support and love in the few precious moment we spent together, especially Stefano and Laura, it is beautifull to see how close you became to my parents. No less I thank Mum and Dad, for how supportive you have always been with me and my wishes, for how much you participated in my joys and my strains and how much of a big part you played in the first ones. I am equally glad of being now close with my fiancé's family for you support to Francesca and me as well.

I finally thank Francesca, first of all for how in these years you supported me whilw living at hundreds of km away, supporting me from the distance and then joining me abroad. Of the last years I am most happy of the time spent with you, and can not wait for spending the rest of my life with you.

Résumé

Le neocortex est tangentiellement subdivisé en zones fonctionnellement spécialisées, constituées radialement de six couches ayant différentes morphologies, connectivités et profils moléculaires.

Au cours du développement, des gènes organisateurs sont exprimés en gradients et déterminent les identités de position des progéniteurs corticaux. Des études récentes suggèrent que certains aspects fonctionnels sont également déterminés dans les cellules post-mitotiques et que l'activité neuronale spontanée pourrait influencer le développement cortical. Comment les gènes postmitotiques exprimées dans le néocortex peuvent déterminer l'identité de position des neurones matures et comment les mécanismes génétiques intrinsèques et les facteurs dépendants de l'activité extrinsèque interagissent les uns avec les autres n'est pas entièrement compris.

Pour étudier le lien entre les facteurs intrinsèques et extrinsèques pendant l'organisation du néocortex, j'ai étudié la fonction du gène organisateur COUP-TFI indépendamment dans les progéniteurs corticaux et/ou les cellules post-mitotiques en utilisant plusieurs modèles de souris.

Dans la première partie de ma thèse, j'ai montré que l'expression de COUP-TFI dans les cellules post-mitotiques est nécessaire et suffisante pour promouvoir l'identité corticale sensorielle et la connectivité entre le thalamus et le cortex. Dans la seconde partie, j'ai utilisé des souris mutantes conditionnelles pour comprendre si COUP-TFI peut promouvoir l'organisation somatosensorielle (S1) via un mécanisme dépendant de l'activité. Mes données ont d'abord indiqué que l'activité corticale spontanée diffère entre les zones motrices et sensorielles au cours du développement, et que COUP-TFI est directement impliqué dans ce processus en régulant la fréquence et la coordination de l'activité neuronale. En conséquence, les neocortex déficient pour COUP-TFI sont initialement innervés par les noyaux thalamiques sensoriels, mais ces connexions ne sont pas maintenues. En outre, l'expression de COUP-TFI est nécessaire pour que les cellules de la couche 4 acquièrent une morphologie étoilées, caractéristique des neurones de la région S1. J'ai trouvé que COUP-TFI régule directement la transcription du gène précoce immédiat Egr1 et d'un sousensemble de canaux ioniques. La ré-expression d'Egr1 dans ces cellules de la couche 4 déficientes pour COUP-TFI leurs permet d'acquérir à nouveau leurs morphologies étoilées.

Cette étude a démontré un nouveau lien entre le motif intrinsèque de transcription et les mécanismes dépendants de l'activité dans l'organisation du cortex somatosensoriel et de son circuit au cours du développement de la souris.

Abstract

The neocortex is tangentially subdivided into functionally specialized areas and radially constituted by six layers with distinct morphologies, connectivity and molecular profiles. During development, patterning genes expressed in gradients determine positional identities within cortical progenitors. Recent evidences suggest that certain functional traits are also determined within post-mitotic cells and that cortical spontaneous neuronal activity might be implicated in these mechanisms. However, how post-mitotic genes expressed in the neocortex can determine the positional identity of mature neurons and how intrinsic genetic mechanisms and extrinsic activitydependent factors interact with each other, is not fully understood.

To investigate the link between intrinsic and extrinsic cues during neocortical organization, I have challenged the function of the area-patterning gene COUP-TFI, in cortical progenitors and/or postmitotic cells by using several mouse models. In the first part of my thesis work, I have shown that COUP-TFI expression in post-mitotic cells is necessary and sufficient to promote somatosensory (S1) cortical identity and proper connectivity between the thalamus and cortex. In the second part, I have used the cortex-specific conditional mouse to understand whether COUP-TFI controls organization of the S1 barrel cortex via an activity-dependent mechanism. My data have first indicated that spontaneous cortical activity differs between motor and sensory neocortical areas during development, and that COUP-TFI is directly involved in this process by regulating the frequency and coordination of neuronal activity. As a consequence, COUP-TFI-deficient cortices are initially innervated by thalamic sensory nuclei, but these connections are not maintained over time, leading to an abruptly reduced barrel cortex. In addition, COUP-TFI expression is required for layer 4 cells to acquire a stellate morphology, a typical trait of S1 layer 4 neurons. By challenging possible targets involved in modulating neuronal activity in the cortex, I found that COUP-TFI directly regulates the transcription of the immediate early gene Egr1 and of a subset of ion channels. Re-expression of Egr1 in layer 4 cells of COUP-TFI mutant cortices allows these cells to acquire back their stellate morphology and to organize thalamic axons into functional barrels of the S1 area.

This study has unraveled a novel link between intrinsic transcriptional patterning and activitydependent mechanisms in the organization of the somatosensory cortex and its circuitry during mouse development. The Central Nervous System (CNS) is composed of a complex of structures that cooperate to collect inputs from the environment and the organism, to elaborate them in order to produce appropriate behavioral and metabolic responses. These structures differ greatly in the different groups of the animal phyla, where different CNS systems have distinct degrees of complexity. The neocortex is an extremely recent structure that evolved only in the mammalian CNS (Aboitiz et al. 2003;[START_REF] Supèr | The early differentiation of the neocortex: a hypothesis on neocortical evolution[END_REF][START_REF] Krubitzer | The evolution of the neocortex in mammals: How is phenotypic diversity generated?[END_REF][START_REF] Kaas | Neocortex in early mammals and its subsequent variations[END_REF][START_REF] Aboitiz | Genetic and developmental homology in amniote brains. Toward conciliating radical views of brain evolution[END_REF]. During mammalian evolution, neocortical dimensions grew at a higher extent compared to other structures, and the number of connections between the cerebral cortex and other CNS structures strongly increased [START_REF] Kaas | Neocortex in early mammals and its subsequent variations[END_REF]. Although in different mammalian species there is a remarkable variability of neocortical size and shape the cerebral cortex maintains its role in collecting and elaborating sensory inputs to generate appropriate behavioral responses (Aboitiz et al. 2003;[START_REF] Krubitzer | The evolution of the neocortex in mammals: How is phenotypic diversity generated?[END_REF][START_REF] Kaas | Neocortex in early mammals and its subsequent variations[END_REF][START_REF] Aboitiz | Genetic and developmental homology in amniote brains. Toward conciliating radical views of brain evolution[END_REF]. The neocortex is constituted by three major cell populations: excitatory neurons, which depolarize their target neurons through release of specific neurotransmitters (mainly glutamate in the cerebral cortex); inhibitory interneurons, which inhibit their target neurons (hyperpolarize their membrane potential); glial cells, which are involved in different trophic support functions, neuronal protection and axon myelination [START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF][START_REF] Halassa | Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior[END_REF].

Index

1.a. The formation of the neocortex during CNS development.

During mammalian development, the CNS is generated from the neural tube, a cylindrical structure of cells that folds from the ectoderm all along the rostro-caudal embryonic axis [START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF][START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF]. Once closed, at approximately E8.5-9.0 in mice, the rostral neural tube give rise to different structures: the rhomboencephalon, the mesencephalon and the prosenchephalon, from which the forebrain originates [START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF][START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF]. Subsequently, around E10 in mice, the rhombencephalon can be further subdivided into myelencephalon, caudally, and metenchephalon rostrally, whereas the prosencephalon can be subdivided into dienchephalon caudally and telencephalon rostrally (Figure i-1). The myelencephalon will give rise to the medulla, the mesencephalon to pons and cerebellum, and the diencephalon to thalamus, the hypothalamus and the neural components of the retina. Finally, the thelencephalon will give birth ventrally to the basal ganglia, dorsally to the paleocortex, neocortex and the archicortex and more rostrally to the olfactory bulbs [START_REF] Kaas | Neocortex in early mammals and its subsequent variations[END_REF][START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF].

While the neocortex will give rise to the cerebral cortex of mammals (which is the seat of higher brain functions such as thoughts, voluntary movements and consciousness), the ganglionic emincence (GE) will further develop into the striatum, an important structure involved in the implementation of voluntary movement (Figure i-2). The paleocortex will give rise to the olfactory and piriform cortex, while from the archicortex originates the hippocampus and enthorinal cortex [START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF][START_REF] Rubenstein | Patterning of the embryonic forebrain[END_REF], two structures involved in important functions: such as such as learning, memory and spatial orientation. At early stages, these five vesicles (myelencephalon, metencephalon, mesencephalon, diencephalon and telencephalon) are all constituted by a thin layer of pseudostratified cells, called neuroepithelium (NE), that highly proliferate to increase the pool of progenitors available for the development of each structure. It is during this developmental phase that dorsoventral patterning of the telencephalon takes place allowing a first subdivision of this structure into pallium (future cerebral cortex) and subpallium (ganglionic eminences), and a rough subdivision of the future cerebral cortex into three distinct regions (paleo-, neo and archicortex).

The neocortex is subdivided in different functional areas along the tangential axis. These areas are innervated and innervate specific brain structures depending from their function. In this introduction, I will describe the major mechanisms involved in neocortical development by putting into evidence some unresolved points regarding its ontogenesis, and highlighting the questions my thesis was aimed to address.

Corticogenesis.

An aspect of neocortical complexity underlying the function of different areas is the diversity of neuronal pools populating these domains. As mentioned above, the neocortex is organized into 6 layers of neurons forming separated and communicating radial functional units during neurogenesis [START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF][START_REF] Reillo | Germinal zones in the developing cerebral cortex of ferret: Ontogeny, cell cycle kinetics, and diversity of progenitors[END_REF]. Progenitors in the developing cortex undergo a series of symmetric (self-renewal) and asymmetric (neurogenic) divisions in order to either increase the tangential and radial size of the future cerebral cortex or give birth to both neurons and glia (at early and late stages respectively), which will migrate radially to populate the cerebral cortex (Figure i-3) [START_REF] Haubensak | Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis[END_REF][START_REF] Cameron | Glial cell lineage in the cerebral cortex: a review and synthesis[END_REF]. Ventricular progenitors synapse with neurons during development, a mechanism that controls several neuronal mechanisms during early phases. The main glial cell populations are oligodendrocytes, astrocytes and microglia, which are primordially involved in support for neurons, axon myelination, neurotransmitter recycling and structural support. Glia cells are now being more deeply investigated thanks to the development of new and more appropriate tools, revealing more important functions [START_REF] Allen | Neuroscience: Glia -more than just brain glue[END_REF]. To understand how principal neurons and glia cells are born and organized, I will summarize below what we know about cortical neurogenesis, or corticogenesis.

2.a. Cortical progenitors in the developing cortex.

At the onset of corticogenesis, the pallium is constituted by neuroepithelial (NE) cells which undergo fast symmetric divisions [START_REF] Cameron | Glial cell lineage in the cerebral cortex: a review and synthesis[END_REF]. These early divisions have the main purpose to increase the pool of multipotent cells, and thus expand the tangential size of the cerebral cortex. As multipotent cells, a NE cell is able to divide and self-renew for a long period of time, and thus expanding the progenitor population and maintaining the potentiality of generating all types of cells which will populate the mature neocortex [START_REF] Haubensak | Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis[END_REF]).

At E9.5-E10.5, NE multipotent cells are initially fate-restricted to become Radial Glial Cells (RGC), a population of progenitors which possess a bipolar morphology [START_REF] Bentivoglio | The history of radial glia[END_REF] and resides in the ventricular zone (VZ), which is the region of the cortex at direct contact with the lumen of the lateral ventricle (Figure i-4).

The first produced neurons are represented by the preplate (PP) cells located at the opposite side of the VZ and in contact with the pial membrane. RGCs have their soma positioned near the ventricle and two processes: the shortest is in contact with the basal lamina at the boundary between VZ and the ventricle lumen, whereas the longest one reaches the pial surface. The RGC population is maintained all along cortical development and its type of division can also give birth to other subsets of progenitors (such as intermediate progenitors and outer radial glia cells), which have the role to further expand the radial thickness of the cortex [START_REF] Stancik | Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex[END_REF][START_REF] Williams | Evidence for multiple precursor cell types in the embryonic rat cerebral cortex[END_REF]. RGC also produce neuron cells by asymmetric divisions, this process is therefore direct neurogenesis, as it does not rely on other types of progenitor cells. On the contrary, types of progenitors, born from RGC, will produce neurons and contribute to the indirect neurogenesis. Among the major RGC marker, there are Pax6 and Nestin, two transcription factors which are quickly downregulated in newborn neurons exiting the cell cycle. Intermediate Progenitor Cells (IPC) are an abundant population of transitory amplifying progenitor cells [START_REF] Kowalczyk | Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex[END_REF].

IPCs are located mostly in the upper VZ and in the Sub Ventricular Zone (SVZ), a structure which lies initially between the VZ and the PP. These progenitors are characterized by short or absent projections and mostly undergo few rounds of symmetric divisions (producing each two progenitors or two neurons) [START_REF] Martìnez-Cerdeño | The role of intermediate progenitor cells in the evolutionary expansion of the cerebral cortex[END_REF]. IPC cells express the transcription factor T-box 2 (Tbr2), which is downregulated in newborn neurons (Figure i-4).

Another subset of progenitors derived from the RGCs is represented by the outer radial glia cells, also called outer subventricular zone cells (OSVZ) in humans, where they are hugely expanded [START_REF] Fietz | OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling[END_REF]). These progenitors are localized at the basal margin of the SVZ, as IPCs do, but possess processes reaching the pia margin, but not the basal lamina of the VZ, have a kind of particular "jumping" behavior when they divide [START_REF] Hansen | Neurogenic radial glia in the outer subventricular zone of human neocortex[END_REF], and importantly do not express Tbr2 a IPCs [START_REF] Stancik | Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex[END_REF].

Another class of cortical progenitors is called Short Neural Precursors (SNP), a set of transiently amplifying progenitors located in the VZ. SNP do not possess a projection directed toward the pia, or they only have a very short one [START_REF] Gal | Molecular and Morphological Heterogeneity of Neural Precursors in the Mouse Neocortical Proliferative Zones[END_REF][START_REF] Stancik | Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex[END_REF]

2.b. Radial migration.

Newborn neurons have to migrate radially to give rise to the cortical plate (CP), which splits the PP into subplate (SP) and marginal zone (MZ), and represents the future cerebral cortex [START_REF] Nadarajah | Ventricle-directed migration in the developing cerebral cortex[END_REF]. At the end of development, the CP will be formed by 6 layers of glutamatergic neurons, in which the MZ will become layer 1 [START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF]. Radial migration of cortical neurons happens mainly in three different ways, which have different dynamics and underlying molecular mechanisms, although it is still debated which is the meaning of each migratory pattern [START_REF] Nadarajah | Two modes of radial migration in early development of the cerebral cortex[END_REF][START_REF] Loturco | The multipolar stage and disruptions in neuronal migration[END_REF][START_REF] Rakic | Mode of cell migration to the superficial layers of fetal monkey neocortex[END_REF][START_REF] Tabata | Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex[END_REF].

One mechanism is that of somal translocation and it is the main one used in direct neurogenesis.

In this case, neurons inherit from their progenitors the apical process to the pia and use it as a puller to progress toward the pial membrane [START_REF] Nadarajah | Two modes of radial migration in early development of the cerebral cortex[END_REF]).

Neurons moving with somal translocation also possess a short projection directed toward the ventricle (Figure i-5). This type of migration progresses at a constant pace with the shortening of the leading process and is common in the early phases of corticogenesis (until E12.5 -E13.5) and will give rise to PN of layers 5 and 6.

Once the soma reaches the projection beneath the pia, the translocation pauses for an hour and then proceeds to the end of it. The average speed of this migration is 60µm/h. A second mechanism is that of cell locomotion, which is at the basis of indirect neurogenesis and will give rise to superficial neurons in layers 4, 3, 2 [START_REF] Nadarajah | Two modes of radial migration in early development of the cerebral cortex[END_REF]. Migrating cells still possess a radially oriented projection (Figure i-5), which does not reach the pia and is on average 30-50µm long [START_REF] Nadarajah | Two modes of radial migration in early development of the cerebral cortex[END_REF]. This process terminates with a growth-cone like structure, and is attached to the filament elongated by RGC toward the pia, which is used as a structure to pull the soma radially. For this reason this mode of radial migration is also called glia-guided migration.

Cell locomotion happens in short and fast interval of myosin Ca ++ regulated contraction, due to this non-continuous movement the average speed is almost half of the somal translocation (35 µm/h). While these two migration modes are mostly aimed to move radially the soma of the differentiating neurons, there is a third type of migration, which is not specifically focused to approach the cell toward the pia [START_REF] Loturco | The multipolar stage and disruptions in neuronal migration[END_REF][START_REF] Tabata | Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex[END_REF].

Both of these modes of migration are conservative for the tangential position of the progenitor: neurons migrate radially maintaining the tangential of their respective progenitors. This third mode is called multipolar migration, as cells undergoing migration extend multiple thin and short processes, which dynamically extend and contract (Figure i-5). The molecular mechanisms underlying this migration are similar to those of glia-guided migration, although the movement is not always directed radially. Multipolar migrating cells can also move tangentially, moving thus away from the initial radial position of their progenitors. Different studies described different speed for this kind of multipolar migration, depending on the experimental conditions. An in vivo study reported a speed of 6.4 µm/h, which is remarkably slower than the other two modes of migration.

Once the leading process of migrating neurons reaches the pia, neurons encounter Cajal-Retzius cells, which populate the MZ. Cajal-Retzius cells express Reelin, which induces migrating neurons to detach from the radial glia scaffold [START_REF] Franco | Reelin Regulates Cadherin Function via Dab1/Rap1 to Control Neuronal Migration and Lamination in the Neocortex[END_REF]. Consequently, earliest born neurons will populate the lowest layers of the cortex, whereas neurons born later populate progressively the most superficial layers of the cortex. The specific timing of birth of upper and lower layer neurons was precisely determined by birthdating experiments [START_REF] Rakic | Neurons in Rhesus Monkey Visual Cortex: Systematic Relation between Time of Origin and Eventual Disposition[END_REF].

These different modes of migration are not used uniquely by specific cell types, although at early stages of corticogenesis (between E11.5 and E13.5 in mice) neurons use almost exclusively somal translocation. Later on, when the developing CP reaches a certain thickness neurons begin using glia-guided migration. Neurons leaving the VZ and SVZ have to move through an intricate structure of fibers in the intermediate zone (IZ) before reaching the CP. Since upper layer neurons, which mainly use cell locomotion, are also evolutionarily more recent, it is hypothesized that this migration mechanism may have evolved as a way for neurons to move more efficiently through a more complex CP structure. Also, multipolar migration is a mechanism that resembles the chemotaxic process, in which cells have to probe the environment in search for the best attracting/repelling cues. This mode of migration could therefore be also a way for migrate through brain structures with an increasing level of complexity.

These modes of migration are conserved among mammals and may have been crucial for allowing the neocortex to evolve to such different levels of complexity, since the same mechanisms are used to create brain structures like the small and lissencephalic mouse neocortex or the big and gyrified human neocortex.

2.c. The radial unit hypothesis.

The radial migration has the consequence of maintaining newborn neurons in the same radial position as their progenitors [START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Rakic | Mode of cell migration to the superficial layers of fetal monkey neocortex[END_REF]. Neurogenesis is therefore a process that does not increase the tangential size of the cortex, but only its thickness. Mouse mutants with altered timing of neurogenic and/or of self-renewing divisions show alterations in radial and/or tangential dimensions [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] Rakic | Decision by division : making cortical maps[END_REF][START_REF] O'leary | Comprehensive Developmental Neuroscience: Patterning and Cell Type Specification in the Developing CNS and PNS[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF]. Even though multipolar migration can slightly change the position of neurons with respect to their progenitors, the tangential translocation is very limited [START_REF] Tabata | Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex[END_REF].

Consequently, the molecular program and the positional information generated by the combination of molecular cues and patterning gene gradients in progenitors is transferred to their offspring [START_REF] Rakic | A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution[END_REF], The radial unit hypothesis was based on this principle, in which radial migration is linked to the protomap hypothesis: cortical progenitors have patterning information, determined by the combination of patterning genes, and the neurons born from each progenitor will migrate radially and organize a functional unit with characteristics determined by their progenitor transcriptional program (Figure i-6). This hypothesis states that the commitment of a neuron to a specific areal fate begins at the level of progenitors, before the last division, creating an important link between the tangential position of neurons and their function. It was also shown that neurons born from the same progenitors synapse prevalently among themselves, creating functional micro-columns which process data radially before sending inputs to other columns for higher level elaboration [START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Elsen | The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map[END_REF].

The mode of radial migration influences also the width of micro-columns. Since thinner functional units imply a higher number of elaboration units in a given cortical tangential domain, the regulation of radial migration is important for the final features of neocortical areas. Humans have very narrow functional units compared to mice, as all primates compared to other mammals [START_REF] Dimidschstein | Ephrin-B1 controls the columnar distribution of cortical pyramidal neurons by restricting their tangential migration[END_REF][START_REF] Abdel-Mannan | Evolution of cortical neurogenesis[END_REF].

However, the differences in input elaboration ability of the neocortex between humans/primates and other mammals are not only due to a tighter regulation of radial migration, but also to the small relative size of human/primate neurons compared to their brain dimension. While in all other mammals, the size of neurons increases with the size of the brain, in primates the size of

Patterning of the developing neocortex.

After the regionalization of the neural tube, the future neocortex is made up of an unorganized neuroepithelium (NE), which is undergoing rapid symmetric division to expand the pool of progenitors [START_REF] Haubensak | Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis[END_REF]. During this period, a network of signals begins and contributes to pattern the NE, which is initially a homogenous epithelium. This will lead to the acquisition of region-specific features and the graded expression of transcription factors (TFs) that will regulate the specification programs of rostral and caudal (motor and sensory, respectively)

areas [START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF][START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Comprehensive Developmental Neuroscience: Patterning and Cell Type Specification in the Developing CNS and PNS[END_REF]). These TFs are important for the size and shape of functional areas and therefore constitute a link between the initial signaling molecules and the final organization of different neocortical functional areas.

These patterning genes play crucial roles in neocortical patterning and are expressed in gradients with no clear-cut domains of expression (Figure i-7).

The earliest morphogenic signals acting on the developing pallium consist of Retinoic Acid (RA), expressed in the meninges and later in the olfactory placode, and of Sonic Hedgehog (Shh), expressed in ventral telencephalon, particularly in the pre-optic area [START_REF] O'leary | Comprehensive Developmental Neuroscience: Patterning and Cell Type Specification in the Developing CNS and PNS[END_REF][START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF][START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. The signaling centers identified so far are localized at the borders of the allocortical ring that is constituted by brain territories surrounding the developing dorsal pallium, a mechanism conserved in all mammalian species (Figure i-2). The allocortical embryonic territories give rise to the hippocampus, in its medial aspect, and to the olfactory cortex in the lateral one [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF].

The allocortical regions are confined with the septum (rostrally) and choroidal roof (caudodorsally), the prethalamic eminence (caudo-ventrally) and the subpallium (rostrally) [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. Three of these territories produce morphogens, which have long been investigated

for their role in cortical patterning. The first Fgf to be released during corticogenesis is Fgf8, at E9 in mice, followed soon by Fgf15, 17 [START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Cholfin | Frontal cortex subdivision patterning is coordinately regulated by Fgf8, Fgf17, and Emx2[END_REF]. Fgf8 is the most studied morphogen in cortical development, where it has been shown to antagonize Hem-expressed morphogens, such as BMPs (Bone Morphogenic Proteins) and WNTs (Wingless-Type MMTV Integration Site Family) [START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF][START_REF] Toyoda | FGF8 acts as a classic diffusible morphogen to pattern the neocortex[END_REF][START_REF] Ohkubo | Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles[END_REF]. On the other hand, Fgf8 establishes a positive loop with Shh, which is crucial to maintain its own expression during early stages of corticogenesis [START_REF] Okada | FGF8 signaling patterns the telencephalic midline by regulating putative key factors of midline development[END_REF]).

Fgf8-mediated antagonizing effect on caudal morphogens is also favored by Chordin and Noggin, which are as well expressed in the ANR. Until E13.5, Fgf8 expression plays a crucial role in organizing and specifying rostral regions of the cortex by promoting rostral areal patterning gens, such as Sp8 [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF]) and repressing caudal areal patterning genes, such as Emx2 and COUP-TFI [START_REF] Zembrzycki | Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain[END_REF]). Fgf8 induces the expression of Foxg1, a TF expressed in all the pallial territory, which regulates forebrain development. Foxg1 promotes indeed the ventral identity as it is a Shh downstream and it represses the Wnt signaling dorsally [START_REF] Danesin | A Fox stops the Wnt: implications for forebrain development and diseases[END_REF][START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF].

The main function of Fgf8 signalling on neocortical patterning is mediated in part by Sp8 expression and converges into organizing a rostral motor identity over a sensory one. This does not only regulates proper thalamic innervation, but also intracortical connectivity, which constitute a major source of inputs for motor cortical areas [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]). These functions have been observed in loss-of-function experiments by examining Fgf8 knock out and hypomorphic mutants, which lead to cortices with a reduced motor domain. Strikingly, in gain-offunction approaches in which Fgf8 is overexpressd by in utero electroporation in rostral regions, the barrelfield sensory domain is reduced, while if it is overexpressed in more caudal regions of the cortex it causes and inversion of the barrelfield orientation [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] Garel | Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants[END_REF][START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF]. Interestingly, overexpression of Fgf8 at early stages (E10.5), causes a duplication of the gradients that typically organizes the neocortex, resulting in a duplication of the neocortex organization [START_REF] Assimacopoulos | Fibroblast Growth Factor 8 Organizes the Neocortical Area Map and Regulates Sensory Map Topography[END_REF].

Fgfr3C is a receptor for Fgf8 expressed in cortical progenitor in a caudal high to rostral low gradient [START_REF] Reid | Differential expression of fibroblast growth factor receptors in the developing murine choroid plexus[END_REF]. Rostral overexpression of the cytoplasmic domain of Fgfr3 results in a reduction of Fgf8 activity, which causes arealization phenotypes similar to those observed in Fgf8 loss-of-function models. Fgf17 is an Fgf8 effector which is important for the specification of prefrontal cortical domains (PFC), a region which in primates is highly expanded and is involved in many of the highly elaborated cerebral functions. In Fgf17 loss-of-function mice, PFC reduction leads to impaired social recognition and behavior. Finally, Fgf15 has a crucial role in cortical neuron differentiation [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF], but its role in areal patterning is not clear.

3.c. WNTs and BMPs: the cortical Hem.

The hem is a portion of the neuroepithelium positioned close to the midline and extending from medial to caudal regions of the cerebral cortex [START_REF] O'leary | Comprehensive Developmental Neuroscience: Patterning and Cell Type Specification in the Developing CNS and PNS[END_REF][START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF][START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. The hem starts secreting BMPs and WNTs from E9 to almost the end of the embryonic life in mice [START_REF] Shimogori | Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex[END_REF]).

Bmp2,4,5 and 7 expression is detected in the hem at E9.5 and becomes shortly after restricted to the dorsal midline. At E10.5, Wnt3a is expressed by the cortical Hem, followed at E11.5 by Wnt5a and 2b. These morphogens act at the level of cortical progenitors in the ventricular zone (VZ),

where their receptors are expressed, and contribute in the specification of the dorso/ventral identity of pallial territories (Figure i-7). They promote dorsal identity and oppose the effect of anti-hem derived morphogens. Hem removal results in reduced cortical epithelium, which acquires features of ventral territories. In particular, ß-catenin activation by Wnts leads to expression of dorsal cortical markers, such as Emx1 and Ngn2. In experiments of ß-catenin reduction, Emx1 and Ngn2 expression decreases, whereas ventral thelencaphalic markers such as Dlx2, Mash1, Gsh2 increase. Moreover, Bmps reduce the expression of Lhx2 and Lhx5 [START_REF] Zhao | Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5[END_REF][START_REF] Monuki | Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-lhx2 pathway[END_REF], two transcription factors broadly expressed in the embryonic VZ in a high caudo-medial to low antero-lateral gradient [START_REF] Bulchand | LIM-homeodomain gene Lhx2 regulates the formation of the cortical hem[END_REF].

3.d. Sfrp2

, EGFs, Tgf-α and Fgf7: the Anti-hem.

The most latero-ventral region of the cortical ventricular zone is called anti-hem. This small region is at the boundary between pallial and subpallial territory (called pallial-subpallial boundary, PSPB), and is an important check-point for thalamo-cortical and cortico-thalamic axons elongation However and despite the variety of secreted morphogens, a specific role in cortical patterning has not yet been ascertained for this structure. Interestingly, the Paired box gene 6 (Pax6) mutant (small eye spontaneous mutation), which displays a reduction of the visual cortical areas, shows impairments in the specification of the anti-hem territory. Therefore, part of Pax6 phenotype could be derived by the loss of these anti-hem derived morphogens.

On top of that, the anti-hem can also regulate arealization due to its role in producting Cajal-Retizius cells. These cells express Dbx1 (Developing Brain Homeobox 1) since E10.5 and migrate tangentially to populate the cerebral cortex in the uppermost layer of the developing cortex. The ablation of Dbx1 expressing cells causes a change of patterning genes expression which leads to shift in cortical areas organization [START_REF] Griveau | A novel role for Dbx1-derived Cajal-Retzius cells in early regionalization of the cerebral cortical neuroepithelium[END_REF]). By comparing neocortical organization between different species, it became clear that the basic organization, in terms of size and reciprocal positioning, of the main functional areas (M1, S1, V1, A1) is basically unaltered [START_REF] Krubitzer | The evolution of the neocortex in mammals: How is phenotypic diversity generated?[END_REF]. Consequently, it was hypothesized that genetic regulation during development should play a critical role in their organization along the mammalian clade [START_REF] Elsen | The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map[END_REF]. Not only the relative size and position of these neocortical areas is similar in species with great difference in size and complexity, but also among species divided by millions of years of independent evolution (such as rodents, dolphins and marsupials).

Mitotic

It was also demonstrated that regionalized cortical marker expression and localization is independent from thalamic innervation, since animal models which lacked thalamo-cortical innervation are still able to correctly express early neocortical area markers [START_REF] Nakagawa | Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input[END_REF].

These data lead Pasko Rakic, a pioneer in this field, to formulate the protomap hypothesis, which states that an independent intrinsic (genetic) mechanism can decide the identify of future cortical areas within progenitor cells [START_REF] Rakic | A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution[END_REF][START_REF] Rakic | Decision by division : making cortical maps[END_REF]) According to this theory, cortical progenitors would acquire, during development, a given areal identity that would be later passed to their progeny, which in turn will constitute the future functional areas. Thus, the tangential size of individual cortical areas is decided by the proliferation of neuronal progenitors that acquired a given areal identity, while the thickness of each area is decided by the number of neurons generated, which once born migrate radially to the cortical plate. Subsequently to the formulation of this theory, a series of studies showed how patterning genes can regulate functional area organization. These genes are expressed from E8.5-9 in murine cortical progenitors in different gradients, as described above. Below, I will go in more details into the function of the 4 most known regulators involved in mouse area patterning at mitotic level:

Sp8, Emx2, Pax6 and COUP-TFI. Also I will discuss how intermediate progenitors may mediate the protomap plan from progenitors to differentiating cells.

4.a. Sp8.

Sp8 (Specificity Protein 8) is a zinc finger transcription factor that is expressed as early as E8-8.5 in the ANR and later from E9.5 in the developing cortex. Sp8 expression gradient follows faithfully Fgf8 gradient, being high rostro-medial to low caudo-lateral and this is due to the direct positive control of Fgf8 on its expression, confirming a strong link between morphogen and transcription factor expression during early corticogenesis [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF].

This link has been demonstrated after Fgf8 overexpression at E11.5, which leads to Sp8 overexpression, and by analyzing Sp8 cortical conditional mutants that recapitulate the Fgf8 hypormorphic cortical phenotype: cerebral cortex shrinkage with lack of the septum and rostral expansion of caudal markers (Ngn2, Pax6, Emx2) [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF]. Nonetheless, Sp8 loss-offunction leads also to a sensory mis-routing of thalamic innervation at E18.5, which does not happen in Fgf8 loss-of-function models, suggesting a specific role for Sp8 in the separation of different sensory territories. Unfortunately, Sp8 mutants die at birth and this does not allow a complete analysis of their area-specifc phenotype. Fgf8 and Sp8 are involved in a positive feedback loop, since Sp8 inactivation can negatively impinge on Fgf8 expression, as shown in Sp8 fl/fl Foxg1-Cre mice, where Sp8 is inactivated in the ANR [START_REF] Zembrzycki | Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain[END_REF]).

In addition, Emx2 can decrease Fgf8 downstream pathway activation by interfering with the interaction of Sp8 with Fgf8 regulatory sequences. Sp8 is at first induced by Fgf8 and creates a positive feedback loop further inducing Fgf8 expression, Emx2 prevents this loop thus indirectly reducing Fgf8 activity [START_REF] Zembrzycki | Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain[END_REF]).

Since Fgf8 can repress

4.b. Pax6.

Pax6 (Paired box 6) is a transcription factor expressed in the developing pallium VZ from E8.5 in a rostro-lateral high to caudo-medial low gradient [START_REF] Nakagawa | Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input[END_REF][START_REF] O'leary | Comprehensive Developmental Neuroscience: Patterning and Cell Type Specification in the Developing CNS and PNS[END_REF][START_REF] Bishop | Regulation of area identity in the mammalian neocortex by Emx2 and Pax6[END_REF]. The Pax6 expression gradient recalls the neurogenic gradient, also because Pax6 expression actually regulates the balance between proliferation and neurogenesis in cortical progenitors [START_REF] Asami | The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex[END_REF]. It promotes the transition from apical to basal progenitors and is involved in regulating the spindle orientation during cell division, one of the features, which differentiate symmetric to asymmetric divisions. Small eye Pax6 (Pax6 sey ) mice possess a spontaneous mutation inducing alterations in Pax6 function, probably a complete loss-of-function, although this is still debated [START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF][START_REF] Georgala | The role of Pax6 in forebrain development[END_REF]).

Pax6 sey/sey mutants have impaired eye formation, a general reduction of the cerebral cortex and the complete lack of thalamo-cortical (TC) innervation. These mutants die at birth and a complete analysis of arealization was thus not feasible, however regionalized cortical markers indicate a rostralization of sensory area (Figure 12345678). Pax6 overexpression, using a YAC (Yeast Artificial Chromosome) construct with a functional Pax6 allele, also failed to confirm these results. In Pax6 YAC mice, the position of the somatosensory area is not altered but its size is slightly increased, as indicated by the induction of pro-neurogenic and pro-apoptotic gene expression affecting progenitors of somatosensory neurons [START_REF] Berger | Conditional activation of Pax6 in the developing cortex of transgenic mice causes progenitor apoptosis[END_REF][START_REF] Manuel | Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization[END_REF].

Finally, Pax6 cortical-specific mutant (Pax6fl/fl Emx1-Cre) only showed a slight reduction of the somatosensory cortex without affecting neighboring areas. These mutants maintain TC axons innervation and survive after birth, contrary to Pax6 sey/sey , suggesting that Pax6 may act on TC axons elongation either earlier than Emx1-cre expression or in other structures apart the cerebral cortex, as Emx1 is not expressed in the thalamus, while the Pax6 sey/sey affects all tissues [START_REF] Piñon | Altered molecular regionalization and normal thalamocortical connections in cortex-specific Pax6 knock-out mice[END_REF]. Thus, the role of Pax6 in neocortical area patterning is still disputable.

4.c. Emx2.

Emx2 (Emtpy spiracles 2) is a TF expressed in mice starting from E8.5 in the rostro-lateral neural plate and later along the pallial VZ in a caudo-medial high to rostro-lateral low gradient [START_REF] Bishop | Regulation of area identity in the mammalian neocortex by Emx2 and Pax6[END_REF]. Accordingly, Emx2 promotes caudal cortical fate and its dose dependent function on caudal area specification is much more evident compared to Pax6 [START_REF] Mallamaci | Area identity shifts in the early cerebral cortex of Emx2-/-mutant mice[END_REF]. Emx2 is a downstream target of Wnt signaling, and as such, it is involved in regulating proliferation; thus its expression leads to an expansion of the occipital cortex [START_REF] Muzio | A mutually stimulating loop involving Emx2 and canonical Wnt signalling specifically promotes expansion of occipital cortex and hippocampus[END_REF]. Emx2 mutant mice show a reduced size of the occipital visual V1 area as well as an increase of the S1 with a corresponding caudal expansion of thalamic VPM axons, although thalamic development does not result affected. The data collected on several Emx2 mutants indicate that this factor act on areal patterning, not only by repressing Fgf8 expression, but also by specifying regional identity in the caudal cortex (Figure i-8) [START_REF] Li | Potential target genes of EMX2 include Odz/Ten-M and other gene families with implications for cortical patterning[END_REF].

As predicted, Emx2 overexpression under the Nestin promoter (Ne-Emx2) induces an opposite areal phenotype compared to Emx2 mutants [START_REF] Hamasaki | EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors[END_REF]). Ne-Emx2 mice show a dose dependent increase in V1 size that did not correlate with an overall cortical size change. Emx2

overexpression also reroutes dLGN axons to the expanded V1 territories, although axons fail to innervate L4 cells, but reach instead the marginal zone in L1. Accordingly, this effect was also observed in Sp8 mutant cortices, in which Emx2 was overexpressed. Collectively, these data show that differences in Emx2 gene dosage strongly affect position and size of neocortical areas.

4.d. COUP-TFI.

COUP-TFI (Chicken Ovalbumin Upstream Promoter Transcription Factor 1) is an orphan nuclear receptor of the steroid/thyroid superfamily [START_REF] Lu | An evolutionary conserved COUP-TF binding element in a neural-specific gene and COUP-TF expression patterns support a major role for COUP-TF in neural development[END_REF]. COUP-TFI acts mainly as a repressor, although in some cases it can promote transcription of its target genes [START_REF] Power | Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1[END_REF][START_REF] Hall | The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids[END_REF]. COUP-TFI function in the CNS development is evolutionarily conserved spanning from ctenophores to mammals [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]).

As for other patterning genes, COUP-TFI is also expressed from early stages of development, E8.5-E9.0 in mice, in cortical progenitors in a caudo-lateral high to rostro-medial low gradient, but in contrast to other patterning genes, COUP-TFI expression is maintained in post-mitotic cells up to adulthood, suggesting that it plays different functions at later stages [START_REF] Zhou | COUP-TFI: An intrinsic factor for early regionalization of the neocortex[END_REF][START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] Qiu | Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon[END_REF]. During cortical development, COUP-TFI proved to play a key role in the organization of sensory area features. COUP-TFI null mice, unfortunately die at birth, most probably due to defects in the development of the ganglion IX, which is important for feeding [START_REF] Zhou | COUP-TFI: An intrinsic factor for early regionalization of the neocortex[END_REF]. COUP-TFI null cortices show impairments in the subplate and L4 organization [START_REF] Zhou | The Nuclear Orphan Receptor COUP-TFI Is Required for Differentiation of Subplate Neurons and Guidance of Thalamocortical Axons[END_REF]) and regionalized markers are strongly affected (Figure i-8) [START_REF] Zhou | COUP-TFI: An intrinsic factor for early regionalization of the neocortex[END_REF].

Since thalamic axons fail to innervate the cortex in COUP-TFI null mice, it was initially not clear whether the sensory areas defect depended on the lack of thalamic innervation or on impairments in the intrinsic mechanisms specifying cortical areas. This question was later addressed by investigating cortical organization in mutants in which inactivation of COUP-TFI was restricted to the cerebral cortex. COUP-TFI fl/fl Emx1-Cre (EmxCKO) mutant mice showed defects similar to those observed in COUP-TFI null mice, even though conditional mice survive until adulthood without major problems and TC axons reach the cerebral cortex [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]). EmxCKO mice not only recapitulated the strong reduction and caudalization of all sensory territories somehow described in the null model, but showed for the first time a huge expansion of rostral motor areas. In terms of thalamocortical connectivity, thalamic axons from core-thalamic nuclei, such as dLGN and VPM, projected towards the caudalized sensory areas in mutant P0 and P7 cortices, whereas matrix-thalamic nuclei, such as POm nad VPMvl (innervating frontal motor regions in wildtype mice) also reach the parietal presumptive S1 (called mS1) in EmxCKO mice, confirming the expansion of rostral motor domains. Barrel-like structures constituting the main territories of normal S1 are strongly reduced and pushed to caudal cortical regions in EmxCKOs, similarly to null mutants [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF].

4.e. Tbr2 and AP2-γ: Intermediate progenitors as mediators of regional identity.

Along the evidence of the primordial map coded within apical progenitors, more recent studies investigated the role of intermediate progenitors (IP) as possible players in functional areas organizations. These evidences show how IP have a determinant role in propagating areal identity from apical progenitors to differentiating neurons [START_REF] Elsen | The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map[END_REF][START_REF] Pinto | AP2gamma regulates basal progenitor fate in a region-and layer-specific manner in the developing cortex[END_REF].

Tbr2 is a gene expressed in all IP in a high rostral to low caudal gradient, its expression regulates the balance between rostral to caudal cortical identity favoring the first. This wasas demonstrated studying mice with a cortical knock out of Tbr2, where caudal identity genes where invaded rostral domains [START_REF] Elsen | The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map[END_REF]. Comparison of microarray of Pax6, Tbr2 and Tbr1 mutants showed that the regional identity was more affected in Tbr2 mutants compared to Pax6 in both CP and SVZ, while Tbr1 mutant affected mainly CP regional identity, which supports the progressive propagation of the protomap from apical progenitors to IP from Tbr2 and finally to CP expressing Tbr1 [START_REF] Elsen | The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map[END_REF].

A different study showed how Tbr2 expression in IP is regulated by AP2γ, a transcription factor expressed in Pax6 cells and Tbr2 cells located in the VZ, but not in the SVZ, suggesting it could be mediating the shift from apical to basal progenitors [START_REF] Pinto | AP2gamma regulates basal progenitor fate in a region-and layer-specific manner in the developing cortex[END_REF]). AP2γ expression begins at E12 in high rostro-lateral to low caudo-medial gradient and further increases at E14 [START_REF] Pinto | AP2gamma regulates basal progenitor fate in a region-and layer-specific manner in the developing cortex[END_REF]. Microarray analisys of AP2γ-/cortex showed a specific effect in the regulation of caudal identity within IP cells. This result was also corroborated with defects in the mature cortex organization and function [START_REF] Pinto | AP2gamma regulates basal progenitor fate in a region-and layer-specific manner in the developing cortex[END_REF], strengthening the hypothesis tha AP2γ regulates caudal fate of IP cells.

Post-mitotic control of cortical development.

In more recent years, researchers have started to investigate the role of post-mitotic genes during cortical development. The developing neocortex displays a set of genes expressed in a regionalized and layer-specific pattern, which suggested a possible specific role. In addition, differentiation of the neocortex proceeds only after the onset of thalamocortical innervation, suggesting that final cortical maturation might be still malleable in post-mitotic neurons and at postnatal phases of

development (Figure i-9).
5.a. Post-mitotic regional-and laminar-expressing genes.

5.a.1. T-box brain 1 (Tbr1).

T-box brain 1 (Tbr1) is a transcription factor of the T-box family. It is expressed during cortical development in early born cortical neurons and later in L6 cortico-thalamic (CT) cells and in a subset of L2 cells [START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF]. During development, Tbr1 downregulates Fezf2 expression in L6 cells, favoring the CT projection fate instead of the SuPN one [START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF][START_REF] Mckenna | Tbr1 and Fezf2 regulate alternate corticofugal neuronal identities during neocortical development[END_REF]. Tbr1 also contributes to the specification of preplate (PP) cells, which are the earliest born cortical glutamatergic neurons [START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF]). The PP is subsequently splitted into subplate (SP) and marginal zone (MZ) by the arrival of new born cortical plate neurons. SP cells are crucial for driving correct thalamocortical innervation and initially organizing the circuit between cortex and thalamus. Tbr1 is thus involved in the correct organization of thalamo-cortical (TC) connectivity as well as callosal projections organization [START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF]). In addition, Tbr1 is expressed by Cajal-Retzius cells located at the MZ and regulates Reelin, essential for radial migration of cortical neurons [START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF]. Recently, Tbr1 has been shown to be expressed in a regionalized manner with a rostral-high to caudal-low expression gradient. It promotes rostral identity by upregulating expression of rostral-specific genes, some of which directly, as it is the case of Auts2, an autism related gene expressed in frontal cortices (Bedogni et al. 2010a)

5.a.2. Related orphan receptor-β (Ror-β).

Ror-β is an orphan nuclear receptor expressed mainly in L4 neurons, but also in L5a at lower levels.

Its expression begins at E15/E16 in L5 and at E18 in L4, it then increases in the sensory cortex during early post-natal days. This progressive increase in L4 turns to be activity-dependent, since input-deprived mice display a lower activation of Ror-β [START_REF] Jabaudon | RORβ induces barrel-like neuronal clusters in the developing neocortex[END_REF]. Layer 4

Ror-β positive cells in the mouse primary somatosensory cortex (S1) are progressively organized into cluster of cells, each topographically connected to thalamic VPM axons carrying sensory input from single whiskers [START_REF] Jabaudon | RORβ induces barrel-like neuronal clusters in the developing neocortex[END_REF]. This parcellation of L4 neurons is important for the correct organization of sensory inputs in S1, as it serves to properly separate topographically distinct axons. Ror-β is therefore upregulated by thalamic activity and is important to both maintain and refine thalamo-cortical circuits by restricting in space L4 neurons dendrites and allowing proper organization of barrels, as observed in acute Ror-β gene manipulations in vivo [START_REF] Jabaudon | RORβ induces barrel-like neuronal clusters in the developing neocortex[END_REF].

5.a.3. Basic helix-loop-helix transcription factor 5 (Bhlhb5).

Bhlhb5 is a transcription factor belonging to the basic helix-loop-helix family, which binds to E-box DNA sequences [START_REF] Xu | Functional and Structural Characterization of the Human Gene BHLHB5, Encoding a Basic Helix-Loop-Helix Transcription Factor[END_REF]. In the developing neocortex, it is expressed in SVZ and CP in a caudo-medial high to rostro-lateral low gradient. Postnatally, Bhlhb5 expression gradient acquires sharper borders and is finally restricted to the L2-5 of primary somatosensory (S1), visual (V1) and auditory areas (A1) at P4 (Joshi et al. 2008a). Bhlhb5 is involved in regulating different features of somatosensory areas by partially regulating S1-specific genes and organizing postsynaptic barrel structures (Joshi et al. 2008a), which is in part performed by creating a repressor complex with Prdm8, (PR Domain Containing 8) a histone methyl transferase expressed in L4 neurons [START_REF] Ross | Bhlhb5 and Prdm8 Form a Repressor Complex Involved in Neuronal Circuit Assembly[END_REF]). In addition, Bhlhb5 is also required in axon targeting of the corticospinal motor tract, since in its absence, the corticospinal motor neurons fail to reach the spinal cord [START_REF] Ross | Bhlhb5 and Prdm8 Form a Repressor Complex Involved in Neuronal Circuit Assembly[END_REF])

(Figure i-9).

5.a.4. Lim homeodomain 2 (Lhx2).

Lhx2 is a transcription factor of the Lim homeodomain family expressed in cortical progenitors in a caudo-lateral high to rostro-medial low gradient [START_REF] Shetty | Lhx2 regulates a cortex-specific mechanism for barrel formation[END_REF]. It is also expressed in postmitotic cells especially in upper layer neurons. Lhx2 expression in progenitors is necessary for the formation of upper layer neurons in S1 and for the topographic organization of segregated barrels in mice [START_REF] Shetty | Lhx2 regulates a cortex-specific mechanism for barrel formation[END_REF]). In the absence of Lhx2, somatosensory barrels are undetectable even if TC afferents seem to innervate the mutant cortex with normal regional specificity.

Electrophysiological recordings reveal a loss of responses evoked by stimulation of individual whiskers, but responses to simultaneous stimulation of multiple whiskers were present, suggesting that thalamic afferents are unable to organize the neurocircuitry for barrel formation.

Thus, Lhx2 function in the cortex is not only required in early patterning formation, but also later in organizing specialized neurocircuitry necessary for neocortical function [START_REF] Shetty | Lhx2 regulates a cortex-specific mechanism for barrel formation[END_REF].

Finally, a recent report showed that in post-mitotic cells, Lhx2 controls neocortical area patterning by regulating downstream genetic and epigenetic regulators that drive the acquisition of sensory properties in CP neurons by changing the balance of topographic connections and favoring the organization of somatosensory areas rather than visual ones [START_REF] Zembrzycki | Postmitotic regulation of sensory area patterning in the mammalian neocortex by Lhx2[END_REF]. Lhx2 is thus essential to maintain functional sensory area properties in cortical neurons post-mitotically, and acts as one of the terminal selector genes in controlling principal properties of neurons [START_REF] Zembrzycki | Postmitotic regulation of sensory area patterning in the mammalian neocortex by Lhx2[END_REF].

5.a.5. Cut-like homeobox 1, 2 (Cux1,2).

Cux1 and Cux2 are homeobox transcription factors specifically expressed in L2/3 and 4 neurons [START_REF] Nieto | Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex[END_REF] [START_REF] Bentivoglio | The history of radial glia[END_REF][START_REF] Cameron | Glial cell lineage in the cerebral cortex: a review and synthesis[END_REF][START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Reillo | Germinal zones in the developing cerebral cortex of ferret: Ontogeny, cell cycle kinetics, and diversity of progenitors[END_REF].

The INs (in mice) are born in the subpallium and migrate to the pallium through tangential migration from the ganglionic emincences during embryonic development [START_REF] Nadarajah | Neuronal migration in the developing cerebral cortex: observations based on real-time imaging[END_REF][START_REF] Corbin | Developmental Mechanisms for the Generation of Telencephalic Interneurons[END_REF][START_REF] Bartolini | Integration of GABAergic interneurons into cortical cell assemblies: lessons from embryos and adults[END_REF][START_REF] Lodato | Excitatory Projection Neuron Subtypes Control the Distribution of Local Inhibitory Interneurons in the Cerebral Cortex[END_REF].

PN type distribution and maturation are crucial for the correct formation of cortical circuits and to this aim, during development, specific time windows for cell migration and specification have to be met (Erzurumlu & Gaspar 2012a;[START_REF] Takasaki | Glutamate Transporters Regulate Lesion-Induced Plasticity in the Developing Somatosensory Cortex[END_REF][START_REF] Datwani | Lesion-induced thalamocortical axonal plasticity in the S1 cortex is independent of NMDA receptor function in excitatory cortical neurons[END_REF].

A series of transcriptional regulators have been identified for their crucial role in PN identity and specification [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]) and will be described in detail below. To describe the development of the different neuronal classes, I will subdivide PNs according to their hodology, namely their afferent and efferent connectivity, and their layer localization.

6.b. Intrathelencephalic neurons.

Intrathelencephalic neurons (IT) are a heterogeneous class of PN sending axons to the ipsi-and/or contralateral cortical hemisphere, striatum, amygdala and claustrum [START_REF] Fame | Development, specification, and diversity of callosal projection neurons[END_REF][START_REF] Leyva-Díaz | In and out from the cortex: development of major forebrain connections[END_REF]. They are evolutionarily more recent and their number and complexity increases in species with higher cognitive capabilities. These neurons are responsible for many of the high-level Input elaboration characterizing the neocortex, as they are involved in processing local inputs before the cortical output is produced, and integrating them to other cortical regions by further increasing the complexity of input processing.

6.b.1. Layer 4 intrathelencephalic neurons.

Of all IT classes, Layer 4 IT (L4;IT) neurons are a special class, as they mainly project radially to L2/3 and L5 and are responsible for starting the excitatory pathway within local circuits (Figure i-12.a and b) [START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Schubert | Cell type-specific circuits of cortical layer IV spiny neurons[END_REF]. In mice, they are born between E13.5 and L4py have a prominent apical dendritic tuft, L4sp neurons have a smaller apical dendritic tuft, whereas L4s have no apical dendritic tuft and spread their dendrites as short branches all around their soma. The latter have been long considered prototypical L4 neurons, but L4 is not exclusively populated by stellate cells in all areas and in all species. V1 of cats is dense of L4s as in S1, while in mice L4s are only found in S1 [START_REF] Smith | Fundamental differences between the thalamocortical recipient layers of the cat auditory and visual cortices[END_REF].

L4;IT receives inputs from mainly two sources: the thalamus and cortical neurons in L2/3 and/or L5. Thalamic axons innervating L4 connect both to interneurons and IT cells; the thalamic nuclei that project to L4 are core type thalamic nuclei belonging to the first order relay nuclei and conveying sensory inputs [START_REF] Reid | Specificity of monosynaptic connections from thalamus to visual cortex[END_REF][START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Jones | Synchrony in the interconnected circuitry of the thalamus and cerebral cortex[END_REF][START_REF] Clascá | Unveiling the diversity of thalamocortical neuron subtypes[END_REF]. L2/3 and L5 neurons are targets of L4 neurons and also the second most relevant source of inputs for L4 neruons themselves. L2/3 and L5 indeed send inputs to L4 as a feedback, although the amount of innervation is less abundant than that of thalamic afferents to L4 [START_REF] Feldmeyer | Excitatory neuronal connectivity in the barrel cortex[END_REF]. It is still not clear what are the genes required for L4;IT specification, although many studies described a set of genes important for L4;IT maturation An important gene is Ror-β, an orphan nuclear receptor, which is crucial for the organization of L4s and of the barrelfield, the most abundant region of the primary somatosensory area which receive topographic innervation from the contralateral whiskers [START_REF] Jetten | Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism[END_REF]. Ror-β and Brn1/2 reciprocally repress each other, as

shown by acute downregulation and upregulation of Ror-β [START_REF] Oishi | Mutually repressive interaction between Brn1/2 and Rorb contributes to the establishment of neocortical layer 2/3 and layer 4[END_REF]. Brn1/2 are initially expressed in all upper layers, and later while Ror-β expression increases specifically in L4, Brn1/2 is restricted to L2/3 [START_REF] Oishi | Mutually repressive interaction between Brn1/2 and Rorb contributes to the establishment of neocortical layer 2/3 and layer 4[END_REF]. Ror-β expression in L4 is very important for the organization of S1, since loss of Ror-β function in mice leads to impaired barrelfield organization (Jabaudon, Shnider, et al. 2012).

Another gene that seems to be involved in layer 4 organization is Prdm8, a DNA methyl transferase PR domain protein, which is specifically expressed in L4 and is necessary for proper organization of the primary somatosensory area (S1) in mice [START_REF] Inoue | Deletion of Prdm8 impairs development of upper-layer neocortical neurons[END_REF]. Finally, Bhlhb5, a transcription factor expressed in L5, 4 and L2/3 [START_REF] Xu | Functional and Structural Characterization of the Human Gene BHLHB5, Encoding a Basic Helix-Loop-Helix Transcription Factor[END_REF]Joshi et al. 2008), is important for the final organization of sensory domains. Part of Bhlhb5 function in L4 might be due to its role in regulating Prdm8 expression [START_REF] Ross | Bhlhb5 and Prdm8 Form a Repressor Complex Involved in Neuronal Circuit Assembly[END_REF]).

6.b.2. IT Commissural Pyramidal Neurons.

IT Commissural Projection Neurons (IT;CPN) are mainly involved in high level input elaboration in the neocortex [START_REF] Thomson | Functional maps of neocortical local circuitry[END_REF][START_REF] Douglas | Neuronal Circuits of the Neocortex[END_REF]. These neurons receive inputs from L4;IT and the thalamus. Their main function is to process inputs from the same subarea before sending outputs to other cortical areas or extracortical targets, therefore providing an important source of data elaboration [START_REF] Petreanu | The subcellular organization of neocortical excitatory connections[END_REF][START_REF] Lefort | The Excitatory Neuronal Network of the C2 Barrel Column in Mouse Primary Somatosensory Cortex[END_REF]. Their abundance varies greatly within mammalian species, but is in general the most abundant category of PN in the neocortex [START_REF] Fame | Development, specification, and diversity of callosal projection neurons[END_REF].

IT;CPN are also widespread distributed among layers, with the majority residing in L2/3 and 4

(around 80% in rodents), and the remaining in layers 5 and 6 (Aboitiz & Montiel 2003;[START_REF] Fame | Development, specification, and diversity of callosal projection neurons[END_REF][START_REF] Leyva-Díaz | In and out from the cortex: development of major forebrain connections[END_REF]. Thus, depending on their location, IT;CPN are mainly born between E14.5 and E15.5 (L2/3), and to a lesser extent between E12.3 and E13.3 (L5) [START_REF] Angevine | Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse[END_REF]. Beside their distribution, these neurons also vary in connectivity. All of CPNs project to homotypic regions of the contralateral cortex through the corpus callosum [START_REF] Caviness | Interhemispheric neocortical connections of the corpus callosum in the reeler mutant mouse: a study based on anterograde and retrograde methods[END_REF]. Therefore, the position of IT;CPNs also defines the contralateral cortical region they project to. But there are different subclasses of CPN projecting also to ipsi and/or contralateral striatum (Figure i-12.a and b) [START_REF] Wilson | Morphology and synaptic connections of crossed corticostriatal neurons in the rat[END_REF][START_REF] Mitchell | Large-scale maintenance of dual projections by callosal and frontal cortical projection neurons in adult mice[END_REF].

As other cortical neuron types, IT;CPNs mature during early development by elongating at first an exuberant number of projections, which are then pruned when correct activation of synapses occurs [START_REF] Mitchell | Large-scale maintenance of dual projections by callosal and frontal cortical projection neurons in adult mice[END_REF][START_REF] Mizuno | Evidence for activity-dependent cortical wiring: formation of interhemispheric connections in neonatal mouse visual cortex requires projection neuron activity[END_REF]. IT;CPN development requires a balance of activity between the two hemispheres, which directly influences their ability to innervate the contralateral cortex [START_REF] Suàrez | Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum[END_REF].

L2 IT;CPN receive innervation from thalamic matrix type nuclei, such as the PoM, while L3 ones receive strong innervation from L4;IT and core thalamic nuclei, making of these neurons an important asset of input elaboration and integration of information coming from different districts of the body [START_REF] Suàrez | Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum[END_REF]. The appearance and the diversity of IT;CPN neurons, although evolutionarily recent, quickly became a landmark of mammals to the point that IT;CPN are the most abundant neurons of the neocortex among all mammalian species (Aboitiz & Montiel 2003).

IT;CPN development is driven by a set of crucial determinants. The most important is Satb2

(Special AT-rich Sequence-Binding Protein 2) [START_REF] Szemes | Isolation and Characterization of SATB2 , a Novel AT-rich DNA Binding Protein Expressed in Development-and Cell-Specific Manner in the Rat Brain[END_REF]), a post-mitotic transcription factor expressed mainly, but not only, in upper layer neurons [START_REF] Alcamo | Satb2 Regulates Callosal Projection Neuron Identity in the Developing Cerebral Cortex[END_REF][START_REF] Britanova | Satb2 Is a Postmitotic Determinant for Upper-Layer Neuron Specification in the Neocortex[END_REF]. Satb2 was the first IT;CPN determinant gene identified and functionally characterized, as described in Satb2-/and Satb2 lacz/lacz mice [START_REF] Alcamo | Satb2 Regulates Callosal Projection Neuron Identity in the Developing Cerebral Cortex[END_REF][START_REF] Britanova | Satb2 Is a Postmitotic Determinant for Upper-Layer Neuron Specification in the Neocortex[END_REF]. On top of that, Satb2 upregulates other IT;CPN associated genes, such as Cux1, and downregulates genes leading to alternative neuronal fate, such as Ctip2, by recruiting the NURD complex on Ctip2 locus [START_REF] Baranek | Protooncogene Ski cooperates with the chromatin-remodeling factor Satb2 in specifying callosal neurons[END_REF][START_REF] Harb | Area-specific development of distinct projection neuron subclasses is regulated by postnatal epigenetic modifications[END_REF]. Recently, it was discovered that Cux1 and 2 are also important for IT;CPN development, connectivity and dendrite arborization, leading to proper innervation of callosal projections [START_REF] Rodríguez-Tornos | Cux1 Enables Interhemispheric Connections of Layer II/III Neurons by Regulating Kv1-Dependent Firing[END_REF][START_REF] Cubelos | Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex[END_REF]. Finally, Brn1

and 2, are expressed in superficial layers and regulate their correct lamination [START_REF] Sugitani | Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons[END_REF].

6.c. Corticofugal Projection Neurons.

Corticofugal Projection Neurons (CFuPN) constitute a wide group of neurons sending their axons to distant targets, which are located outside of the cerebral cortex (striatum, thalamus, pons, spinal cord). This group of neurons is evolutionary more ancient and establishes some of the longest connections in the CNS. Contrary to IT neurons, their connectivity is strongly correlated to their laminar distribution, as neurons located in different layers have distinct projection patterns [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF][START_REF] Fame | Development, specification, and diversity of callosal projection neurons[END_REF][START_REF] Gupta | Life is a journey: a genetic look at neocortical development[END_REF].

6.c.1. Subplate neurons.

Subplate neurons (SP) are a transient population of CFuPNs localized in the deepest layers of the neocortex. They are the first neurons born during neurogenesis (in mice around E11-E11.5) [START_REF] Mcconnell | Subplate neurons pioneer the first axon pathway from the cerebral cortex[END_REF]). At early stages, SP send axons to the developing thalamus which help thalamo-cortical axons pathfinding [START_REF] Deng | Corticothalamic and thalamocortical pathfinding in the mouse: Dependence on intermediate targets and guidance axis[END_REF]. SP project also axons to L4;IT creating a temporary circuit helping the establishment of thalamocortical connectivity during late stages of thalamocortical axon innervation [START_REF] Hanganu | Cellular mechanisms of subplate-driven and cholinergic input-dependent network activity in the neonatal rat somatosensory cortex[END_REF][START_REF] Luhmann | Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex[END_REF]), both of these topics will be discussed in further details in chapter 10.b and 10.c.

6.c.2. Corticothalamic Neurons.

Corticothalamic Neurons (CT), unlike SP, are a permanent neuronal population projecting to the thalamus (Figure i-12.c) [START_REF] Deng | Corticothalamic and thalamocortical pathfinding in the mouse: Dependence on intermediate targets and guidance axis[END_REF]. These neurons are born early after SP, between E11.5 to E12.5 in mice, and populate mainly L6 (and in a small part L5b) [START_REF] Angevine | Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse[END_REF][START_REF] Mcconnell | Cell Cycle Dependence of Laminar Determination in Developing Neocortex[END_REF]. Their axons project through the Internal Capsule (IC), which is located lateral to the thalamus [START_REF] Miller | The early development of thalamocortical and corticothalamic projections[END_REF], before reaching their targets in the thalamus.

CT receive rather few inputs from the thalamus, but are highly innervated by high order cortical areas, namely secondary and associative cortical areas [START_REF] Vàlez-Fort | The stimulus selectivity and connectivity of layer six principal cells reveals cortical microcircuits underlying visual processing[END_REF][START_REF] Harris | The neocortical circuit: themes and variations[END_REF]. CT also receive inputs from local L4 neurons and other local CFuPNs, as well as all types of thalamocortical projections [START_REF] Harris | The neocortical circuit: themes and variations[END_REF]. CT neurons target thalamic nuclei that innervate their local cortical areas in a topographic manner. This reciprocal topographic innervation is the base of an important fine-tuning of inputs relayed by the thalamus to the cortex [START_REF] Price | The development of cortical connections[END_REF][START_REF] Harris | The neocortical circuit: themes and variations[END_REF]. One of the major genes involved in CT specification is Tbr1 [START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF], which represses Fezf2 expression (FEZ Family Zinc Finger 2) [START_REF] Mckenna | Tbr1 and Fezf2 regulate alternate corticofugal neuronal identities during neocortical development[END_REF], a transcription factor involved in the specification of another important neuronal class:

the subcerebral projection neurons.

6.d. Subcerebral Projection neurons.

The second category of cortical PN elongating its axons along the IC are the Subcerebral Projection Neurons (SuPN) [START_REF] Miller | The early development of thalamocortical and corticothalamic projections[END_REF]. Their axons, once passed the IC, target different subcerebral regions such as the spinal cord, the pons, the medulla (Figure i-12.d) [START_REF] Lai | SOX5 Controls the Sequential Generation of Distinct Corticofugal Neuron Subtypes[END_REF][START_REF] Koester | Development of Projection Neuron Types , Axon Pathways , and Patterned Connections of the Mammalian Cortex[END_REF][START_REF] O'leary | Cortical axons branch to multiple subcortical targets by interstitial axon budding: Implications for target recognition and "waiting periods[END_REF]. This category of neurons populates mainly L5 and, in mice, they are born around E13.5. SuPN can be mainly subdivided into three types [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF];

Angevine & Sidman 1961): Cortico Spinal Motor Neurons (CSMN), cortico-bulbar and cortico-tectal PN. The first group projects to the spinal cord and have collateral projections to the striatum and the red nucleus. These neurons initiate voluntary movement and are therefore mainly populating neocortical motor areas [START_REF] Terashima | Anatomy, development and lesion-induced plasticity of rodent corticospinal tract[END_REF][START_REF] Shreyer | Topographic sequence of outgrowth of corticospinal axons in the rat: a study using retrograde axonal labeling with Fast blue[END_REF][START_REF] Jones | Growth and Maturation of the Rat Corticospinal Tract[END_REF]. These are well-characterized PNs, since they are affected in neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, a disease that characterized by the degeneration of motor neurons in the CNS, leading to a progressive muscular atrophy. The second group of SuPN (cortico-culbar PN) projects axons to the pons and medulla and are also involved in the implementation of voluntary movement. The last category of neurons, which resides mainly in the visual cortex, innervates the tectum and the superior collicollus and is involved in saccadic eye movements.

6.e. Fate specification of SuPN and CT neurons.

Over the past years, different studies investigated the role of post-mitotic regulation between subcerebral (SuPN) and cortico-thalamic (CT) neurons fate.

Fezf2 (FEZ Family Zinc finger 2) is one of the most important genes for SuPN specification (Figure i-15) [START_REF] Rouaux | Fezf2 directs the differentiation of corticofugal neurons from striatal progenitors in vivo[END_REF][START_REF] Mckenna | Tbr1 and Fezf2 regulate alternate corticofugal neuronal identities during neocortical development[END_REF][START_REF] Chen | The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex[END_REF]). This TF is expressed in a set of deep-layer neurons from their first specification up to adulthood and is necessary and required to promote SuPN fate. Nonetheless, Fezf2 is also expressed in a set of SP and CT neurons [START_REF] Hirata | Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons[END_REF]. In different studies it was reported how Fezf2 specifies SuPN fate over that of CPN and CT neuron fate. Fezf2 loss-of-function leads to SuPN identity loss, although cortical progenitors still produce L5 populating neurons, which acquire IT;CPN features, such as Satb2 expression and connections to the contralateral cortex through the corpus callosum [START_REF] Chen | The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex[END_REF].

Fezf2 also induces Ctip2 (COUP-TFI Interacting Protein 2) expression, another important TF controlling SuPN connectivty [START_REF] Chen | The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex[END_REF]).

Ctip2, just as Fezf2, is expressed strongly in L5 and is required for correct SuPN development. [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]. It was hypothesized that this dynamic is related to biologically significant events, aiming at finalizing the fate restriction of specific neuron types [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF].

Nonetheless, we recently demonstrated that Ctip2 expressing neurons are a heterogeneous population of whose connectivity is modulated by a combination of different TF even in adults.

The multiple combination of TF expressed is indeed associated with specific morphologica, electrophysiologic and projection profiles [START_REF] Harb | Area-specific development of distinct projection neuron subclasses is regulated by postnatal epigenetic modifications[END_REF]. Moreover, we showed that COUP-TFI regulates the balance of the different neuronal subpopulations by regulating the expression of Lmo4 a transcriptional regulator that prevents Satb2 mediated Ctip2 repression in layer 5 neurons.

This mechanism, and hypothetically other similar to this one, allows the existence of cortical neurons with a mixed transcriptional profile, increasing the cell-diversity of cortical neurons. (c) Changes in expression of these key regulators can cause boundaries between subtypes to shift, with neurons partially or completely acquiring features characteristic of other subtypes. In some mutants (for example, Satb2-null (Satb2-/-) and Sox5-/-), neurons acquire CFuPN identity generally rather than a welldefined CThPN or SCPN identity. The boundaries between CFuPN and deep-layer or superficial-layer CPN (represented by dashed lines) may shift independently of one another. Couptf1 cKO, chicken ovalbumin upstream promoter transcription factor 1-conditionalknockout (Couptf1fl/ fl; Emx1-Cre) mice; Ctip2, COUP-TF-interacting protein 2; FEZF2, fez family zinc finger 2; SATB2, special AT-rich sequence binding protein 2; SOX5 (SRY (Sex Determining Region Y)-Box 5), SRY-box containing protein5; TBR1, T-box brain protein 1 (taken from [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF].

The tangential organization of cortical areas.

The mentioned classes of neurons are differentially represented in the various tangential domains (called functional areas) in which the cerebral cortex is subdivided; it is this cytoarchitectural diversity that defines the different functions of distinct neocortical areas. Although the tangential dimension of the neocortex varies greatly among species, its general structure and organization is maintained among the different classes of mammals (Aboitiz et al. 2003;[START_REF] Krubitzer | The evolution of the neocortex in mammals: How is phenotypic diversity generated?[END_REF].

The Neocortical areas have been subdivided into different functional categories. Primary or low order areas are functional areas that elaborate topographically and in a time accurate manner, sensory inputs relayed by core-type thalamic projections that reach mainly L4;IT neurons and to a lesser extent L5 neurons [START_REF] Harris | The neocortical circuit: themes and variations[END_REF]. These nuclei usually convey somatosensory, visual and auditory inputs. The primary areas are, from the most rostral to the most caudal regions of the cortex, the primary motor area (M1), the primary somatosensory area (S1), the primary auditory area (A1) and the primary visual area (V1) in mice (Figure i-17) [START_REF] Rash | Area and layer patterning in the developing cerebral cortex[END_REF][START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. S1, V1 and A1 are similarly organized, as their main function is to gather sensory information of a specific modality. From primary areas, sensory inputs are elaborated with topographic specificity, and subsequently recruited to high order areas (namely secondary and associative areas), which further increase the elaboration of sensory inputs prior organizing behavioral responses [START_REF] Zampieri | Mapping sensory circuits by anterograde transsynaptic transfer of recombinant rabies virus[END_REF]. S1 areas receive axons from the Ventro-postero-medial nuclei (VPM), which convey somatosensory information from the skin, skeletal muscles and joints [START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Wimmer | Dimensions of a projection column and architecture of VPM and POm axons in rat vibrissal cortex[END_REF]. V1 area receives inputs, not only at the level of L4 but also of L6, from the dorsolateral geniculate nuclei (dLGN) which receives inputs from the retina [START_REF] Nassi | Parallel processing strategies of the primate visual system[END_REF]. A1 area is innervated by the medio-ventral geniculate nuclei (MGv) which convey information from the cochleae and inner ears [START_REF] Kimura | Auditory thalamic nuclei projections to the temporal cortex in the rat[END_REF]. M1 areas have few core-type innervations, but receive most of their innervations in upper layers from other neocortical areas

(Figure i-18
). These areas are defined as primary motor areas as they are populated by corticospinal neurons, which innervate interneurons in the contralateral the spinal cord, initiating voluntary movements [START_REF] Koester | Development of Projection Neuron Types , Axon Pathways , and Patterned Connections of the Mammalian Cortex[END_REF][START_REF] Armand | Cells of origin of crossed and uncrossed corticospinal fibers in the cat -A quantitative horseradish peroxidase study[END_REF]. Nonetheless they receive matrix type innervation from the postero medial nucleus (Po) [START_REF] Harris | The neocortical circuit: themes and variations[END_REF]. Secondary or higher order areas are deputed to higher elaboration of inputs that were previously processed in primary areas. These areas receive inputs from many other cortical regions and from matrix-type thalamic nuclei, such as the Po that convey mainly nociceptive information, and from the ventro-lateral part of the VPM, which is also innervated by nociceptive tracts [START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Pouchelon | Patterning of pre-thalamic somatosensory pathways[END_REF].

Prototypic cortical circuits.

The complexity and specificity of the interactions between cortical areas is extremely elevated, just as the amount of input and output connections. Nonetheless, research in the field managed to pinpoint key features of neocortical circuit organization.

In primary sensory areas, peripheral sensory inputs reach L4;IT neurons after being collected, elaborated and relayed by basal ganglia at first and core thalamic nuclei afterwards [START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Wimmer | Dimensions of a projection column and architecture of VPM and POm axons in rat vibrissal cortex[END_REF][START_REF] Kimura | Auditory thalamic nuclei projections to the temporal cortex in the rat[END_REF][START_REF] Binzegger | A Quantitative Map of the Circuit of Cat Primary Visual Cortex[END_REF]). Core thalamic nuclei axons synapse on both L4;IT and interneurons, which synapse on L4;IT back. The stimulation from thalamic axons creates an activation of L4;IT, followed by their hyperpolarization, driven by interneuron activity stimulated by the same thalamic axons. This simple mechanisms allows to initiate L4;IT activity from thalamic stimulation in an extremely timed-controlled pattern by first activating L4;IT and inhibiting them afterwards, allowing them to be stimulated by further inputs [START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Wehr | Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex[END_REF][START_REF] Wilent | Dynamics of excitation and inhibition underlying stimulus selectivity in rat somatosensory cortex[END_REF].

L4;IT send axons to L2;3 CPN neurons which further process the information and send inputs to lower layers or to other ipsi-or contro-lateral cortical areas. Lower-layer neurons in L5 of primary sensory areas send axons mainly to the striatum and only scarcely to the spinal cord [START_REF] Harris | The neocortical circuit: themes and variations[END_REF]. L6 CT neurons send inputs to the thalamus, providing a source of feedback to thalamic nuclei from the cortex [START_REF] Wang | Functional alignment of feedback effects from visual cortex to thalamus[END_REF]. Primary sensory areas L2/3 neurons strongly connect to motor domains and secondary sensory areas as well, initiating cortical activity in both of these regions [START_REF] Yamashita | Membrane potential dynamics of neocortical projection neurons driving target-specific signals[END_REF]

Endogenous activity of neocortical neurons during development.

During development, both progenitors and cortical neurons undergo a set of very tightly and rapidly regulated events, spanning from proliferation waves, migration, morphological remodeling and vesicle release. One of the main regulators of these fast processes in neurons is the

9.a. Calcium-dependent pathways modulate cortical proliferation.

Cortical proliferation is regulated in part by Ca ++ oscillation, which operates through different signaling mechanisms. One of these mechanisms is the activation of voltage-gated Ca ++ channels, which act independently on cortical progenitors in function of the extracellular environment [START_REF] Loturco | GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis[END_REF] by creating waves of synchronized Ca ++ oscillations that coordinate cortical proliferation. This process is regulated in part through gap junctions or hemichannels, which directly release molecules to the extracellular space. Release of ATP is also involved in mobilizing

Ca ++ by activating purinergic P2Y receptors [START_REF] Lin | Purinergic signaling regulates neural progenitor cell expansion and neurogenesis[END_REF][START_REF] Scemes | Reduced expression of P2Y1 receptors in connexin43-null mice alters calcium signaling and migration of neural progenitor cells[END_REF]). This synchronicity is not only regulated by signaling molecules but also by direct current diffusion between neighboring cells through gap junctions, in a model of diffusion similar to that of small-world networks [START_REF] Smedler | Network analysis of time-lapse microscopy recordings[END_REF][START_REF] Barabási | Network biology: understanding the cell's functional organization[END_REF].

9.a.1. Cortical neurons migration is controlled by Calcium activity.

The process of cortical neuron migration is strongly dependent on Ca ++ mobilization. Cell locomotion uses myosin contraction to help cortical neurons moving forward from the VZ to the pia, similarly to what happens in skeletal muscles [START_REF] Tsai | A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration[END_REF]. Somal translocation is driven by cytoskeleton remodeling and consequent process shortening, which also needs Ca ++ . While cortical progenitor migration and proliferation Ca ++ pathways require ATP signaling and P2Y

receptor expression, post-mitotic cortical cells migrate independently from ATP signaling. Postmitotic neurons lose P2Y and start their migration after expression of N-type Ca ++ channels [START_REF] Komuro | Selective role of N-type calcium channels in neuronal migration[END_REF][START_REF] Tabata | Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex[END_REF].

Cortical neuron migration is a strictly timing-regulated process, suggesting the existence of a mechanism of synchronization between groups of migrating neurons. Gap junctions are not used during neuronal migration of postmitotic cells, differently from what happens during synchronization of cortical progenitors. NMDA and GABA A are the main players in regulating cortical neuron migration through Ca ++ pathway. In particular, NMDA receptor activity regulates cortical neuron migration, whereas GABA activity controls interneuron migration by hindering cortical neuron migration [START_REF] Behar | Glutamate Acting at NMDA Receptors Stimulates Embryonic Cortical Neuronal Migration[END_REF][START_REF] Behar | GABA receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex[END_REF].

9.a.2. Calcium signaling and dynamics of axon elongation.

During the '80s it was first observed that electric activity is capable of modulating axon elongation dynamics through a Ca ++ regulated mechanisms [START_REF] Cohan | Suppression of neurite elongation and growth cone motility by electrical activity[END_REF][START_REF] Mattson | Components of neurite outgrowth that determine neuronal cytoarchitecture: influence of calcium and the growth substrate[END_REF]).

Interestingly, this process does not depend on synapse formation, as Ca ++ waves are observed before synapse formation. This process was also demonstrated in TC axonal elongation, which occurs during embryonic development from E13.5 to E15.5 (in mice). TC neurons undergo a specific wave of spontaneous activity, which activates transcription of the guidance receptor In both cases these molecules induce an increase of Ca ++ concentration in the growing side [START_REF] Song | Signal transduction underlying growth cone guidance by diffusible factors[END_REF].

Other evidence corroborated the model of the complementary role of neuronal activity and molecular signaling during axon elongation and map formation in sensory systems. In the retina, ganglion cells have waves of spontaneous activity that are important for creating topologicallymapped connection to the tectum [START_REF] Yates | Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development[END_REF]). This process happens through an initial overabundance of tectal connections from retinal cells, which are then pruned to select a more precisely set of mapped connections. In mice devoid of the Ephrin-β2 receptor, ganglion cells have waves of spontaneous activity, which are not coordinated between neighbouring cells, resulting in a less precise pruning process and a less precise mapping of connections. It was later demonstrated that the rate of spontaneous activity in ganglion cells directly influences the dynamics of branching and pruning, independently from the retinotopic map formation, suggesting that spontaneous activity might be involved more in these late events (branching and pruning) than in early patterning and mapping processes [START_REF] Benjumeda | Uncoupling of EphA/ephrinA signaling and spontaneous activity in neural circuit wiring[END_REF].

Similar mechanisms of axon branching and pruning have also been described in IT;CPN neurons, which elongate and innervate contralateral hemispheres of the cortex. This process happens in early post-natal days in mice and its efficiency depends on the balance of activity between the two hemispheres. Callosal neurons genetically targeted with the inward rectifier channel pKir2.1 fail to properly innervate the contralateral cortex, but when both of the CPN hemispheres are targeted by pKir2.1 expression, the efficiency of contralateral innervation is restored [START_REF] Suàrez | Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum[END_REF].

The rate of firing and contralateral innervation is also controlled by a CPN intrinsic transcriptional program: Cux1 directly regulates Kv1 expression in CPN neurons, which controls CPN firing rate and in turn their efficiency to innervate proper contralateral targets (Rodríguez-Tornos et al.

2016).

10. Establisment of thalamo-cortical circuits in the somatosensory area.

Thalamo-cortical circuit formation is a process that strongly depends on sensory-based activity, which progressively establishes and selects synapses between thalamic axons and cortical neurons (Erzurumlu & Gaspar 2012b). In the mouse neocortex, the mature primary somatosensory area is

10.a. Thalamo-cortical innervation between VPM and S1.

The topological map of whiskers innervation is maintained from the periphery to the brainstem and to the thalamus prior to reaching the neocortex. The Trigeminal Nucleus Principalis (PrV) innervation from the trigeminal nerve takes place around E13.5 to E15.5, although barrellets, clusters of cells which receive and project topologically whisker innervations from the trigeminal nucleus to the VPM thalamic nucleus, become distinguishable only after birth, around P1 [START_REF] Pouchelon | Patterning of pre-thalamic somatosensory pathways[END_REF]. PrV neurons target the VPM around E15.5, almost 3 days after VPM neurons birth, which takes place from E11 to E13 in the developing dorsal thalamus.

10.b. Guidance of TC trajectories from the thalamic nuclei to the developing cortex.

VPM neurons start early the innervation of the cortex, sending axons along the ventral thelencephalon from E13.5. These axons reach the developing cortex around E15.5 where they wait a couple of days before innervating cortical neurons. As previously discussed, this process depends on VPM neurons endogenous activity, which promotes axons elongation [START_REF] López-Bendito | Thalamocortical development: how are we going to get there?[END_REF].

Thalamic activity is not the only factor determining the elongation of thalamic axons toward the cortex; TC axons have to pass a series of structures which play each a role in leading their elongation. One of the early hypothesis, based on anatomical observation of developing TC and CT projection was that of the "handshake hypothesis" [START_REF] Molnàr | Mechanisms controlling the guidance of thalamocortical axons through the embryonic forebrain[END_REF]. This hypothesis stated the importance of descending projection from the cortex to guide incoming TC axons, although this hypothesis found both supportive and contrary data since when it was first stated in 1990 (Molnàr & Blakemore 1991;[START_REF] Molnár | Development of signals influencing the growth and termination of thalamocortical axons in organotypic culture[END_REF][START_REF] Adams | Complexities in the thalamocortical and corticothalamic pathways[END_REF][START_REF] Molnàr | Mechanisms controlling the guidance of thalamocortical axons through the embryonic forebrain[END_REF]). On the other hand, it was demonstrated that intrinsic transcriptional programs within the thalamus can influence TC axons pathfinding [START_REF] Mcconnell | Subplate neurons pioneer the first axon pathway from the cerebral cortex[END_REF]). Pax6, Gbx2, Emx2 and Neurog2 can indeed regulate the segregation of thalamic nuclei and regulate the targeting of TC axons [START_REF] Jones | Pax6 is required for the normal development of the forebrain axonal connections[END_REF][START_REF] Bulfone | Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries[END_REF][START_REF] Seibt | Neurogenin2 specifies the connectivity of thalamic neurons by controlling axon responsiveness to intermediate target cues[END_REF][START_REF] López-Bendito | Role of Emx2 in the development of the reciprocal connectivity between cortex and thalamus[END_REF] . It still remains to be demonstrated whether pools of TC neurons are topographically specified and which TF regulates this process.

On another level TC axons travel through the prethalamus, a region located ventrally to the developing thalamus, and the ventral thelencephalon, which will give rise to the hypothalamus, before passing the pallial-subpallial boundary (PSBP) [START_REF] Molnàr | Mechanisms controlling the guidance of thalamocortical axons through the embryonic forebrain[END_REF]. The prethalamus has indeed projections that grow toward the thalamus when TC axons extend in this regions, and so does the ventral thelencephalon [START_REF] Molnár | Mechanisms underlying the early establishment of thalamocortical connections in the rat[END_REF][START_REF] Braisted | Thalamocortical axons are influenced by chemorepellent and chemoattractant activities localized to decision points along their path[END_REF]. At the same time, the ventral thelencephalon expresses high level of Slit1/2, which have strong repellent effect to TC axons, allowing them to route toward the pallium [START_REF] Braisted | Thalamocortical axons are influenced by chemorepellent and chemoattractant activities localized to decision points along their path[END_REF][START_REF] López-Bendito | Robo1 and Robo2 Cooperate to Control the Guidance of Major Axonal Tracts in the Mammalian Forebrain[END_REF].

Being repelled by the ventral telencephalon, TC axons travel through the subpallium. This process is also regulated by a set of GABAergic neurons born in LGE and migrating into the MGE (López-Bendito et al. 2006). These cells are called corridor cells [START_REF] Deng | Corticothalamic and thalamocortical pathfinding in the mouse: Dependence on intermediate targets and guidance axis[END_REF] and express a membrane bound form of Neuregulin-1, ligand of ErbB4, which is in turn expressed by TC axons [START_REF] Bielle | Slit2 activity in the migration of guidepost neurons shapes thalamic projections during development and evolution[END_REF].

Another important migration cue is mediated by the expression of Zic2 allows neurons located close to the midline to not cross to the other side of the brain and project ipsilaterally. This process influence thalamo-cortical neurons as well and operates by inducing the expression of EphA4 [START_REF] Escalante | Zic2-dependent axon midline avoidance controls the formation of major ipsilateral tracts in the CNS[END_REF] 10.c. The innervation of VPM TC axons in the postnatal cortex.

Cortical innervation of TC axons is a process that takes place after birth when L4 is formed. At early postnatal stages, TC axons are guided to their target also by a fine control between Ephrins and Eph recepors. The most important for TC axons innervation are Ephrin-A4, A5 and B3, while most important Eph receptors are EphA3, A4 and A7 (Figure i-22). VPM and dLGN, both core-type thalamic nuclei, express these receptors, which are expressed at a high rostro-medial to a low caudo-medial gradient between E13-E15 [START_REF] Dufour | Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes[END_REF]. VPM and dLGN and send their axons to cortical layers which strongly express Ephrin-A5 in L4, mediating an attractive interaction with the receptors expressed. Ephrin-B5 has instead a repelling effect on VPM and dLGN axons, as it is expressed strongly in L5, L3/2 and the amygdala [START_REF] Mann | Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections[END_REF][START_REF] López-Bendito | Thalamocortical development: how are we going to get there?[END_REF]. Ephrins guidance relevance is most likely to end after axons reach their target layers, terminating at around P4, when also structural plasticity of VPM axons ends [START_REF] Durham | Effects of neonatal whisker lesions on mouse central trigeminal pathways[END_REF]. b a ndeed, in the time window between P2-P4, whisker lesions resuls in TC axons rearrangement: VPM axons corresponding to whiskers in close proximity to the lesioned ones will innervate S1 regions that would have otherwise been innervated by axons related to the latter.

Consequently, the final organization of single barrel structures in S1 appears in the cortex around P5-P7 and happens as a consequence of two distinct events. On one side, the separation of two neighboring clusters depends on the organization of the septa structures, narrow regions in L4 separating two barrels, that receive Po instead of VPM afferents [START_REF] Furuta | Septal Neurons in Barrel Cortex Derive Their Receptive Field Input from the Lemniscal Pathway[END_REF]. Septa neurons project cortico-cortically in larger areas compared to barrel neurons [START_REF] Kim | Barrels and septa: Separate circuits in rat barrel field cortex[END_REF].

On the other side, the presence of barrel or stellate cells, which organize dendritic receptive fields within the barrel region assuming a stellate morphology (Espinosa et al. 2009)(Figure i-24); This part will be further described in the following paragraph.

Finally, from P10 to P14 synapses between VPM TC axons and L4 and between L4 to L3/2 undergo a critical plasticity period. This period directly influences the specificity of the receptive field of these neurons. L4 receptive maps are the first to mature at around P12, while L3/2 neurons mature later at about P14 in rats [START_REF] Stern | Rapid development and plasticity of layer 2/3 maps in rat barrel cortex in vivo[END_REF]. This process depends strongly on experienceinduced synaptic plasticity, as sensory deprivation in these time windows directly hampers the receptive field specificity [START_REF] Lendvai | Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo[END_REF]. Sensory deprivation not only changes spatial organization of inputs, but also specific firing patterns of L3/2, such as the transition from a phasicfast adapting pattern to a regular less adapting one [START_REF] Maravall | Development of intrinsic properties and excitability of layer 2/3 pyramidal neurons during a critical period for sensory maps in rat barrel cortex[END_REF]).

Moreover, co-culture experiments of developing thalamus and somatosensory cortex showed that L4 innervation of VPM axons depends on the spontaneous activity of both thalamus and cortex [START_REF] Uesaka | Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching[END_REF]. Embryonic dorsal thalamus and early post-natal somatosensory cortex in coculture progressively increase their rate of firing activity in the first 2 weeks in vitro in a synchronized pattern. Thalamic axons progressively grow into the co-cultured cortex in a process which is disrupted when applying blockers of Na+ currents and NMDA receptors [START_REF] Uesaka | Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching[END_REF]).

Evoked activity plays therefore an important role in the final organization of the circuit between VPM and somatosensory cortex.

10.d. Subplate neurons relay information between thalamic axons and Layer 4 neurons.

One of the established functions of SP neurons is that of amplifying thalamic inputs in the developing cortex [START_REF] Hoerder-Suabedissen | Molecular diversity of early-born subplate neurons[END_REF]. Due to the transient nature of this function, which becomes unnecessary after synapses between thalamic and cortical cells have been established, SP neurons are gradually eliminated after P10. In more mammals with larger and more complex cortices, such as primates, the number of SP neurons is higher than in mice, although their number peaks during embryonic development and reduces before birth (Hoerder-

Suabedissen & Molnár 2015).
SP axons mostly reach L4 neruons with which they form chemical or electrical synapses. Targets of SP cells are not only immature neurons, but also neighboring SP neurons, which whom they form a gap junction columnar syncytium during early post-natal days in mice [START_REF] Luhmann | Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex[END_REF].

SP neurons receive afferents from the thalamus and elicit an amplifying activity to cortical neurons through different synaptic mechanisms, which depend on the SP type [START_REF] Hanganu | Cellular mechanisms of subplate-driven and cholinergic input-dependent network activity in the neonatal rat somatosensory cortex[END_REF][START_REF] Kilb | Glycine receptors mediate excitation of subplate neurons in neonatal rat cerebral cortex[END_REF][START_REF] Luhmann | Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex[END_REF]. During post-natal maturation, SP axons in S1 in the period of barrel clusters organization. At P0, SP axons are randomly dispersed in in the volume of L4 and, organizing both inside and outside the clusters of thalamic axons. Later on, at P6, when clusters can be visualized, SP axons appear to organize inside clusters together with TC axons and later on at P10, SP axons are excluded from the inside of the cluster. This dynamic process suggests a temporary support role between TC axons and L4 neurons prior to the circuit maturation [START_REF] Piñon | Dynamic integration of subplate neurons into the cortical barrel field circuitry during postnatal development in the Golli-tau-eGFP (GTE) mouse[END_REF].

10.e. L4 Stellate cell origin and function during S1 cortical development.

In mice, stellate cell formation in S1 is a feature that strongly depends on the organization of the thalamo-cortical circuit. These cells are a specific feature of the S1 organization and appear only after TC axons connects to L4. The dendritic organization of stellate cells is responsible for the precise localization of VPM axons in separated clusters.

Stellate cell morphology is regulated intrinsically by the expression of Ror-β, which determines the clustering of neurons into barrel structures in the developing cortex (Jabaudon, Shnider, et al. 2012). Ror-β expression and S1 identity are regulated by patterning genes, as observed in COUP-TFI mutant models, indicating that intrinsic cortical properties can regulate stellate cell formation even though they only acquire this feature after VPM axons synapse on L4 [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]).

Several experimental evidences demonstrate that S1 identity and stellate cell organization depend directly on the activity of VPM afferents. Two separate papers show that blocking VPM activity by inactivating the metabotropic glutamate receptor 5, mGlur5, in cortical neurons and the vesicular glutamate transporters Vglut1 and Vglut2 in the thalamus, lead to the complete loss of VPM stimulation on the cortex [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF][START_REF] Ballester-Rosado | mGluR5 in cortical excitatory neurons exerts both cell-autonomous andnonautonomous influences on cortical somatosensory circuit formation[END_REF].

In these mouse models, the barrel organization in S1 is strongly affetcted and barrels lack a clear separation. Most importantly, Vglut1/2 inactivation does not affect Ror-β expression, although it results in the loss of stellate morphology and ectopic acquisition of pyramidal morphology in S1 L4 neurons [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF]). Thus, not only intrinsic genetic programs in cortical neurons, but also the activity of the VPM is essential for the maturation process of the barrel cortex.

Thalamic innervation is also important in determining the primary versus secondary identity of the cortical target regions, independently from the intrinsic cortical program, suggesting that postmitotic cortical neurons can maintain a certain degree of plasticity postnatally. This was mainly demonstrated by comparing the transcriptomic profile of L4 neurons in control cortex and in mice in which the VPM nucleus was genetically ablated. In this model, VPM axons are replaced by Po axons and the transcriptomic programs of S1 L4 neurons results to be similar to that of S2, which is normally innervated by Po axons (Pouchelon et al. 2014).

Immediate early genes and sensory areas.

It is well reported that neuronal stimulation has a strong effect on transcriptional activity [START_REF] Kaczmarek | Sensory regulation of immediate-early gene expression in mammalian visual cortex: implications for functional mapping and neural plasticity[END_REF][START_REF] West | Biological Functions of Activity-Dependent Transcription Revealed[END_REF]. While some of the fastest neuronal responses do not depend on novel mRNA transcription, others are longer lasting, do depend on activated mRNA transcription and often involve structural modifications of neurons. Changes in transcriptional activation have become a target for studying processes of adaptation of sensory maps in function of different sensory experience [START_REF] Erzurumlu | Neural activity: Sculptor of "barrels" in the neocortex[END_REF]. The investigation of the molecular mechanisms responsible for this process led to the identification of the class of Immediate Early Genes (IEG), a set of genes which is expressed in a few hours after stimulation and can encode different set of proteins, such as growth factors, cell-surface receptors, protein kinases, and transcription factors [START_REF] Sukhatme | Early transcriptional events in cell growth: the Egr family[END_REF][START_REF] Staiger | Immediate-early gene expression in the barrel cortex[END_REF][START_REF] Curran | Fos: An immediate-early transcription factor in neurons[END_REF]. Primary sensory areas have a rather enriched expression pattern of some of these IEG genes, which have been investigated in the mammalian visual and somatosensory systems [START_REF] Kaczmarek | Sensory regulation of immediate-early gene expression in mammalian visual cortex: implications for functional mapping and neural plasticity[END_REF][START_REF] Filipkowski | Inducing gene expression in barrel cortex--focus on immediate early genes[END_REF].

Some studies compared the expression of IEG transcription factors in different paradigms of sensory stimulation to study whether the activation of IEG genes can be correlated with the learning and plasticity ability of mammalian cortices [START_REF] Wallace | The effect of vibrissa deprivation pattern on the form of plasticity induced in rat barrel cortex[END_REF][START_REF] Glazewski | The role of alpha-CaMKII autophosphorylation in neocortical experience-dependent plasticity[END_REF][START_REF] Dubroff | Use-dependent plasticity in barrel cortex: intrinsic signal imaging reveals functional expansion of spared whisker representation into adjacent deprived columns[END_REF]), but do not correlate with general neuronal metabolic dynamics [START_REF] Dragunow | The use of c-fos as a metabolic marker in neuronal pathway tracing[END_REF].

Some of the IEG transcription factors, strongly associated with sensory areas, are Arc, cFos and Egr1, which display different expression patterns and timing, most likely used by cortical neurons to obtain appropriate set of responses to different stimulation [START_REF] Filipkowski | Inducing gene expression in barrel cortex--focus on immediate early genes[END_REF][START_REF] Staiger | Immediate-early gene expression in the barrel cortex[END_REF]).

11.a. Arc.

Arc (activity-regulated cytoskeleton-associated protein) is a gene that was isolated from seizure stimulated hippocampus methods. It is expressed in the neocortex from P8, peaking at P21 in mice. Arc expression is upregulated after 30 minutes from seizure induction and remain upregulated for 8 hours after [START_REF] Lyford | Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites[END_REF]. It is expressed in the neocortex from P8, peaking at P21 in mice. Arc mRNA localizes in the dendrite terminals ant the resulting protein associates with F-actin suggesting its role in modifying dendrite morphology [START_REF] Lyford | Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites[END_REF].

11.b. cFos.

cFos is a transcription factor that forms heterodimers with other transcription factor, such as Jun.

In the mature S1, cFos is normally expressed in scattered patterns and at low concentrations, and strongly increases after sensory stimulation [START_REF] Mack | Induction of transcription factors in somatosensory cortex after tactile stimulation[END_REF][START_REF] Melzer | Stimulus-dependent expression of immediate-early genes in rat somatosensory cortex[END_REF]. cFos expression strongly increases in L4 and to a lesser extend also in L5, and is also specifically localized in cortical regions directly activated by whisker stimulation in rodents [START_REF] Bisler | Expression of c-Fos, ICER, Krox-24 and JunB in the whisker-to-barrel pathway of rats: Time course of induction upon whisker stimulation by tactile exploration of an enriched environment[END_REF].

Consequently cFos downstream pathway activation regulates experience dependent neuronal plasticity events (Sassone-Corsi 1995; [START_REF] Barth | Upregulation of cAMP response element-mediated gene expression during experience-dependent plasticity in adult neocortex[END_REF].

11.c. Egr1.

Egr1 (Early growth response 1) is a transcription factor of the zinc finger family involved in regulating the balance of differentiation and mitogenesis. Egr1 is also known under other acronyms, such as Krox-24, Zif-268, NGFI-A and ZENK [START_REF] Veyrac | The transcription factor Zif268/Egr1, brain plasticity, and memory[END_REF]. Differently from cFos, Egr1 basic expression in sensory areas does not depend on sensory-evoked stimulations [START_REF] Mack | Induction of transcription factors in somatosensory cortex after tactile stimulation[END_REF]. It is endogenously expressed in L4 and L6 neurons, but nevertheless dependent on thalamic activity, since disruption of thalamic inputs in to the cortex leads to strong Egr1 downregulation [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF]). Egr1 expression is not unique to these layers, as it can be detected in other layers as well, although at lower levels.

Different studies have reported that Egr1 function is linked to NMDA, AMPA and L-type Ca ++ channels activity [START_REF] Worley | Constitutive expression of zif268 in neocortex is regulated by synaptic activity[END_REF][START_REF] Wang | Role of kainate/AMPA receptors in induction of striatal zif/268 and preprodynorphin mRNA by a single injection of amphetamine[END_REF][START_REF] Murphy | L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes[END_REF]) and that its expression increases after birth, peaking after the first 2 weeks. The exact role of Egr1 in the sensory cortex is still unclear, although it has been associated to key features of neuronal plasticity. In the hippocampus and, particularly, the dentate gyrus, Egr1 expression is necessary in granule cells for long term potentiation (LTP) [START_REF] Cole | Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation[END_REF][START_REF] Wisden | Differential expression of immediate early genes in the hippocampus and spinal cord[END_REF]). In vivo, behavioral studies of mice null for Egr1 have unraveled its role in memory learning in different paradigms [START_REF] Veyrac | The transcription factor Zif268/Egr1, brain plasticity, and memory[END_REF]).

Egr1 regulation is downstream of the MAPK/ERK, JNK pathway and is able to downregulate its own transcription, allowing thus a tight activity-dependent temporal regulation of its own transcription [START_REF] Karin | Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus[END_REF][START_REF] Yang | Temporal recruitment of the mSin3A-histone deacetylase corepressor complex to the ETS domain transcription factor Elk-1[END_REF].

12. Link between neuronal activity, COUP-TFI and Egr1.

Different studies highlighted the link between neuronal activity, COUP-TFI and Egr1 expression. In the olfactory bulb and in mesenchimal cells, the transcriptional regulator COUP-TFI can control Egr1 expression [START_REF] Bovetti | COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons[END_REF][START_REF] Tsai | Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): coming of age[END_REF]. These results show that COUP-TFI protein can bind the Egr1 locus by forming a complex with Sp1 protein, which recognizes a sequence at the 5' of Egr1 sequence [START_REF] Pipaón | COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site[END_REF]). In the olfactory bulb, protocols of acute odour stimulation increases COUP-TFI in dopaminergic cells, which regulates Egr1 expression, resulting in a defective response of Tyrosine Hydroxilase expression [START_REF] Bovetti | COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons[END_REF]). The odour stimulating protocol induces the expression of COUP-TFI and Egr1 alike in the same group of cells and in COUP-TFI loss-of-function models the increase of Egr1 expression from basal conditions to stimulated condition is inhibited. Thus in the olfactory bulb, Egr1 expression increase in activity dependent condition requires COUP-TFI expression, which may be necessary to regulate Egr1 locus to be promptly expressed upon stimulation [START_REF] Bovetti | COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons[END_REF]).

More recently it was also demonstrated that COUP-TFI expression in the neocortex is affected in bilateral eye enucleated mice. In these experiments, mice where blinded at birth and analyzed at adult state. In enucleated mice visual area size was reduced, while S1 area was significantly increased. Also, V1 areas had reduced expression of COUP-TFI, in all layers and Ror-β in L4 [START_REF] Abbott | The effects of lifelong blindness on murine neuroanatomy and gene expression[END_REF]. As in the previsou study, COUP-TFI expression is regulated by the sensory experience, increasing evidence that this TF is regualated by mechanisms involved in neuronal activity downstream pathway.

In the neocortex it is although not very clear whether and how COUP-TFI regulation by neuronal activity has specific functions which differ by the regular expression of this TF. Considering the amount of control this expression of this gene can have on general cortical organization [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF], investigating this mechanism may reveal more insight on how cortical neurons organize to have specific responses to sensory evoked activity.

Overall aim of this work.

My Ph.D. thesis aims in understanding the influence of post-mitotic intrinsic programs and electric activity on the organization of functional areas in the developing neocortex. Transcriptional regulators expressed in cortical progenitors are known to play crucial roles during cortical area patterning. In spite of that, few studies have investigated how transcriptional information, defining area-and laminar-specific properties, are translated from progenitors to cortical neurons and how developing neurons interact with activity-dependent intrinsic and extrinsic signals.

To address these topics, I have first investigated whether patterning information can also influence cortical area organization within post-mitotic cells instead of progenitors, as mainly suggested by the literature. To this purpose, I have dissected the role of the areal patterning gene COUP-TFI, expressed in both mitotic and post-mitotic cortical cells, independently in the two cell populations by using a whole range of transgenic mouse lines. Areal and laminar identity, as well as connectivity between the thalamus and cortex, have been assessed in mice in which COUP-TFI is absent in both cortical progenitors and their progenies, or only in post-mitotic cells.

The analysis of these mice has allowed me to go further and investigate whether neuronal activity can influence the intrinsic properties of cortical cells during the subdivision of the developing neocortex into functional areas. Spontaneous activity, which is present early in the embryonic brain, and experience-driven activity during the postnatal period are both critical for circuit development. Several studies have highlighted the essential contribution of electrical activity to the development of topographic organization of cortical circuits. Although intrinsic genetic mechanisms are key to sculpture the primordium of the neocortex, extrinsic activity-dependent factors might also control those events, which will ultimately shape neocortical organization and cell-type specification during corticogenesis.

Since COUP-TFI expression seems to respond to external sensory stimuli and to control cortical sensory identity and TC circuitry, I used the cortical conditional mouse to understand whether and how endogenous spontaneous activity contribute to the generation of a primitive connectivity map between the thalamus and cortex. The hypothesis is mainly based on the evidence that layer IV cells of the somatosensory cortex are mis-specified and do not receive thalamic afferents in the absence of COUP-TFI. As a consequence, no barrel cortex is formed in the somatosensory mutant cortex.

Genomic DNA extraction.

Genotyping samples from live animals were collected from earmarks at the age of P21, while for younger animals and embryos genotyping samples were taken from the tip of the tail after euthanasia. Samples were dissolved incubating for 2h minimum at +58°C in rocking agitation in 500µl Lysis buffer and 2mg/ml Proteinase-K. After tissue dissociation the genomic DNA was precipitate adding 500µl of Isopropanol and mixed by inversion. The precipitate was collected via centrifugation for 10' at 14kg. The supernatant was removed by aspiration and the pellet wash washed with 500µl of 70% ethanol, centrifuged 10' at 14kg and let dry. The pellet was then resuspended in H 2 O mq, 70µl from earmarks and 400µl for post-mortem tissues.

2. Mutant Mice lines and Genotype.

2.a. COUP-TFI fl/fl Emx1-IRES-Cre.

In order to assess the role in cortical patterning of the overall COUP-TFI expression in progenitor (aagcaatttggcttcccctgg) recognizes a sequence upstream the 3 rd exon in the floxed region. As a consequence wt allele will generate a 250bp amplicon, while COUP-TFI fl allele will generate a 350bp amplicon. ARM402 and EX351 were used at 1µM concentration, while ARM531 primers were used at 2µM concentration. Therefore COUP-TFI +/+ will produce only a 250bp amplicon, COUP-TFI fl/+ will produce both a 250bp and a 350bp amplicon and COUP-TFIfl/fl will only produce a 350bp. All PCR genotyping reactions were performed using Green Taq (PROMEGA) 2x Mix, which contains the Taq Polymerase with a proper reaction buffer at 2x of the working concentration.

The total volume for each reaction used is 10µl, of which 1µl is constituted by the genomic DNA extracted (from 100ng to 500 ng of DNA), and 9µl of the PCR mix. The program used for amplification was as follows.

To identify the presence of Cre alleles the primers used were the Cre FW (cccgcagaacctgaagatgt)

and Cre Rev (tgatcctggcaatttcggct) primers at a concentration of 1µM, which anneal inside the Cre recombinase sequence. In the same reaction were also used the

2.c. KO;iz/hCOUP-TFI.

KO;iz/COUP-TFI mice have been genotyped using the primers for the COUP-TFI fl allele previously described, as these primers not only recognize COUP-TFI + and fl allele, but also COUP-TFIallele, in which the third exon has already been removed in all cells. PCR reactions with ARM531, EX351

and ARM402 primes the COUP-TFIallele produce an amplicon of about 450bp when annealing on a COUP-TFIallele. The Nex-Cre allele was detected as previously described and the CMV-floxstop-flox-hCOUP-TFI allele was detected with a PCR reaction using the TG-fw (gctttctggcgtgtgacc)

and TG-rev (attaagggccagctcattcc) primers used at a concentration of 1µM in the same mix used for the previous reactions [START_REF] Wu | Generation of ES cells for conditional expression of nuclear receptors and coregulators in vivo[END_REF]. The reaction was carried with the following program:

95°C 10' 95°C 30'' 29x 58°C 45'' 72°C 30'' 72°C 8'

Agarose Gel electrophoresis.

Agarose ultrapure (Invitrogen) was dissolved in TBE buffer (Tris Base 89mM, Boric Acid 89mM, EDTA 2mM, pH 8.3 in H 2 O milli-q. Before cooling and solidification of the solution I added Ethidium

Bromide which is able to absorb UV light and emits at 590nm, which increases 20fold when intercalating DNA or RNA. BET agarose gel allows detecting DNA and RNA at a sensibility of 5ng.

The TBE buffer is also used to fill the vat that contains the gel, allowing the electricity to run from the negative to the positive electrode. The electric field pulls the DNA, which is negatively charged toward the positive electrode. When used to visualize DNA or RNA smaller than 1kb the percentage of agarose used was 2%, in case it was bigger I used 1%. The electrophoresis was run applying an electric field of 100V for the 1% agarose gel and of 120V for the 2%agarose gel.

Standard ladder of DNA 1kb and 100bp (Promega) were used to compare the length of samples.

Gel images were taken on a standard UV transilluminator.

Post-mortem brain dissection and fixation.

In order to collect embryonic murine brains I recorded plug dates observed after an overnight mating, counting the morning of the plug date as embryonic date 0.5 (E0.5). Once reached the desired embryonic stage pregnant female mice were euthanized by cervical dislocation. Embryonic heads where rapidly dissected in ice cold PBS and fixed with PFA4% for 1h at +4°C in gentle agitation, then brains where fully dissected afterwards after being rinsed and equilibrated in ice cold PBS.

Post natal brains where collected after intracardiac PFA4% perfusion. Mice were anesthetized with pentobarbital 11µg/µl using 100 µl each 10g of weight. Incisions were made to expose the earth from the chest cage, the right atrium was cut and PFA4% was injected in the left ventricle. The amount of PFA injected was 4ml for P0 mice, 5ml at P7, 8ml at P16 and 10ml at P21. After the perfusion, brains were dissected and fixed in PFA4% at +4°C in gentle mixing. For tissues used for in situ hybridization, whole mount hybridization or DiI post-mortem injection the fixation was carried out for an overnight, samples used for immunohistochemistry were fixed for 2 hours.

Cryostact fixed samples cutting.

P0 and P7 brains to be used in Immunofluorescence were cryoprotected by gradually equilibrating the brains in PBS 30% sucrose at +4°C in gentle rocking, with intermediate steps at 10 and 20% sucrose. Samples were washed briefly in Optimal Cutting Temperature (OCT) embedding medium and completely embed in it to be frozen with dry ice before storing at -80°C until cutting.

OCT block were cut using the Cryostat (Leica Cryostat) in which they were first equilibrate at -21°C for 1h and then cut at a different width depending on the stage: 12 µm for E12.5, 16 µm for E15.5, 18µm or P0, 20 µm for P7 and P16. The slices were placed on polarized slides (Thermo Scientific), let dry over night at room temperature (RT) and then stored at -80°C before using. All the washings and the incubations of the slides were made in proper shanks.

Vibratome fixed samples cutting.

Samples of P16 and P21 brains to be used in immunohistochemistry and of P7 brains to be used for post-mortem DiI, were embedded in 4% agar in PBS a. Agar was dissolved in PBS by heating and later equilibrated at 60°C. Fixed samples in PBS were dried and 60°C 4%agar PBS was poured in the sample well. Prior to the solidification by cooling, the samples were positioned in the desired orientation within the agar block to facilitate the desired cutting angles. Agar blocks samples were fixed with superglue on the cutting block of a Leica Vibratome 100M (Leica) and cut while kept in ice cold PBS. Slices where cut at 100µm for immunohistochemistry and at 150µm for DiI.

Cryosection immunohistochemistry.

Cryosection slides stored at -80°C were let dry 1h at RT and processed antigen retrieval. Slides were incubated in Unmasking Buffer (Sodium citrate 85mM, pH6.0) in shanks, which were submerged in old Unmasking Buffer. Samples were boiled in a microwave oven for 15'', then a second time with new Unmasking buffer only until the boiling point and cooled in ice for 10'.

Samples were then washed three times in PBS 10' at RT.

For BrdU immunofluorescence, samples were denaturated. Slides were washed briefly in PBS at RT and Post-Fixed with PFA 4% at RT 15' to increase the resistance of the slices. Slides were washed in PBS for 10' at RT and incubated 30' at +37°C in Denaturation buffer (PBS 1x, HCl 6.3%), and then again washed three times in PBS 10' at RT.

For Serotonin immunofluorescence samples were incubated with ammonium chloride 50mM in PBS at RT after the unmasking process.

Samples were then blocked for aspecific primary binding sites by incubating them for at least 1h at RT in blocking solution (PBS, 0.1% Triton, 10% Goat Serum). In case primary antibodies were developed in goats, the goat serum was substituted with new-born calf serum. Slides were incubated with the primary antibodies diluted in 200 µl of PBS 0.1% Triton, 3% Goat Serum.

After primary antibody incubations slides were washed with PBS, 3 times, for 10' and incubated with secondary antibody diluted in 200 µl of PBS 0.1% Triton, 3% Goat Serum. Incubation occurred over 2h at RT.

After the incubation the secondary antibodies were washed in the same way of the primary antibodies, and finally the slides were mounted with the custom mounting medium (90% glycerol, 1% N-propil gallate in H 2 O mq. 1/10000 Hoechst) and stored at -20°C until acquisition.

Vibratome sections immunohistochemistry.

Vibratome cut slices were stored in PBS at +4°C and incubated in Permeabilization buffer (10% Goat Serum, 3% BSA, 0.1% Tween-20 in PBS) at +4°C in gentle agitation for 1 over-night up to over 2 days. Permeabilization buffer is used in 1ml per well in a 12 well plate or 2ml per well in a 6 well plate. This process blocks aspecific binding sites that would otherwise aspecifically trap antibodies.

Then slices were incubated overnight with primary antibodies diluted in Permeabilization buffer overnight at +4°C in gentle agitation. After that, slices were washed in PBS 0.1%Tween-20 (PBSt) 6 times for 30' at +4°C in gentle agitation. Slices were then incubated with secondary antibodies diluted in Permeabilization buffer and washed in PBSt 6 times for 30' at +4°C in gentle agitation and finally mounted in slices in custom mounting medium and stored at -20°C until acquisition.

For samples with DiI injection the protocol for immunohistochemistry is adapted by using PBS instead of PBSt and swapping the Permeabilization buffer with Digitonin 1mg/ml in PBS for the blocking procedure and Digitonin 0.5mg/ml for antibody incubation procedures. 12. Escherichia coli electrocompetent cells transformation with plasmidic DNA.

Escherichia coli cells were grown in 800ml of Luria-Bertani Broth (LB) (bacteriptone 1%, NaCl 1%, Yeast extract 0.5% in H 2 O mq), once reached the logarithmic phase in the growth curve, corresponding to A 600 the solution was spinned at 4000rpm at +4°C to eliminate the LB and resuspend the pellet of cells in 100ml of chilled washing solution (10% glycerol in H 2 O mq) to wash them. This solution was spinned at 9000rpm at +4°C for 15' for 3 times, suspending cells in 3ml of the washing solution which is then divided in 50µl aliquotes stored at -80°C. Each aliquote obtained could be used for electroporating cells with 10ng of desired plasmidic DNA after equilibrating the cells in ice for 5' and then adding the DNA and letting it diffuse at least 1' at RT.

The electroporation is performed using 4 pulses at 1800V, creating temporary holes in the membrane walls, allowing the plasmidic DNA to enter cells. After this process cells are incubated for 1h at +37°C in LB to let them recover after the electroporation shock, then are plated and incubated over night at +37°C in LB-agar petri dishes containing the proper antibiotic in order to select the cells that acquired the plasmid. The single colonies grown could be picked and grown in order to expand the single clones that included the plasmidic DNA during the electroporation.

Plasmidic DNA extraction from Escherichia coli cells.

The protocol used to extract the plasmidic DNA from the E. coli cells were those of the kit miniprep, maxi-prep and maxi-prep endo free (QUIAGEN) provided by the manufacturer. Cells were grown respectively in 5ml LB 8-6h for the mini, 200ml LB for the midi and 400ml LB for the maxi, for 14-16 h at + 37°C. All the three protocols provided by the manufacturer apply an alkaline lysis to the cells followed by either precipitation or filtration on chromatographic columns or by precipitation and centrifugation.

RNA digoxigenin probes synthesis.

TOPO-A plasmids containing the open target sequence for the desired gene was linearized using a restriction enzyme that cuts the plasmid in a single site positioned either at the 5' of the target.

The reaction was carried with 15µg of DNA of the plasmid using 5µl of the proper 5x buffer and 3µl of the enzyme in H 2 O mq for a total volume of 50 µl. The reaction was carried initially for 2h at + 37°C and verified in an agar gel. The linearized plasmids were purified on MicroSpin (Roche) columns using the protocol provided by the manufacturer.

Sequence amplification and plasmidic cloning.

To amplify sequences for targeted genes sequences of the UTR around 250 bp were selected, designing primers of about 20 bp and 64°C melting temperature. Sequences were amplified with PCR reaction made in 50µl volumes with 25µl of GreenTaq mix, 1µM primers, 1 µl of cDNA template. The sequence was confirmed on an agarose gel and extracted with microspin columns (Invitrogen) following the manufacturers protocol. Purified sequences were cloned into a TOPO-A-II plasmid following the manufacturer protocol. The ligated plasmid was electroporated in E. coli cells and amplified.

Chromatin Immuno Precipitaion, ChIP.

Protein sepharose-A resin (SIGMA) was washed 3 times in ice cold PBS in gentle agitation for 30' at After the denaturation samples were spun at 8000g for 2' to separate surnatant from the resin, moving the previous in new tubes for each sample. The process was repeated twice to make sure of clearing all resin from the samples. To separate DNA fragments from the crosslinked proteins 12 µl of NaCl 5M were added to each sample and were incubated overnight at +65°C. RNA was removed from samples adding 1µl of RNAse-A 10mg/ml and incubating for 30' at +37°C, the reaction was blocked adding 6µl of EDTA 0.5M. Then all proteins were digested adding 12µl of Tris HCl pH 8.0 and 1µl of proteinase K 10mg/ml for 1-2h at +45°C. Samples were purified by PCA extraction: 300 µl of PCA were added to each samples, vortexed for 10' and spin for 1' at 14kg.

After moving the liquid fraction in new tubes 300 µl of Chloroform were added to the samples repeating the previous step. The new liquid fraction obtained was then precipitated adding 33µl of C 2 H 3 NaO 2 , 1µl of glycogen and 1ml of ethanol 99% and freezing overnight at -20°C and resuspending the samples in 20µl of H 2 O mq.

Real-Time PCR relative quantification.

Real-Time PCR was used to relatively quantify samples from cDNA preparation and ChIP enriched chromatins preparations (rtq-PCR). In both cases reactions were carried out on a Lightcycler II 480

(Roche) and a KAPA bioscience Syber Green master mix. Each sample was prepared at a total volume of 20µl where 10µl consisted in the 2x Syber Green master mix, 2µM of Fw and Rev primers, 0.5 µl of sample in H 2 O mq. Each sample was done in triplicate. Single mRNA reactions on cDNA samples each sample was done in triplicate at n=3 in all of the genetic conditions examined.

The housekeeper gene used was GAPDH.

For ChIP samples reactions a standard curve of amplification was added to the samples performing reactions using as templates purified extracts of the amplicon obtained with the primers used.

These amplicons were previously amplified, extracted from gel and used at different dilutions in order to cover the range of quantity present in the unknown samples. Post Hoc analysis on variance of expression within two independent groups was performed using T-test analysis on Excel. Variance in three independent groups was evaluated trough single way ANOVA test on Excel. RNA for microarray and real time-PCR (rtq-PCR) was obtained from E12.5, E15.5, P0 and P7 neocortices in three biological samples for each genotype analyzed. Samples were dissected in ice cold PBS, dissecting neocortical tissue and removing meninges. Tissues collected were snap frozen in liquid nitrogen and stored at -80°C until extraction. RNA was extracted using the RNeasy Mini kit (Qiagen) following manufacturer's instructions. cDNA was synthesized starting from 1 mg of total RNA using Superscript III First-Strand Synthesis System for rtq-PCR (Invitrogen) following manufacturer's instructions.

19. DiI retrolabeling.

19.a. Post-Mortem DiI injection.

Lipophilic crystals were placed into post-mortem P0 and P7 brains. After 2 h fixation in 4% PFA, crystals of DiI' (DiI C18(3); Molecular Probes) and DiD' (DiI C18(5); Molecular Probes) were inserted into defined cortical regions. To perform thalamic injections of tracer molecules fixed brains were cut in the two hemispheres and embed in 4% agar PBS. Agar blocks were cut in order to create a 45°C angle between the midline of the hemisphere and the edge of the agar block toward the lateral part of the hemisphere. Samples were cut on the vibratome, cutting the samples from the caudo-medial region toward the rostro-lateral one, using the angle created in the agar block. This setting allows to expose dorsal thalamic nuclei from the medial side keeping intact the internal capsule and therefore thalamic axons toward the cortex.

To precisely inject DiI in the selected thalamic nucleus, DiI molecules were dissolved in DMSO 100% by loading 20 µl in a 0.5ml tube, dipping a clean pipette cone in the DiI powder and dissolving the crystals stuck to the pipette cone in the DMSO. The DiI in DMSO was loaded in a

Nanoject II Microinjector glass pipette. Operating the Nanoject with a micromanipulator over a stereomicroscope, DiI/DMSO was injected in the selected thalamic nucleus, injecting 3x9.2nl.

After 2 weeks in 2% PFA at 37 °C or 1.5 months at+4°C to allow lipophilic dyes diffusion, brains (three for controls and three for each mutant) were cut at the vibratome to obtain 100-150 mm thick slices, which were mounted as described above. Three controls and three mutant brains were processed and analyzed for each tracing experiment described in the text.

19.b. Stereotaxic axonal tracing injections.

Live positioned in the center of the λ, the x, y, z parameters were set to zero. The needle was then moved to the desired x-y coordinate and the skull was then perforated with a dental drill. At this point the needle was inserted at the desired depth.

Once assuring the tip of the needle was not blocked and fluently able to allow the flow of injection, DiI or CTB were injected in the brain waiting 2 minutes before removing the needle, in order to prevent backward diffusion of injected liquid. Then the skin was stitched with surgical suture silk making "U" shaped stiches. All operated mice were treated with analgesic Buprenorphine 0.015µg/µl using 100µl for 10g of weight, injecting it under the neck skin. Mice were kept at +37°C until full recovery from anesthesia and observed daily thereupon. Brains were collected 4-7 days after injection.

For P7 brains samples, pups were injected at P4 after anesthesia on ice. Injections were performed with either CTB 6x9.2nl or DiI 12x9.2nl. P16 and P21 samples were obtained injecting pups at either P13 or P16 respectively. Pups were anesthetized with intraperitoneal injection of Ketamine/Xylazine 10µg/µl using 100µl per 10g of weight.

Injected samples were collected after PFA4% perfusion and consequently fixed 2h in PFA4% at +4°C.

Coordinates used were as follows: Cortical tissue from the parietal region of the neocortices from control and EmxCKO brains collected at E18.5, P1 and P2. Parietal cortical tissue was rapidly dissected and freed from meninges under sterile conditions in ice cold HBSS with high glucose. Samples were digested with papain (0,5 mg/ml) dissolved in HBSS plus DNAse (0,1 mg/ml).

age P4 to P7 P13 to P16 P16 to P21 axis X Y Z X Y Z X Y Z S1 / / / B -0.94 B +3 -1 B -1.34 B +3 -1 VPM / / / B
Isolated cells were plated at density of 1200 cells/mm2 onto the Multi-Electrode-Array (MEA, previously coated with poly-DL-lysine and laminine). To assure dissociated neurons to adhere at the center of the chip by using a spacer ring made of Sylgard 184 (Dow Corning) with 4.5 mm internal diameter (11 mm external diameter). Chips were incubated with 1% penicillin/streptomycin, 1% glutamax, 2.5% fetal bovine serum, 2% B-27 supplemented neurobasal medium in a humidified 5% CO 2 atmosphere at 37°C and spacer rings were removed after 4 hours.

Each MEA chip was covered with a fluorinated ethylene-propylene membrane (ALA scientific, Westbury, NY, USA) to reduce medium evaporation and maintain sterility. Each chip was repeatedly recorded at days in vitro (DIV) 6, 9, 15, 20.

Multisite extracellular recordings were performed using the Multi-Channel Systems (Reutlingen, Germany) MEA-system. This system uses a 64 electrodes array (TiN/SiN) over an 8x8 square grid.

Electrodes were spaced with 200 µm and had a 30 µm electrode diameter. Data were acquired using MC_Rack software (Multi-Channel Systems Reutlingen, Germany). The spike detection threshold was set at 215 mV and sampling at 10 kHz. All recording were performed in a nonhumidified incubator at +37°C and with 5% CO 2 perfusion. Cells were allowed to stabilize in the incubator for 90 seconds before starting the recording session. Recordings of spontaneous activity were performed for 6'.

To perform bursts analysis we used the Neuroexplorer software (Nex Technologies, Littleton, MA, USA) after spike sorting operations. Considering a burst as a group of spikes with decreasing amplitude [START_REF] Harris | Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells[END_REF], we set the threshold of at least 3 spikes and a minimum burst duration time of 10 ms. Interval algorithm specifications were set to start burst (0.17 sec) and maximum interval to end burst (0.3 sec) recording in 0.02 s bins [START_REF] Gavello | Leptin counteracts the hypoxia-induced inhibition of spontaneously firing hippocampal neurons: A microelectrode array study[END_REF].

We performed two different approaches for the burst analysis: considering the contribution of each recording channel and calculating a mean value for each MEA over the active recording channels. The parameters extracted during the analysis were: frequency, number of bursts and burst duration. Cross-correlation probability vs time diagrams were constructed by means of Neuroexplorer software (Nex Technologies, Littleton, MA, USA). Post Hoc analysis on variance within two independent groups was performed using T-test analysis on Excel.

In Utero Electroporations.

In utero electroporations were performed as previously described in [START_REF] Saito | Efficient gene transfer into the embryonic mouse brain using in vivo electroporation[END_REF] applying circumstantial customization. We prepared the DNA mix to be injected using DNA 1mg/ml, 2 µl FASTGREEN (Sigma) in a total volume of 20µl reached by adding H 2 0 mq sterile. DNA mix were leaded in 75-mm glass capillary tubes (Drummond Scientific, Broomall, PA), which were previously pulled with a micropipette puller P-97 (Sutter Instrument, Novato, CA) under the following conditions: pressure, 500; heat, 800; pull, 30; velocity, 40; time, 1. The pulled capillaries were filled with DNA mix and broken at 60 µm external diameter by pinching with sterile forceps.

Loaded capillary were loaded on a Femtojet microinjector (Eppendorf), adjusting injection pressure and holding pressure to allow for proper injection.

Prior surgery all instruments were sterilized at over 200°C for 10'. E13.5 pregnant mice were anesthetized with Ketamine/Xylazine 10µg/µl using 100 µl per 10 g of weight. Anesthetized mice were shaved in the abdomen and cleaned with 70% ethanol and immobilized with standard tape over a +37°C warmed mat and applied with eye hydrating gel droplets. A 3 cm midline laparotomy was performed, cutting separately the derma and the peritoneum. Sterile gauzes with holes slightly larger than the laparatomic cut were applied over it to subsequently extroflect uteri over a clean surface. With help of an optic fiber light source the forebrain was visualized in order to properly inject 1µl of DNAmix in a single telencephalic lateral ventricle. Electric pulses were delivered using forceps-type electrodes CUY650P3 (NEPAGENE, Japan), holding the injected forebrain between the electrodes, orienting the positive electrode toward the upper lateral side of the injected ventricle and the negative electrode at the opposite side. Four electric pulses (37V, 50 ms each with 1s lags in between ad 5% decay rate between each pulse) were delivered using a NEPA21 electroporator (NEPAGENE, Japan).

After each injection uteri were hydrated with prewarmed sterile saline solution and maintaining electrode forceps in sterile PBS. Uteri were extroflected one half at a time to allow the remaining half to be maintained wet and warm in the meanwhile. After electroporating all possible embryos all uterine horns were repositioned in the abdominal cavity, taking care of repositioning uteri, intestine and bladder in the correct position. The abdominal wall and skin were sewed up with surgical sutures, using "X" stitches for the first and "U" stitches for the second.

Mice were peritoneal injected with Gentamicin 10 µg/ml (Sigma) 100µ per 15 g of weight to prevent infections. Operated mice were also treated with injection under the skin of the neck of Buprenorphine 0.015µg/µl using 100µl for 10g of weight. Mice were kept at +37°C until recovery and observed over the following days. Brains of electroporated pups were collected at P16.

Thalamo-cortical slices field recording.

P8, P16 and P21 mice were anesthetized with isoflurane (Axience) inhalation and rapidly decapitated. Brains were rapidly dissected and incubated in ice cold artificial cerebro-spinal fluid (ACSF: 125mM NaCl, 25mM NaHCO3, 2.5mM KCl, 7mM MgCl 2 , 2mM CaCl 2 , 1.25mM NaH 2 PO 4 , 10mM glucose, pH7.4) and perfused constantly with 95%O 2 ,5%CO 2 . To obtain thalamo-cortical slices obtained with a MicromHM650V vibratome (Thermo Fisher Scientific) using procedures previously described in [START_REF] Agmon | Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex[END_REF][START_REF] Agmon | Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex[END_REF]. Brains were manually cut rostrally at a 55° angle between the midline and the lateral cutting edge. The rostral cut side of the brain was used to glue the brains on the cutting plate and slices were cut in ice cold ACSF perfused with 95%O 2 , 5%CO 2 . Slices obtained were 600 µm thick and contained thalamic structures, internal capsule and parietal cortex, allowing maintaining thalamo-cortical connections between the VPM and the S1 in the same slice. After cutting slices were maintained at 37°C for 1 h before recordings in interface chamber (Campden Instruments) with constant bubbled ACSF perfusion. Recording were performed with recording electrodes in glass pulled pipettes filled with buffer saline solution. Electrodes were inserted in L4 and L6 of S1 recording with DAGAN EX4-400 amplifiers. Electrical stimulations were applied with bipolar electrodes (FHC Inc.), a SIU91A isolator (Cygnus Technologies), pCLAMP software, and a DigiData 1420 digitizer. Sampling rate was 10 kHz and data were low-pass filtered at 4 kHz.

Trace analysis to detect monosynaptic amplitude and delay time from stimulation were calculated with Clampfit software. Post Hoc analysis on variance within two independent groups of variable was performed using T-test analysis on Excel.

Image acquisition.

Images from immunofluorescence on cryosections and in situ hybridization experiments were acquired using the LEICA DM6000 microscope.

Image quantifications.

Cell counting and colocalization analysis on immunofluorescence experiments on wide field microscopes acquired images were performed either via manual counting, using object counter on Phothoshop CS6 or using the Imagej JACoP plugin [START_REF] Bolte | A guided tour into subcellular colocalization analysis in light microscopy[END_REF]. Embryonic cortical slices were counted manually, over a cortical radial section of similar size between all samples.

E12.5 embryos images were quantified in 200µm wide slices, E14.5 and E15.5 embryos images were quantified over 400µm wide slices. P0 cortical counting were performed subdividing radial sections of the cortex in 6 equally spaced bins. P7 cortical counting were performed subdividing radial sections of the cortex in 10 equally spaced bins each bin was analyzed for specific protein detection, colocalization and total number of nuclei with DAPI staining. To determine the bin correspondence with cortical layers they were compared the expression of layer specific markers such as Ctip2 (L5), Cux1 (L2/3 -4), Ror-β (L4), Tbr1 (L6), Fog2 (L6). Quantification of single cells count and co-localization evaluation where made on 3 animals on the average of 3 images representing the same anatomic area.

Post Hoc analysis on variance within two independent groups was performed using T-test analysis on Excel. Variance in three independent groups was evaluated trough single way ANOVA test on Excel.

Radial and tangential intensity plots were quantified using a custom made program on the Matlab 3' -CDS.rev (previously described), which would amplify a 2kb amplicon in case of ligation, without discerning the frame orientation, a PCR reaction with cDK5B.FW (AGGACTAAACGCGTCGTGTCC), which binds at the cDK5 promoter and a Egr1 3' -CDS.rev primer which would give a 3kb amplicon in case of in frame ligation and a final PCR reaction with the cDK5B.FW + Egr1 5' CDS.FW primers which would give a 3kb amplicon in case of ligation in the wrong frame.

The reaction was carried in 10µl volume with 5µl GreenTaq mix, 1µm of forward and reverse primer, 1 µl of plasmid template in H 2 O mq. The program used was the following:

96°C 6' 96°C 1' 10x 63°C 1'30'' 72°C 3' 96°C 1' 25x 63°C 45'' 72°C 3' 72°C 8'
Selected plasmids were also verified by sequencing from the cDK5B.FW primer. The verified plasmid contained a full Egr1 CDS plus 5' 3'UTR, which was amplified with the Endofree-Maxiprep kit (Quiagen) and used in the in utero electroporations experiments as cDK5::Egr1::IRES::GFP.

Ca ++ imaging time lapse recording.

Pups at P1 and P4 were rapidly decapitated after anesthesia in ice. Brains were dissected in ice cold ACSF high Mg ++ constantly bubbled in 95%O 2 , 5%CO 2 . Horizontal slices were cut in a Leica Vibratome 100M in ice cold ACSF high Mg ++ constantly bubbled and transferred in ACSF to incubate for 1h at +37°C in constant bubbling. Consequently slices were incubated in ACSF 0.63%

Oregon Green BAPTA and incubated in +37°C in constant bubbling. Slices which contained samples of motor, somatosensory and visual areas were selected to be imaged. Imaging was performed loading slices in a 35mm diameter petri dish with a 1,3mm glass bottom in which bubbled ACSF was constantly perfused. Slices were immobilized with nylon net in the petri dish and let to equilibrate 30' before image acquisition. Time lapse images were acquired on a Spinning Disk microscope (Olympus / Andor technology), sampling in each slice a rostral a somatosensory and a visual region 2 times over 15 minutes at 150 ms per frame using a 40x objective.

Ca ++ imaging analysis.

Acquired images were analyzed using a defined and automated workflow to allow reproducibility To compare the synchronicity rate in the analyzed time lapse I used a custom algorithm which analyzed each threshold time series and compared with every other single threshold signal. In this algorithm, signals were scanned along the time line and for each bursting event it was asked whether in any other ROI signal there was a bursting event in the time range of 2s. If it was the case the two time series were considered synchronized for that specific event, which increased the synchronicity counter for that ROI signal. This analysis was performed over all the time length, comparing each ROI to all other ROI. At first, this algorithm calculated a matrix of synchronicity counters, where each counter indicated the amount of synchronous events for each couple of tonic ROI. At the end of the analysis the synchronicity counter counted all synchronous events in all cells of the time lapse. To compare this score with other time lapse it was divided by the amount of possible synchronous events there could have been found in the time lapse, namely the total amount of bursting event found multiplied for the total amount of tonic ROI. The clustering data was then compared with the Pearson coefficient score of the same datasets used during the clustering algorithm. This process compared how the synchronicity index for every pair of tonic ROI matched with the tangential distance, obtaining a score from -1 to 1 where -1 indicates complete complementarity between synchronicity distance and tangential distance and 1 indicates complete correlation between the previous parameters.

Unfortunately the clustering of cell pairs using correlation and tangential distance did not provide robust data, most likely due to the insufficient size of the images acquired, which did not allow strongly distinguishing the rate of synchronicity of cells within different functional columns. (EmxCKO), a conditional knock out in which COUP-TFI is inactivated in all cortical precursor cells from E10.5 , and the D6/COUP-TFI, in which COUP-TFI is overexpressed in rostral and medial regions when normally expression is at its lowest. All of the aforementioned models have altered COUP-TFI expression levels in cortical progenitors and their post-mitotic progenies, preventing us from understanding its specific functions in these two groups of cells. Thus, the goal of the first part of my Ph.D. work was to understand whether an areal patterning gene, such as COUP-TFI, could play distinct roles in progenitors and post-mitotic cells during the process of arealization.

Results

On this behalf, I crossed the COUP-TFI floxed mice [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]) with a mouse line expressing Cre recombinase under the control of the Nex gene (also called NeuroD6 or MATH2) [START_REF] Goebbels | Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice[END_REF]. Nex is a transcription factor expressed in cortical neurons exiting the cell cycle. In mice, Nex is activated from E11 in the first neuronal cells that differentiate in the pallium, and is constantly upregulated in successive waves of newborn neurons as part of their process of differentiation. Since Nex is expressed during neurogenesis, its early expression follows the rostro-lateral high to caudo-medial low neurogenetic gradient. In view of the fact that the Nex coding sequence was replaced with the Cre sequence, this allele was used only in heterozygosis, in order to avoid complete loss of Nex function even if it was shown to be redundant during cortical development [START_REF] Goebbels | Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice[END_REF][START_REF] Wu | Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone[END_REF].

Specific COUP-TFI post-mitotic cortical deletion in NexCKO mice.

Since many aspects of cortical development depend on proper thalamic innervation [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF],Erzurumlu & Gaspar 2012a), it was of key importance to show that the Nex-Cre was not acting in the thalamus. Thus, I analyzed Cre expression in NexCKO embryonic brains at E15.5: Cre is expressed strongly in the CP of the cortex and partially also in the SVZ and IZ of the neocortex, but it is completely absent in other regions of the forebrain, especially in the dorsal thalamus, (Figure To further corroborate these data, I also performed a 24h BrdU pulse from E13.5 to E14.5. This experiment shows that post-mitotic COUP-TFI loss in NexCKO does not alter significantly the total number of cells exiting the cell cycle. The co-staining of BrdU and Ki-67 showed that the amount of cells still in cell cycle (BrdU+/Ki-67+) and of cells exiting the cell cycle (BrdU+/Ki-67-) is not altered (number of cells in 400μm section, using the average of three representative images of three Hence, COUP-TFI loss was confirmed in the CP, IZ and partially SVZ, but not in apical progenitors of the VZ, nor in IPs of the SVZ.

1

Area identity impairments in post-mitotic COUP-TFI loss-of-function brains.

As previously introduced, COUP-TFI protein is expressed in a caudo-lateral high to rostro-medial low gradient from E9 onward. COUP-TFI expression gradient is maintained in the majority of cortical progenitors and neurons during corticogenesis (Figure 1 To confirm whole mount data, I also performed in situ hybridization (ISH) with similar riboporbes on sagittal sections of control, EmxCKO and NexCKO cortices in order to simultaneously visualize laminar and areal organization. Indeed, while whole mount ISH label cortical boundaries and assess dimensions of single areas, they provide no information about the laminar expression of marker genes. Cad8 is strongly expressed in upper layers in both the rostromedial and the caudolateral domains in P7 control brains, and in lower layers all along the somatosensory cortex, in good accordance with the data observed in whole mounts. EmxCKO and NexCKO mutants express Cad8 along the whole rostro-caudal axis, but it is sharply decreased caudally where shrunken sensory areas still persist. Lmo4 is a co-transcriptional regulator of the LIM domain only family [START_REF] Deane | Tandem LIM domains provide synergistic binding in the LMO4:Ldb1 complex[END_REF]) (Figure 1-4), and in control P7 cortices has a rostral expression that spans among all layers, while in other areas it is detected only in upper layers.

In EmxCKO and NexCKO brains, Lmo4 is expressed in all cortical layers along the rostro-caudal axis of the cortex with the exception of a small caudal region, which corresponds to the putative sensory domains. Ror-β, on the contrary, is strongly expressed in L4 in S1 P7 cortices and to a lesser extent in motor and visual domains. In accordance with the data obtained so far, in both In summary, the marker analysis done so far show that post-mitotic expression of COUP-TFI is necessary for the proper development of sensory domains, since in both the full cortical and postmitotic mutants, sensory domains are similarly reduced in size and caudally shifted. Because of the acquisition of S1 of a motor-like identity in COUP-TFI mutants, we called this area mS1 (for motorized S1).

Laminar organization of the neocortex requires COUP-TFI expression in postmitotic cells.

To further analyze the laminar organization of the parietal (S1) cortex, I used a series of markers expressed in distinct layers and with a different distribution between areas. The counting of positive cells along the radial axis was performed by subdividing the cortex into ten equally spaced radial bins. Ctip2 is a transcription factor expressed in subcerebral projection neurons of Layer 5 [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. In S1 cortex, Ctip2+ cells are limited to a restricted domain in layer 5 corresponding to layer 5b, while the motor cortex has an enlarged layer 5 and thus a higher number of Ctip2+ cells, in line with its output function. In layer 6, Ctip2 is expressed at a lower extent in a subgroup of neurons, whereas expression of Ctip2 in upper layers corresponds most probably to interneurons generated in the subpallium and migrating to the pallium, as recently reported [START_REF] Nikouei | BCL11B/CTIP2 is highly expressed in GABAergic interneurons of the mouse somatosensory cortex[END_REF]. Layer 5 Ctip2 expressing neurons are radially expanded in NexCKO cortices, similarly to EmxCKO ones, as previously reported [START_REF] Faedo | COUP-TFI coordinates cortical patterning, neurogenesis, and laminar fate and modulates MAPK/ERK, AKT, and β-catenin signaling[END_REF][START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF] It is important to note that layer 5 expansion in NexCKO brains is not related to increased proliferation, as previously demonstrated in BrdU-labeled cells between E13.5 to E14.5 (Figure 1-

2.a).

The Ctip2+ cell distribution in mutant mS1 cortices resembles the laminar organization of control motor areas and is in good accordance with the observed acquisition of a motor identity in presumptive S1 mutant cortices. Ctip2 expression is also increased in layer 6 neurons resulting in a co-expression of Ctip2 with layer 6 markers, such as the transcription factors Tbr1 (Bedogni et al. 2010;[START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF]) and Fog2 (Friend Of GATA 2) [START_REF] Chen | Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex[END_REF]. Both Tbr1 and Fog2 are important for layer 6 development in terms of laminar identity and axonal connectivity, and at P7 shows a significant increase in the number of these neurons in bins 2 (P ANOVA=0.01) and 3 (P ANOVA=0.005), corresponding to layer 6 of both CKOs. (c) P7 M2 coronal sections double-labeled with Ctip2 (layer 5) , layer 6 markers (Tbr1, FoxP2, and Fog2) and callosal marker (Satb2). (d) Corresponding counting plots show that the number and distribution of Ctip2+/Tbr1+, Ctip2+/FoxP2+, Ctip2+/Fog2+, Ctip2+/Satb2+ cells in layers 6 are similarly altered in both CKOs (red line, control, green line, EmxCKOs, and blue line, NexCKO, in the plots). However, the percentage of Fog2+, Foxp2, Tbr1, Satb2 cells in layer 6

(bins 1 to 2) of NexCKOs resulted overall more similar to control than to EmxCKO brains (bin 1: P = 0.6 vs P = 0.03; bin 2: P = 0.28 vs P = 0.01, respectively). Scale bars = 600µm. P control vs NexCKO: (*) P≤ 0.05, (**) P≤ 0.01, (***) P≤ 0.005; P EmxCKO vs NexCKO: (@) P≤ 0.05, (@@) P≤ 0.01, (@@@) P≤ 0.005; P control vs

Altered sensory thalamo-cortical connectivity supports areal and laminar impairments of COUP-TFI mutants.

To understand whether molecular changes in gene expression are supported by altered arealspecific connectivity features, I have investigated the thalamo-cortical connectivity in both EmxCKOs and NexCKOs. Thalamo-cortical axons are elongating from dorsal thalamic nuclei during embryonic development, and reach the internal capsule at E13 and the cortex around E16 [START_REF] Rakic | Prenatal genesis of connections subserving ocular dominance in the rhesus monkey[END_REF][START_REF] Lund | Development of the geniculocortical pathway in rats[END_REF]. After 2-3 days in subplate, they enter the CP and reach the appropriate layer targets. First, I examined the distribution of the serotonin transporter 5HT (5-Hydroxytryptamine Transporter), originally secreted by axons of the raphe magno nucleus, and subsequently uptaken by thalamo-cortical axons in the sensory cortex where they organize in a dense structure in layer 4, and particularly in barrels in S1 (Fiure 1-8) [START_REF] Li | How do barrels form in somatosensory cortex?[END_REF].

Immunofluorescence (IF) with a specific anti-serotonin transporter antibody on sagittal cryosections of P7 control, EmxCKO and NexCKO brains revealed no distinct barrel-like structure of 5-HT thalamo-cortical axons in the mS1 cortex, which can instead be identified more caudally in the occipital cortex. Here, thalamo-cortical axons are organized in a much more reduced space which corresponds to the part of the cortex where layer 5 (Ctip2+) radial distribution resembles that of a normal sensory area (Figure 12345678).

COUP-TFI expression in post-mitotic cells represses rostral mitotic genes.

The gene set analysis also revealed a mild enrichment of transcripts expressed in the rostral VZ (Figure 1-10). Since loss of COUP-TFI in NexCKO brains does not directly affect progenitor cells, this effect might be to a feedback from post-mitotic cells to progenitors, as it was already described in other studies [START_REF] Seuntjens | Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors[END_REF]). E15.5 is a development period in which expression of the principal patterning genes start to be downregulated; therefore, I decided to quantify the transcript of the principal area patterning genes at E12.5, i.e. when most of them are highly expressed.

Results

Magrinelli Elia -Ph.D. Thesis et al. 2013[START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF][START_REF] Zembrzycki | Genetic interplay between the transcription factors Sp8 and Emx2 in the patterning of the forebrain[END_REF]. Even if Sp8 is considered important in rostral specification, gain-of-function of Sp8 in rostral cortices induces a reduction and not an expansion of presumptive motor area [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF]). Thus, upregulation of Sp8 in COUP-TFI mutant progenitors unlikely fully justifies the massive upregulation of rostrally-expressed genes observed in the absence of COUP-TFI. Unfortunately, the KO;iz/hCOUP-TFI mice die at birth like COUP-TFI nulls, since in both cases COUP-TFI is lacking in ganglion IX and pups cannot properly feed [START_REF] Qiu | Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization[END_REF]). This obliged me to limit my analysis to perinatal stages.

My data also show that while COUP-TFI expression levels in

To validate these mice, I first analyzed the protein profile of hCOUP-TFI in the iz/hCOUP-TFI brains previously mentioned, that COUP-TFI is efficient in promoting somatosensory identity rather than a general sensory identity, and correlate with the expression pattern of Lmo4 in KO;iz/hCOUP-TFI.

It is important to note that in KO;iz/hCOUP-TFI brains thalamic nuclei lack COUP-TFI expression (as is the case for KO brains), which may influence the process of cortical innervation and therefore prevent a complete rescue of a correct somatosensory innervation from the VPM to the parietal cortex. Notably, control and KO;iz/hCOUP-TFI brains express Ctgf also in a rostro-ventral region of the cortex that is not labeled in NexCKO and COUP-TFI KO.

These data confirm that COUP-TFI post-mitotic expression can autonomously confer positional information to incoming TC axons.

COUP-TFI expression in rostral cortical post-mitotic cells can modify their motor identity into a sensory one.

As discussed, KO;iz/hCOUP-TFI overexpress COUP-TFI rostrally, where usually COUP-TFI is usually not expressed or expressed at low level. Since COUP-TFI is a key regulator of sensory identity specification, I analyzed the effect COUP-TFI overexpression in single cells of rostral regions of KO;iz/hCOUP-TFI. As shown above, Lmo4 in situ hybridization on P0 sagittal sections shows high expression of Lmo4 in rostral cortex, whereas Lmo4 is strongly reduced in rostral regions of KO;iz/hCOUP-TFI brains.

Cad8 has a similar pattern of expression, while Bhlhb5 as an opposite one. In COUP-TFI KO, as previously indicated, parietal cortical regions strongly express Lmo4 and Cad8 and not Bhlhb5.

Thus, the mS1 cortex of COUP-TFI mutants expresses the typical markers of control motor areas.

On the contrary, KO;iz/hCOUP-TFI brain frontal regions acquire a pattern of expression similar to the S1 of controls, since Bhlhb5 is strongly expressed in upper layer, while Cad8 and Lmo4 are downregulated (Figure 1 Part 2.

Regionalized endogenous and evoked activity is controlled by COUP-TFI.

During proliferation and after the emergence from the proliferation phase, cortical progenitors and neurons undergo an endogenous activity based on spontaneous Ca ++ currents [START_REF] Yuste | Neuronal Domains en Developing Neocortex[END_REF][START_REF] Uhlén | Calcium signaling in neocortical development[END_REF]. This endogenous process regulates different mechanisms during development and is progressively downregulated when experienced evoked activity reaches cortical neurons after birth [START_REF] Uhlén | Calcium signaling in neocortical development[END_REF][START_REF] Corlew | Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones[END_REF][START_REF] Mao | Dynamics of spontaneous activity in neocortical slices[END_REF]. Sensory inputs brought by thalamic axons to cortical neurons during the latest phase of cortical development are the main source of evoked acitivity [START_REF] López-Bendito | Thalamocortical development: how are we going to get there?[END_REF].

Endogenous activity is progressively reduced as evoked activity is established [START_REF] Andreae | Independent Vesicle Pools Underlie Different Modes of Release during Neuronal Development[END_REF][START_REF] Corlew | Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones[END_REF]), although no evidence indicates how these processes influence each other and how they may contribute in the organization of functional areas. Thalamic activity directly influences cortical organization, as observed in models with specific interruption of the thalamocortical synapse transmission, in which cortical sensory areas fail to properly organize [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF][START_REF] Ballester-Rosado | mGluR5 in cortical excitatory neurons exerts both cell-autonomous andnonautonomous influences on cortical somatosensory circuit formation[END_REF]Erzurumlu & Gaspar 2012b). The thalamic double KO of Vglut1-2 and the cortical KO of 5HT and mGlur3 all display disorganization of the barrel cortex and impairments in layer 4 neuron specification [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF][START_REF] Ballester-Rosado | mGluR5 in cortical excitatory neurons exerts both cell-autonomous andnonautonomous influences on cortical somatosensory circuit formation[END_REF]. It is known that L4 neurons in the somatosensory cortex require thalamic inputs to acquire the stellate morphology, which further influences somatosensory area topological organization (Jabaudon, Shnider, et al. 2012;Pouchelon et al. 2014). These data highlight the importance of thalamic activity during the acquisition of somatosensory neuronal attributes, but how these specific features are acquired during development to properly respond to sensory evoked activity, is still not clear.

To investigate how intrinsic properties of cortical neurons can influence endogenous activity and thus the subsequent integration of thalamo-cortical inputs, I decided to use again the EmxCKO mouse model in which COUP-TFI is solely inactivated in cortical neurons but not in thalamic nuclei.

As also discussed in chapter 12 of the introduction, there are preliminary data indicating how COUP-TFI could influence neuronal activity during development. Hence, differently from the COUP-TFI constitutive KO model, EmxCKO mice do not have any intrinsic problems in thalamocortical axons elongation, providing a good paradigm to study how thalamic axons interact with cortical territories affected by alteration in their intrinsic genetic program. Importantly, our tracing experiments show strong misrouting of thalamic afferents to the mutant EmxCKO cortex, and COUP-TFI does respond to activity-dependent cues during maturation of specific cell types, as previously reported [START_REF] Abbott | The effects of lifelong blindness on murine neuroanatomy and gene expression[END_REF]. Thus, the combination of these observations led me hypothesize that COUP-TFI could be involved in "electrical pre-patterning" of the somatosensory cortex, before responding to sensory inputs from the periphery.

Differences in endogenous activity rate and synchronicity between neocortical regions in early post-natal cortices are modulated by COUP-TFI function.

To start investigating the endogenous activity of cortical neurons at early post-natal days, I recorded endogenous Ca ++ activity via time-lapse acquisitions in acute horizontal cortical slices with the help of a fluorescent Ca ++ indicator (Syber Green BAPTA). Horizontal planes allow analyzing, on the same slice, representative regions of motor, somatosensory and visual areas with the aim of finding specific differences and excluding, as much as possible, potential variability among these areas in different samples (Figure 2.1-a). In each experiment, I have analyzed control and EmxCKO brains from the same littermates. Endogenous Ca ++ activity peaks at P0 in mice and progressively decreases while evoked activity begins [START_REF] Corlew | Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones[END_REF]), I thus performed these experiments at P1 and P4, after having genotyped the pups, and evaluated mainly frequency of Ca ++ bursts and synchronicity of these events. To measure the frequency of endogenous activity, I calculated the frequency of Ca ++ intake bursts in a given timeline on all imaged cells. At P1, the frequency of endogenous activity in control brains is progressively higher from the rostral to the caudal regions, indicating a regionalization of endogenous activity (Figure 2-1.b). Accordingly to previous investigations, the general rate of endogenous activity decreases in all recorded areas at P4, resulting in a flattened gradient [START_REF] Corlew | Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones[END_REF][START_REF] Mao | Dynamics of spontaneous activity in neocortical slices[END_REF].

By comparing the rate of activity between EmxCKO and control brains, I observed a disruption of the endogenous activity gradient at P1 resulting in an abnormally higher rate of activity in motor cortex and a significantly lower rate of activity in somatosensory and visual cortices in EmxCKO mutants. At P4, the endogenous activity in S1, which in controls strongly decreases at this stage, remains significantly higher in EmxCKO brains.

It is well established that synchronized neuronal activity helps in strengthening connection between cells and represents thus an important parameter in the organization of early endogenous circuits. At early post-natal days, cortical neurons are organized into temporary circuits depending either on immature synaptic signaling or on gap junctions [START_REF] Easton | Genetic elimination of GABAergic neurotransmission reveals two distinct pacemakers for spontaneous waves of activity in the developing mouse cortex[END_REF]).

To assess whether spontaneous synchronous bursting can change between cortical areas and whether it is modulated by COUP-TFI, I evaluated the synchronicity of Ca ++ burst events between cells recorded in the same time-lapse imaging. I isolated signals from each cell selecting manually region of interest (ROI), I then extracted the fluorescence intensity for every time frame and evaluated the occurrence of Ca ++ burst events. For each cell and for each burst event, I looked at the next closest event in every other ROI signal time series, thus creating a matrix of event matches. Every match was filtered, keeping only events with a distance of less than 2 seconds. To each pair of ROI time series, a value between 0 (no correlation with any other cell) and 1 (full correlation of all the recorded events in the time lapse) was assigned. The total amount of correlated events for all cells in a particular time lapse was divided by the total number of possible matches and the number of cells, normalizing the synchronicity rate recorded in a time lapse both for the amount of events present one cell is doing and the amount of possible connections it can have with the total of the cells present in the acquired image. These divisions allow obtaining a score ranging from 0 to 1.

At P1, there are no differences in the amount of synchronicity between the three cortical regions in control brains, whereas at P4 both motor and somatosensory regions have a significantly higher degree of synchronicity compared to visual areas (Figure 2-1.c). In EmxCKO brains, while the synchronicity at P1 is similar to control brains, P4 mutant brains depict a similar synchronicity between areas but a significantly lower synchronicity in the somatosensory area when compared to the same region of control brains (Figure 2-1.c).

These data indicate a significant difference in the endogenous activity between presumptive cortical areas at birth, which was not previously reported. In particular, my data show that frequency and synchronicity of activity established in the putative somatosensory area at P4, seem to be regulated by embryological COUP-TFI function.

Patterns of endogenous firing and synchronicity are affected in somatosensory cortical neurons of COUP-TFI mutants.

To further investigate the endogenous electrical behavior of cortical neurons in the somatosensory cortex of control and COUP-TFI mutants, I had the opportunity to record in collaboration with A. Marcantoni at the University of Torino, Italy, the activity of cortical dissociated neurons on Multi-Electrode-Array (MEA) chips (Figure 2-2). The Ca ++ imaging data provide snapshot information of how endogenous activity of cortical neurons in slices proceeds at a given time point. To have more direct information on how neuronal circuits progressively evolve, it is important to directly observe and record the establishment of neuronal networks and how they change over time. In MEA chips, it is possible to observe the electrical behavior of dissociated neurons up to several weeks, whereas cortical acute slices are viable for no more than 6-12h.

Moreover, electrodes are preferable and quite reliable, since Ca ++ fluorescent revelators tend to have possible cytotoxic effects with time due to their function as chelators (Myung Bok Wie et al.

2001).

In these experiments dissociated neurons were plated in neurobasal medium and incubated at 37°C with 5% CO 2 air perfusion. In these conditions, cortical neurons display an endogenous activity and create progressively first connections, and then circuits with other neurons. In normal conditions, MEA recordings display an initial immature asynchronous and phasic behavior, which progressively evolves into a tonic firing mode with a high degree of synchronization of recordings among the different electrodes, indicating the establishment of strong and stable synaptic circuits between plated neurons (Figure 2-2) [START_REF] Arnold | Microelectrode array recordings of cultured hippocampal networks reveal a simple model for transcription and protein synthesis-dependent plasticity[END_REF]. It is therefore possible to measure the rate of activity and the tendency of creating stable circuits during time in different conditions. Besides analyzing the degree of network synchronization we also monitored the firing pattern; by plotting each Inter spike interval (IEI) on the x-axis (IEI i ) against its succeeding IEI duration (IEI i+1 ) on the y-axis. We noticed that in control neurons the L-shaped distribution typical of burst firing was more evident when compared to EmxCKO (Figure 2 My previous data show that an intrinsic patterning gene, such as COUP-TFI, can influence the endogenous activity of cortical neurons during development. COUP-TFI, in particular, seems to act in modulating endogenous activity in the somatosensory cortex, in line with its high expression levels in this region (Alfano reveiw, 2013). However, the observations obtained on the endogenous activity in cortical slices and neurons do not give us enough insights into how endogenous activity of cortical neurons influences their ability to interact with specific external stimuli. Thus, the successive question is: how does intrinsic cortical activity correlate with sensory-evoked activity originating from the periphery and translated by thalamo-cortical connections? The establishment of the thalamo-cortical circuit and in particular of the circuit between the VPM thalamic nuclei and the primary somatosensory (S1) cortex is a dynamic process, which starts during the first postnatal week in the mouse. Just before birth, VPM axons reach their target cortical region but innervate layer 4 only around P4-P5, as discussed in the instruction chapter 10 (Erzurumlu & Gaspar 2012b).

The initial TC axons input target both to SP and L4 neurons directly, in a system that has SP neurons provide a relay for TC inputs toward L4 neurons until approximately P10, as also explained in the introduction. Our previous data show that COUP-TFI mutant somatosensory cortex acquire a motor-like identity and fail to form distinct barrels in the parietal cortex, even if a tiny and caudally-shifted barrelfield-like structure could be distinguished in the occipital pole close to the visual cortex. However, it was still unclear from these data whether all VPM afferents misproject to this ectopic barrel-like structure, or whether VPM axons project also to the parietal cortex but fail to form functional barrels because of altered cortical intrinsic activity previously described. To investigate the establishment and maintenance of these thalamo-cortical connections in the S1 cortex of EmxCKO mice and the functionality of this circuit, I first decided to label the barrel cortex with specific cellular markers and then trace somatosensory connectivity by in vivo fluorescent labeling during the first 3 weeks of age (this paragraph).

To properly compare the retrograde labeling I first performed immunostaining at P7, P16 and P21

with antibodies specific for the serotonin receptor (5HT) and the glutamate transporter Vglut2 in In summary, the tracing analysis showed that somatosensory thalamic inputs are strongly affected and only partially innervate their cortical target regions in the absence of cortical COUP-TFI. In particular, TC axons seem to completely retract from the mS1 between P16 and P21, a period in which pruning plays an important role in the maintenance of stable connections (Figure 2-12).

This does not seem to be the case for the ectopic barrel domain, which maintains thalamo-cortical connectivity even at later stages. (rose) [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF] These results suggest that COUP-TFI expression in cortical neurons is crucial for the correct establishment and strengthening of thalamo-cortical connections during postnatal stages of development. VPM axons can stimulate cortical neurons in the mS1 mutant cortex during early stages, but this circuit is not maintained at late postnatal stages. Among the Glutammate AMPA receptors I selected Gria1 expressed in L5 and L2/3 at P4, Gria2

expressed in all cortical neurons, Gria3, highly expressed in L6, L2/3 and Gria4 at P4 is localized in L5,6.

Among Kainate receptors: Grik1 (expressed since E18.5 in L5 neurons), Grik2 (expressed since E18.5 in L4), Grik3 (expressed since E18.5 in SP and L6 neurons) and Grik5 (expressed at E18.5 in SP, L5and L2/3 neurons).

The NMDA glutamate receptors selected were the followings: Grin1, which is expressed at P0 in L4, 2/3 and Grin2b that is expressed in SP, L5 and L2/3 neurons (Figure 2 f.c. 1.37 ± 0.07, Kcnh1: f.c. 0.75 ± 0.11, Kcnk2: f.c. 0.72 ± 0.10).

Finally, glutammate receptors of different families appeared as well altered in the absence of COUP-TFI: the AMPA Gria2 receptor was downregulated at P0, but not at P7 (control=1, EmxCKO: Gria2: f.c. 0.76 ± 0.17). The Kainate Grik1 receptors were upregulated at P0 and downregulated at P7 (control=1, EmxCKO: Grik1 P0: f.c. 1.73 ± 0.26, Grik1 P7: f.c. 0.66 ± 0.12).

Finally, the NMDA receptor Grin1 was donwregulated at P7 in EmxCKO cortices (control=1, EmxCKO: Grin1: f.c. 0.73 ± 0.09) (Figure 2-16.c).

It is interesting to notice that some of these channels in EmxCKO brains have a different trend of expression compared to control brains at P0 compared to P7. Hcn1 is significantly downregulated in EmxCKO at P0 but not at P7. Its expression at E18.5 is limited to upper layers, while a P4 is enlarged to other layers (Figure 2

COUP-TFI directly regulates membrane ion channels.

The expression levels alteration of the genes mentioned in the previous chapter could be under the direct or indirect function of COUP-TFI. Thus, I next investigated whether some of these could be putative direct targets of COUP-TFI protein. COUP-TFI is able to bind to a direct repeat sequence (DR) [START_REF] Anon | COUP-TF : A long lasting experience in forebrain assembly ( Under correction )[END_REF]. This would corroborate the hypothesis that patterning genes can directly influence the electrophysiological profile of cortical neurons to shape cortical circuitries during development. To this aim, I first searched for conserved binding sites of COUP-TFI in the genetic loci of the molecules found differentially expressed in EmxCKO cortices by using the ECR browser tool (http://ecrbrowser.dcode.org/).

Thanks to this in silico approach, I was able to identify putative binding sites, identified as direct repeats (DRs) on the following genetic loci: Cacna1g (1 conserved DR sequence in close proximity of the 5' UTR), Cacna2d1 (1 conserved DR at around 2kb from the 5' UTR), Cacna2d3 (1 conserved DR at about 100kb from the 3' UTR), Grin2b (1 conserved DR at about 15kb from the 3' UTR), Hcn1

(1 conserved DR at around 25kb from the 5'UTR), Kcna1b (2 conserved DR, one at around 3kb from the 5'UTR, one at close proximity of the latter), Kcnd2 (4 conserved DR, one at close proximity of the 5'UTR), Kcng3 (1 conserved DR at around 15kb form the 5' UTR), Kcnj6 (1 conserved DR site in the 5'UTR), Kcnk2 (1 conserved DR at around 20kb form the 3' UTR), Kcnmb4

(2 conserved DR sites, one at around 8kb from the 3'UTR and one in its close proximity), Kcnt2 contributes to spontaneous rhythmic activity in the brain and has been found mutated in patients with early infantile epileptic encephalopathies [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF][START_REF] Tsay | HCN1 Channels Constrain Synaptically Evoked Ca2+ Spikes in Distal Dendrites of CA1 Pyramidal Neurons[END_REF]). Overall, changes of expression of these molecules strengthen our hypothesis that an areal patterning gene, such as COUP-TFI, can regulate the machinery necessary for cortical neurons to properly respond to external stimuli, which are critical for the establishment and maintenance of early cortical circuits.

Altered Egr1 expression in EmxCKO brains correlates with the impairments in

the establishment of thalamo-cortical circuits.

Synaptic neuronal activation triggers a series of intracellular pathways that regulate RNA transcription and translation as well as post-translational modifications [START_REF] West | Biological Functions of Activity-Dependent Transcription Revealed[END_REF]. A category of transcription factors called immediate early genes (IEG) Is constituted by a series of genes whose transcription is directly and rapidly induced by different cellular stimuli [START_REF] Flavell | Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system[END_REF][START_REF] Sukhatme | Early transcriptional events in cell growth: the Egr family[END_REF][START_REF] Veyrac | The transcription factor Zif268/Egr1, brain plasticity, and memory[END_REF]. Synaptic communication is one of the mechanisms activating immediate early gene translation and this, in turn, is crucial to mediate important events in the CNS, such as synaptic plasticity, LTP and LTD [START_REF] Wisden | Differential expression of immediate early genes in the hippocampus and spinal cord[END_REF][START_REF] Cole | Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation[END_REF]). In the cortex two immediate early genes are expressed in early post-natal days, such as cFos, Arc (Activity-Regulated Cytoskeleton-Associated Protein) and Egr1. Arc is an activity regulated cytoskeleton associated protein whose expression is regulated by neuronal activity [START_REF] Lyford | Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites[END_REF].

Differently from Egr1 and cFos, Arc mRNA is translocated to the dentrites and regulates translated in loco where it associated [START_REF] Steward | Synaptic activation causes the mRNA for the leg Arc to localize selectively near activated postsynaptic sites on dendrites[END_REF]. Arc function has not yet been clearly understood, although it most likely involves the interaction with polymerizing actin. Arc expression in adult cortex and hippocampus is strongly upregulated in protocols of LTP and seizures [START_REF] Fosnaugh | Activation of arc, a putative "effector" immediate early gene, by cocaine in rat brain[END_REF][START_REF] Tan | The activity-regulated cytoskeletal-associated protein arc is expressed in different striosomematrix patterns following exposure to amphetamine and cocaine[END_REF].

In the developing cortex Arc is expressed at P4 in all layers with a medial-high to caudal-low gradient (Figure 2 It was recently shown that COUP-TFI induces Egr1 in dopaminergic olfactory bulb interneurons after olfactory stimulation, and that in its absence these neurons fail to express Egr1 and the mature dopaminergic marker TH [START_REF] Bovetti | COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons[END_REF]. Moreover, COUP-TFI protein directly interacts with the Egr1 locus in an urogenital mesenchymal cell line by forming a regulatory complex with Sp1 [START_REF] Pipaón | COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site[END_REF]). Thus, Egr1 likely seems to be a direct target of COUP-TFI in activity-dependent processes. To investigate whether Egr1 expression is also directly regulated by These data support the hypothesis that COUP-TFI expression is necessary for the acquisition of the stellate morphology independently of TC axons and that COUP-TFI directly regulates Egr1 expression in a cellautonomous way during L4 specification.

Consequently, I hypothesized that Egr1, which acts both as an immediate early gene and is also regulated

COUP-TFI expression in post-mitotic cortical cells is necessary for functional areas organization.

During these last 15 years, a plethora of studies converged in the identification and characterization of a set of factors, including morphogens and transcriptional regulators, required in patterning the size and position of functional areas during neocortical development [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF][START_REF] O'leary | Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex[END_REF]. These genes mainly include transcription factors expressed in distinct rostro-caudal and latero-medial gradients in cortical progenitors, defining a 2D map in which each coordinates is defined by the specific combination of gene expression levels. According to the radial unit model, cortical neurons acquire positional information from the progenitors and translate them into several features that will define functional cortical areas in the adult brain [START_REF] Rakic | Decision by division : making cortical maps[END_REF].

In previous studies, genetic manipulation of mitotic genes in the cortex demonstrated how genes expressed in progenitors could affect the relative size of presumptive areas (also called protoareas) by altering the positional identity information that cortical neurons would inherit from their respective progenitors. However, it was not clear how the positional information encoded by patterning genes could be translated to differentiating neurons. This was mostly due to the fact that areal patterning genes, such as Pax6, Sp8, Emx2, are expressed only in mitotic cells and that constitutive mutants for these genes die at birth well before arealization is fully accomplished [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF][START_REF] Hamasaki | EMX2 regulates sizes and positioning of the primary sensory and motor areas in neocortex by direct specification of cortical progenitors[END_REF][START_REF] Georgala | The role of Pax6 in forebrain development[END_REF]. COUP-TFI was an exception in this regard, since it is expressed both in progenitors and post-mitoic neurons and the expression gradient is maintained even after birth. The first studies dealing with the full KO of COUP-TFI showed a severe arealization defect at birth [START_REF] Zhou | COUP-TFI: An intrinsic factor for early regionalization of the neocortex[END_REF]), but was unable to distinguish whether this was due to its role in mitotic and/or post-mitotic cells, or even to the severe lack of thalamic inputs. Still, the cortical conditional knock-out did not distinguish its function in mitotic versus post-mitotic cells [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]), but implied that, because of the severe phenotype, COUP-TFI might act in both cell types during the process of area patterning. The findings that factors expressed in cortical post-mitotic cells could also influence specific properties of cortical neuron specification in distinct layers, and thus act on areal organization, was drastically changing a dogma in which progenitors were the major actors in cell fate commitment and specification (Joshi et al. 2008b;Bedogni et al. 2010;[START_REF] Ross | Bhlhb5 and Prdm8 Form a Repressor Complex Involved in Neuronal Circuit Assembly[END_REF].

Nevertheless, these studies mainly focused on loss-of-function experiments and failed to properly demonstrate a direct control on area identity. Indeed, post-mitotic conditional knock outs in which sensory areas organization was strongly affected have been described [START_REF] Ballester-Rosado | mGluR5 in cortical excitatory neurons exerts both cell-autonomous andnonautonomous influences on cortical somatosensory circuit formation[END_REF], but these models were associated more with a strong impairment of sensory inputs from the thalamus rather than to an intrinsic transcriptional genetic program instructing cortical area identity prior to thalamic innervations.

The first aim of my project was thus to directly demonstrate via a combination of loss-and gain-offunction approaches that a strong areal patterning gene such as COUP-TFI could act independently in mitotic and post-mitotic cortical cells (Alfano & Magrinelli, Harb, Hevner, & Studer, 2014). By first deleting COUP-TFI expression in post-mitotic cells and analyzing the effects on area specification during early embryonic and later during post-natal development, we were able to demonstrate by genomic gene set analysis first at E15.5, but also by expression of area and laminar markers as well as TC topographic connectivity at P7, that COUP-TFI was mainly acting in post-mitotic cells not just by translating progenitor information, but instead by directly impinging a sensory identity in early neurons. In particular, COUP-TFI is acting in this way first by specifying layer 4 cells, the thalamic recipient cells that are strongly represented in somatosensory area but only weakly in frontal/motor area and second by restricting the specification time of layer 5 cells, and the output cells that instead are well represented in motor areas but only weakly in somatosensory regions. By doing so, in absence of COUP-TFI, the S1 area acquires several motorlike features, i.e. an almost inexistent layer 4 and an expanded layer 5.

Nevertheless, our data also unraveled a post-mitotic to mitotic feedback in which COUP-TFI and Sp8 expression are upregulated in progenitor cells, when COUP-TFI is deleted only in post-mitotic neurons. However, we do not believe that this feedback effect is sufficient to justify the huge areal phenotype we have described [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF]).

Sp8 overexpression in progenitors failed to show any expansion of the rostral region and on the contrary, the motor area is even reduced [START_REF] Sahara | Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning[END_REF]. Similarly, overexpression of COUP-TFI only in progenitors has again the opposite effect (reduced motor area), as seen in Fgf8

hypomorphs in which COUP-TFI is rostrally expanded [START_REF] Faedo | COUP-TFI coordinates cortical patterning, neurogenesis, and laminar fate and modulates MAPK/ERK, AKT, and β-catenin signaling[END_REF]. Finally, Emx2 and Pax6

are not particularly changed in mutants where COUP-TFI is deleted in post-miotitc cells, concluding again that the feedback effect on progenitors does not seem to really contribute to the severe areal phenotype described in this work.

Nonetheless, it would be interesting to investigate whether post-mitotic cortical mutants based on later deletions of transcription factors, such as COUP-TFI or Lhx2, at a time-point when no postmitotic to mitotic feedback can influence cortical development anymore, can reproduce the same phenotype. In addition, since Sp8 function has proven to be determinant for the organization of motor domains (only in loss-of-function), and COUP-TFI is more specific for sensory identity, it would be interesting to investigate the areal phenotype of a full or partial combined loss-offunction of both, COUP-TFI and Sp8. This investigation would allow revealing the plasticity of the cortical program in acquiring characteristics depending on cortical extrinsic inputs, such as the thalamic innervation. There is indeed evidence supporting the fact that cortical neurons have an intrinsic protomap independently from thalamic axons innervation [START_REF] Hevner | Cortical and thalamic axon pathfinding defects in Tbr1, Gbx2, and Pax6 mutant mice: Evidence that cortical and thalamic axons interact and guide each other[END_REF]. As Sp8

and COUP-TFI play important roles in creating the sensory and motor identity before the arrival of TC axons it would be interesting to observe what type of cortical identity prevails in absence of these two strong intrinsic reulators.

A recent study in which, similarly to COUP-TFI NexCKO mutants, the transcription factor Lhx2 is inactivated in Nex expressing cells [START_REF] Zembrzycki | Postmitotic regulation of sensory area patterning in the mammalian neocortex by Lhx2[END_REF] confirms the importance of postmitotically expressed genes in the organization of distinct areas and proposes a two-step model of area patterning. In progenitors patterning genes prespecify sensory area blueprints. These informations are sequentially sustained by the function of a series of transcription factors to maintain and translate the blueprints into functional area properties in post-mitotic cells. This implies that the expression of these genes only in progenitors (and not in post-mitotic cells) is not sufficient to control areal properties on their own. These data also support the importance of a transcriptional program starting after the mitotic phase during areas organization. These programs would have to maintain areal and laminar identity in cortical neurons translating the 2D positional identity up to a 3D positional identity program, which will take into consideration also the cell-type specification and connectivity pattern of distinct cortical layers. Uncovering the dynamics and the diversity of early and late postmitotic transcriptional program could indeed highlight important features necessary to create cell diversity of cortical neurons.

A second aim of this project was to test whether post-mitotic transcriptional program modifications were sufficient to reprogram functional areas identity over the program inherited from progenitors. To this purpose, we overepxressed COUP-TFI only in post-mitotic cells by using the same Nex-Cre mouse, previously used for the post-mitotic deletion, with the ultimate aim to re-activate COUP-TFI expression in a COUP-TFI null background in which progenitors fail to express COUP-TFI. Considering the previously demonstrated importance for COUP-TFI function in defining sensory areas, this model allowed to observe whether sensory area identity could be induced over a different identity inherited from progenitors.

We were astonished to find out that COUP-TFI expression in post-mitotic cells was sufficient not only to rescue the full phenotype of COUP-TFI null mice, but also to independently establish area and laminar hallmarks of sensory areas as well as a characteristic VPM connectivity profile in already programmed motor regions. Indeed, this model also provided a non-endogenous overexpression of COUP-TFI in rostral cortical domains, where COUP-TFI is normally not expressed, allowing us to evaluate its independent role in already programed motor cells. These data strongly indicated that cortical areas identity and thalamic topography can be still modified in post-mitotic cells by overriding the genetic program in progenitor cells.

Unfortunately, this model did not allow us studying cortical development after birth, therefore it was not possible to observe how well sensory areas were developing at later post-natal stages, and whether for example, ectopic barrels, could have been generated in rostral areas. Since VPM axons ectopically projected to this re-programmed sensory region, I predict that this would be the case.

TC axons topography organization and maintenance depend on different

mechanisms.

2.a. Area Identity of early post-mitotic cortical neurons controls initial TC axons topography.

My work suggests that TC axons topography, innervation and maintenance may be driven by different mechanisms with a certain degree of independence. For example, loss of post-mitotic COUP-TFI expression leads the VPM to ectopically innervate caudal regions, which are normally not a target of this nucleus and are instead targeted by a different core thalamic nucleus, the dLGN, which conveys visual inputs to the primary visual area [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. Nevertheless, at P7 the VPM nucleus innervates the presumptive barrel cortex as well, even with different degrees of efficiency. How comes that the same nucleus can project to two different targets with different efficiencies?

Previous data showed that EphrinA5 is expressed in EmxCKO brains at E18.5, even if its pattern is caudalized when compared to control brains [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]). EphrinA5 is important for regulating the laminar localization of core TC axons within the different cortical layers [START_REF] López-Bendito | Thalamocortical development: how are we going to get there?[END_REF], suggesting that caudally shifted EphrinA5 expression might contribute to the ectopic caudal innervation of the VPM in COUP-TFI mutants. However, before innervating the cortex, TC axons need to wait at the level of the subplate and subplate cell organization is significantly affected in COUP-TFI mutants, as supported by the altered Ctgf expression gradient. This defect was observed both at P0 and later at P7. Subplate cells play an important role in the formation of the initial circuit between TC axons and cortical neurons [START_REF] Piñon | Dynamic integration of subplate neurons into the cortical barrel field circuitry during postnatal development in the Golli-tau-eGFP (GTE) mouse[END_REF][START_REF] Mcconnell | Subplate neurons pioneer the first axon pathway from the cerebral cortex[END_REF], and their correct specification may indeed prove important for the initial topographic positioning of TC axons along the tangential axis of the cortex. This is also supported by the rescue of TC axons in KO;iz/hCOUP-TFI. In these mice, the Ctgf gradient of expression is comparable to that of control brains which correlates with how VPM axons target less caudal domains as in KO;iz/hCOUP-TFI brains.

However, in the null mice in which COUP-TFI was re-expressed only in post-mitotic cells, we did not obtain a full TC connectivity rescue. This is probably due to the total lack of COUP-TFI expression in the thalamus, which has been reported to be required in the correct specification and pathfinding of sensory thalamic neurons (Shen-Ju Chou et al. 2013). Thus, even if COUP-TFI was missing in the thalamus, part of the VPM axons could surprisingly reach their correct targets in the cortex, strongly suggesting that the cues within the cortex, and most probably within the subplate cells, are fundamental in attracting TC axons to the right cortical region. Nonetheless, these results clearly indicate that COUP-TFI expression in post-mitotic cortical cells is necessary and sufficient to initially guide VPM axons to their correct targets. The dual VPM projections that we observed in EmxCKO mice might thus due to the caudalization of the SP gradient that most probably can attract VPM axons to the occipital cortex and to a less extent to the mS1. This observation is also in line with previous data in which Fgf8 was overexpressed at early stages to influence the shift of VZ progenitors that will give birth to Sp neurons, or at the contrary a truncated soluble form of the respective receptor Fgfr3c was overexpressed. These study showed how TC axons follow the gradient of the modified SP neurons, indicating how this population plays and important role in directing TC axons topography [START_REF] Shimogori | Fibroblast growth factor 8 regulates neocortical guidance of area-specific thalamic innervation[END_REF]. These data might contribute in further understanding the role of subplate cells during the topographic organization of thalamic inputs, since these transient cells have dramatically expanded during evolution and may have been crucial for the evolution of more complex and interconnected cortices.

2.b. Cortical neuronal activity is involved during the process of TC circuit maintenance.

My data show that despite the ectopic VPM innervation to caudal areas of EmxCKO cortices, these mutants have some VPM neurons innervating the mS1 at P7. However, these connections are progressively lost during the first two post-natal weeks, when the development of the VPM-S1 circuit becomes independent from subplate cells and establishes instead activity dependent plasticity [START_REF] Dubroff | Use-dependent plasticity in barrel cortex: intrinsic signal imaging reveals functional expansion of spared whisker representation into adjacent deprived columns[END_REF]. In EmxCKO brains, VPM nuclei are not directly affected by COUP-TFI deletion, since the Emx-Cre line does not act in thalamic nuclei [START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]). Thus, thalamic inputs normally project towards the mS1 in these mice, but find a cortical environment, which is not suitable for establishing stable synapses. Indeed, I have shown that the mS1 cortex does respond to thalamic stimulation at P7 but that this response progressively diminishes up to P21. The reason of why this happens is still not clear.

Previous studies have demonstrated that cortical maturation is a process that depends on the properties of cortical excitatory neurons but also inhibitory interneurons [START_REF] Lodato | Excitatory Projection Neuron Subtypes Control the Distribution of Local Inhibitory Interneurons in the Cerebral Cortex[END_REF][START_REF] Southwell | Intrinsically determined cell death of developing cortical interneurons[END_REF][START_REF] Bonifazi | GABAergic hub neurons orchestrate synchrony in developing hippocampal networks[END_REF][START_REF] Le Magueresse | GABAergic Interneurons Shape the Functional Maturation of the Cortex[END_REF]. During cortical development, interneurons mature passing through specific steps and consequently influence the maturation of cortical neurons, providing trophic factors and pacing the establishment of early cortical circuits. Interneurons not only singularly regulate excitatory neurons activity, but also form a network of inhibition between different interneuron types, whichultimately regulates excitatory neurons activity [START_REF] Pfeffer | Inhibition of inhibition in visual cortex: the logic of connections between molecularly distinct interneurons[END_REF].

The distribution of GABAegic interneurons gas not been well characterized in our mouse model.

Interneurons do reach and invade the cortex since they are not affected by the mutation, but, most probably, their distribution is affected in the cortex due to the altered laminar structure.

Thus interneurons might abnormally interfere with pyramidal cortical neurons in EmxCKO mice. In addition, we still have not taken into account the role of the progressive shift of GABA dependent activity, from the initial excitatory function, to the mature inhibitory one, which depends on the upregulation of specific Cl -pumps that change the overall ratio of Cl -concentration within developing cortical neurons [START_REF] Bonifazi | GABAergic hub neurons orchestrate synchrony in developing hippocampal networks[END_REF][START_REF] Ikeda | Differential development of cation-chloride cotransporters and Cl -homeostasis contributes to differential GABAergic actions between developing rat visual cortex and dorsal lateral geniculate nucleus[END_REF]Kovàcs et al. 2014).

Further investigation on these subjects may help in better understanding the electrical properties required for establishing and maintaining the circuit between TC axons and cortex, and, importantly, the role of interneurons in this process by taking into account the Cl -gradient inversion during the maturation of cortical circuits.

Correlation of spontaneous cortical activity and thalamic evoked activity during development.

Previous studies already showed a link between spontaneous cortical activity and experiencedependent activity in the cortex, and their reciprocal correlation [START_REF] Tritsch | The origin of spontaneous activity in the developing auditory system[END_REF][START_REF] Tang | Spontaneous calcium transients in developing cortical neurons regulate axon outgrowth[END_REF][START_REF] Blankenship | The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina[END_REF][START_REF] Schneggenburger | Molecular mechanisms governing Ca2+ regulation of evoked and spontaneous release[END_REF]. Spontaneous activity in cortical neurons depends on the expression of a specific set of components regulating the passive and active ion transport. These modulators of activity allow neurons to have spontaneous ions currents with internally regulated rhythms, as well as synaptically-evoked ion currents that help translating electrical currents into transcriptional activities [START_REF] Tritsch | The origin of spontaneous activity in the developing auditory system[END_REF][START_REF] Tang | Spontaneous calcium transients in developing cortical neurons regulate axon outgrowth[END_REF][START_REF] Blankenship | The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina[END_REF][START_REF] Schneggenburger | Molecular mechanisms governing Ca2+ regulation of evoked and spontaneous release[END_REF]. Spontaneous activity of excitatory neurons during cortical development depends on these internally regulated mechanisms, as well as on the interneuron distribution and function, and the regulation of extracellular ion concentration [START_REF] Bartolini | Integration of GABAergic interneurons into cortical cell assemblies: lessons from embryos and adults[END_REF]Kovàcs et al. 2014).

My data strongly suggest that intrinsic transcriptional regulation of cortical neurons can modulate spontaneous activity of cortical neurons before the onset of sensory-evoked activity. COUP-TFI expression in cortical neurons influences the rate of spontaneous activity and their ability of creating synchronous networks during early post-natal days. Synchronous spontaneous activity is an important feature in the organization of cortical circuits during development [START_REF] Kirkby | A role for correlated spontaneous activity in the assembly of neural circuits[END_REF], therefore this mechanisms could provide some insight into how transcriptional modifications, often at the basis of congenital cognitive impairments, may act on the synchronicity of electrical events in the developing cortex. Much is known on the transcriptional regulation of cell type diversity and on the mechanisms controlling activity in the cortex, but few studies tried to connect these two aspects [START_REF] Rodríguez-Tornos | Cux1 Enables Interhemispheric Connections of Layer II/III Neurons by Regulating Kv1-Dependent Firing[END_REF][START_REF] Harb | Area-specific development of distinct projection neuron subclasses is regulated by postnatal epigenetic modifications[END_REF].

Several studies on cell diversity have mainly focused on identifying key transcription factors and characterizing their regulation. For how crucial these studies proved to be in defining the general organization of specific functional areas during cortical development and across different species, they could not unravel the fine and intrinsic features that differs distinct cortical cell types. In this work, I have identified some of the players of electrical spontaneous activity that might be under the direct or indirect function of the transcriptional regulator COUP-TFI. Investigating how patterning genes regulate these characteristics that allow developing neurons integrating into functional cortical circuits, would help understanding the complexity of cortical development and function. Furthermore COUP-TFI is a gene that has been associated with human cognitive disease especially linked to the childhood. In particular COUP-TFI haploinsufficiency has been associated

with ptic atrophy and developmental delay [START_REF] Bosch | NR2F1 mutations cause optic atrophy with intellectual disability[END_REF], and its deletion has been associated also wyth syndromic deafness [START_REF] Brown | NR2F1 deletion in a patient with a de novo paracentric inversion, inv(5)(q15q33.2), and syndromic deafness[END_REF]. Also, a series of cognitive disabilities, such as mental retardation, epilepsy and periventricular heterotopia has been linked to a deletion containing the COUP-TFI locus [START_REF] Guerrini | Periventricular heterotopia, mental retardation, and epilepsy associated with 5q14.3-q15 deletion[END_REF].

Immediate early gene regulation and function in the development of

thalamo-cortical sensory circuits.

Immediate early genes have been described as important relays in controlling cellular responses to specific stimulations [START_REF] Filipkowski | Inducing gene expression in barrel cortex--focus on immediate early genes[END_REF][START_REF] Curran | Fos: An immediate-early transcription factor in neurons[END_REF]. In the central nervous system, this group of genes was abundantly studied in the previous decade as a way to connect transcriptional modifications to some of the plasticity mechanisms and synapse strengthening observed in classic electrophysiological and behavioral tasks [START_REF] Bisler | Expression of c-Fos, ICER, Krox-24 and JunB in the whisker-to-barrel pathway of rats: Time course of induction upon whisker stimulation by tactile exploration of an enriched environment[END_REF]. cFos has in particular been related to plasticity mechanisms in L4 neurons of S1 during acute stimulation protocols [START_REF] Sukhatme | A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization[END_REF][START_REF] Dragunow | The use of c-fos as a metabolic marker in neuronal pathway tracing[END_REF][START_REF] Curran | Fos: An immediate-early transcription factor in neurons[END_REF].

In this project, I observed that Egr1 expression is strongly correlated to L4 core thalamic nuclei inputs and innervation in the mouse neocortex, and that this mechanism might be directly regulated by COUP-TFI. Egr1 expression in L4 neurons is required in the acquisition of the stellate morphology, a morphological feature acquired mainly upon innervation of S1 L4 neurons from the VPM thalamic nuclei.

Previous reports have indeed demonstrated that beside VPM-specific thalamic activity, a set of other characteristics specific of S1 area, are necessary for shaping the stellate morphology of L4 neurons, such as the expression of Ror-β [START_REF] Li | Laminar and columnar development of barrel cortex relies on thalamocortical neurotransmission[END_REF][START_REF] Ballester-Rosado | mGluR5 in cortical excitatory neurons exerts both cell-autonomous andnonautonomous influences on cortical somatosensory circuit formation[END_REF]Pouchelon et al. 2014). Thus, both the thalamic inputs and the cortex need to co-ordinate each other for the establishment and maintenance of a proper TC circuit.

In this project, I showed that spontaneous activity was affected in the developing cortex and that

Egr1 was not induced in L4 during the process of thalamic innervation, interfering in this way with the activity-evoked response originating from the VPM. As a consequence, thalamic axons tried to somehow innervate the cortex but were unable to maintain this connection after P16. In particular COUP-TFI, and consequently Egr1 expression in L4 neurons, allows these cells to acquire the stellate morphology upon VPM TC axons innervation.

In spite of that, it is still necessary to define the precise role of Considering the role of subplate cells for the correct TC connectivity and the early spontaneous activity gradient we observed, it can be hypothesized and explored that SP neurons early activity may contribute to their axonal extension [START_REF] Mire | Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth[END_REF][START_REF] Yamamoto | Shaping brain connections through spontaneous neural activity[END_REF]. This dynamic has already been described in the thalamus, yet no specific study addressed this topic in the subplates, especially considering eventual regional differences. Previous studies also already established that the patterning gene COUP-TFI can regulate dendrite extension and axonal elongation in cortical neurons [START_REF] Armentano | COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth[END_REF] Nevertheless, my data nicely complement the previous literature showing that loss of stellate cells and impaired barrel organization is due to affected thalamic activity [START_REF] Sukhatme | A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization[END_REF][START_REF] Dragunow | The use of c-fos as a metabolic marker in neuronal pathway tracing[END_REF][START_REF] Curran | Fos: An immediate-early transcription factor in neurons[END_REF], by proposing that cortical spontaneous activity in the cortex also needs to be properly set up prior receiving thalamic inputs. Thus, as previously reported, spontaneous and evoked activities are reciprocal and complementary events that need to be properly coordinated during cortical development and maturation. My results are also in line with the already established role of patterning genes role in defining cortical area functions;

patterning gene may indeed control the electrical properties of cortical neurons allowing them to be prepared for external inputs, by regulating a series of specific ion transporters and regulators and/or influencing the pathways activated upon neuronal activity.

In This will allow me to evaluate whether Egr1 can help forming stable synapses between VPM and S1 neurons or whether it can induce the stellate morphology independently from the formation of synapses with VPM axons. Finally, it is still important to find out whether the distribution of interneurons is altered in EmxCKO mutants and consequently how this could have an effect on the spontaneous activity properties of glutamatergic pyrmaidal neurons.

  of the developing neocortex. p. 32 3.a. Rectinoic Acid: meninges and olfactory placode. p. 34 3.b. Fibroblast growth factors: the anterior neural ridge. specification of SuPN and CT neurons. p. 58 7. The tangential organization of cortical areas. p. 61 8. Prototypic cortical circuits. p. 65 9. Endogenous activity of neocortical neurons during development. p. 68 9.a. Calcium-dependent pathways modulate cortical proliferation. p. 69 9.a.1. Cortical neurons migration is controlled by Calcium activity. p. 69 9.a.2. Calcium signaling and dynamics of axon elongation. p. 70 10. Establisment of thalamo-cortical circuits in the somatosensory area. p. 71 10.a. Thalamo-cortical innervation between VPM and S1. p. 73 10.b. Guidance of TC trajectories from the thalamic nuclei to the developing cortex. p. 73 10.c. The innervation of VPM TC axons in the postnatal cortex. p. 74 10.d. Subplate neurons relay information between thalamic axons and Layer 4 neurons. COUP-TFI post-mitotic cortical deletion in NexCKO mice. p. 131 1.3. Area identity impairments in post-mitotic COUP-TFI loss-of-function brains. p. 136 1.4. Laminar organization of the neocortex requires COUP-TFI expression in post-mitotic cells. p. 141 1.5. Altered sensory thalamo-cortical connectivity supports areal and laminar impairments of COUP-TFI mutants. p. 147 1.6. COUP-TFI post-mitotically promotes the expression of caudally-enriched genes in the cortex. p. 152 1.7. COUP-TFI expression in post-mitotic cells represses rostral mitotic genes. p. 152 1.8. KO;iz/hCOUP-TFI is a transgenic line that allows re-expression of COUP-TFI in post-mitotic but not in progenitor cells. p. 156 1.9. Post-mitotic COUP-TFI overexpression reverses the feedback regulation of mitotic patterning genes. p. 160 1.10. Post-mitotic COUP-TFI overexpression is sufficient to specify sensory identity and endogenous and evoked activity is controlled by COUP-TFI. p. 174 2.2. Differences in endogenous activity rate and synchronicity between neocortical regions in early post-natal cortices are modulated by COUP-TFI function. p. 175 2.3. Patterns of endogenous firing and synchronicity are affected in somatosensory cortical neurons of COUP-TFI mutants. p. 179 2.4. Thalamic inputs from the VPM fail to form proper barrels in the COUP-TFI mutant S1 cortex. p. 186 2.5. COUP-TFI mutant S1 cortices are initially activated by VPM stimulation but fail to respond to external stimuli at P21. p. 203 2.6. Glutamate receptors and ion channels are differentially expressed in the EmxCKO cortex. p. 210 2.7. COUP-TFI directly regulates membrane ion channels. p. 216 2.8. Altered Egr1 expression in EmxCKO brains correlates with the impairments in the establishment of thalamo-cortical circuits. p. 219 2.9. COUP-TFI and Egr1 cell autonomously regulate stellate cell morphology acquisition.

Figure i- 1 .FigureFigure 1 - 1 .Figure 1 - 2 .Figure 1 - 3 .Figure 1 143 Figure 1 - 6 .Figure 1 - 7 .Figure 1 - 8 .Figure 1 - 9 .Figure 1 -Figure 1 - 158 Figure 1 - 159 Figure 1 -Figure 1 - 163 Figure 1 -Figure 1 -Figure 1 -Figure 2 - 3 .Figure 2 - 4 .Figure 2 - 5 .Figure 2 - 6 .Figure 2 - 9 .Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure 2 -Figure

 11112131143161718191115811591116311123242526292222222222 Figure i-1. Organization of CNS development. p. 20 Figure i-2. Subdivision of Pallial and Subpallial territories. p. 22 Figure i-3. Sequential inside-out neurogenesis of cortical neruons. p. 24 Figure i-4. Diversity of cortical progenitors. p. 26 Figure i-5. Radial migration of differentiating neurons. p. 28 Figure i-6. The radial unit model. p. 31 Figure i-7. Patterning genes expression in the developing cortex. p. 33 Figure i-8. Patterning genes regulation and role on cortical areas organization. p. 38 Figure i-9. Mitotic and post-mitotic expressed gened regulating arealization. p. 46 Figure i-10. Laminar organization and excitatory neurons diversity in the neocortex. p. 50 Figure i-11. Interneuron migration from the ganglionic eminences to the cortex. p. 51 Figure i-12. Subdivision of differently projecting cortical neurons. p. 53 Figure i-13. Morphological diversity of neocortical neurons. p. 54 Figure i-14. Molecular diversity of Subplate cells. p. 57 Figure i-15. Molecular control of different cortical projection neurons. p. 60 Figure i-16. Functional areas subdivision in the human neocortex. p. 62 Figure i-17. Organization of cortical areas and thalamo-cortical innervation. p. 63 Figure i-18. Diversity of thalamo-cortical axons laminar distribution. p. 64

Figure

  Figure i-1. Organization of CNS development.

Figure i- 2 .

 2 Figure i-2. Subdivision of Pallial and Subpallial territories.

Figure i- 3 .

 3 Figure i-3. Sequential inside-out neurogenesis of cortical neruons.

Figure i- 4 .

 4 Figure i-4. Diversity of cortical progenitors.

Figure

  Figure i-5. Radial migration of differentiating neurons.

  neurons does not relate to the size of the brain (Herculano-Houzel et al. 2014). Finally, there is evidence that also different areas of the same brain have different sizes of micro-colums (Benavides-Piccione et al. 2006). In general, primary sensory areas have smaller microcolumns compared to higher order or motor areas (Benavides-Piccione et al. 2006).

Figure i- 6 .

 6 Figure i-6. The radial unit model.

Figure i- 7 .

 7 Figure i-7. Patterning genes expression in the developing cortex.

  and pathfinding[START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF][START_REF] Sansom | Gradients in the Brain: The Control of the Development of Form and Function in the Cerebral Cortex[END_REF]. During embryonic development, this region is a source of morphogens. The name anti-hem was chosen due to the secretion of molecules, such as the secreted frizzled protein 1 and 2 (Sfrp1,2)[START_REF] Assimacopoulos | Identification of a Pax6-dependent epidermal growth factor family signaling source at the lateral edge of the embryonic cerebral cortex[END_REF], known to antagonize Wnt signaling triggered by the hem. These molecules are homologues of the Wnt receptors and thus interfere with the corresponding Wnt signaling. The anti-hem also produces other morphogens, such as Tgf-α, Fgf7 and EGFs (Figurei-7).

  Patterning genes and the protomap hypothesis. While morphogens are expressed at the boundaries or outside the pallial territories, cortical progenitors in the neocortical VZ express a series of regionalized transcription factors. Their graded expression patterns along the rostro-caudal and latero-medial axis of the cortex somehow match with the gradient of morphogens and the rough organization of future functional areas (Cholfin & Rubenstein 2008; Rash & Grove 2006; Aboitiz & Zamorano 2013; O'Leary & Nakagawa 2002; Alfano & Studer 2013). These transcription factors are mostly expressed in VZ and SVZ progenitors in a combination of gradients predicting the future functional area organization. The final size and positioning of functional areas is indeed strongly altered after overexpression or downregulation of these factors (Figure i-8).

Figure i- 8 .

 8 Figure i-8. Patterning genes regulation and role on cortical areas organization.

Figure i- 9 .

 9 Figure i-9. Mitotic and post-mitotic expressed gened regulating arealization.

Figure i- 10 .

 10 Figure i-10. Laminar organization and excitatory neurons diversity in the neocortex.

Figure

  Figure i-11. Interneuron migration from the ganglionic eminences to the cortex.

Figure i- 12 .

 12 Figure i-12. Subdivision of differently projecting cortical neurons.

Figure i- 13 .

 13 Figure i-13. Morphological diversity of neocortical neurons.

  SP also possess unique transcriptional programs and markers, such as Ctgf (Connective Tissue Growth Factor), Nurr1 (Nuclear Receptor Related 1), Moxd1 (Monooxygenase, DBH-Like 1) and Cplx3 (Complexin 3) which are also shared within different species (Figure i-14) (Hoerder-Suabedissen & Molnár 2013). Tbr1 (T-box brain 1) is a transcription factors expressed in early corticogenesis, being specific for both SP and L6 neurons[START_REF] Hevner | Tbr1 regulates differentiation of the preplate and layer 6[END_REF]). This gene is important for SP specification resulting in impaired thalamocortical projections to the basal thelencephalon.

Figure i- 14 .

 14 Figure i-14. Molecular diversity of Subplate cells.

Figure i- 15 .

 15 Figure i-15. Molecular control of different cortical projection neurons.

  dimension and organization of each area depend on the specific charachteristics of the different mammalian classes. Particular exception in the geometric organization of functional areas can be found in mammalian species with particular peripheral sensory systems, such as the platypus bill and the echolocation in bats. The number of areas varies greatly from one animal to the other and depends strongly on the dimensions and complexity of the brain. Up to 200 areas have been identified in the human brain, each with specific functions operating with the unique goal of elaborating sensory inputs in progressively more complex and refined computations, starting from primary, to secondary and associative areas (Figure i-16).

Figure

  Figure i-16. Functional areas subdivision in the human neocortex.

Figure

  Figure i-17. Organization of cortical areas and thalamo-cortical innervation.

Figure i- 18 .

 18 Figure i-18. Diversity of thalamo-cortical axons laminar distribution.

  ; Harris & Shepherd 2015) (Figure i-19 and 20).

Figure

  Figure i-19. Prototypical organization of cortical circuits.

Figure i- 20 .

 20 Figure i-20. Principal distribution of input-output routes of cortical neurons.

intracellular

  Ca ++ pathway, first demonstrated in 1959 by Koketsu et al. Single cells are competent in controlling their own levels of Ca ++ concentration, which needs to be tightly regulated due to its important role in cell survival (Orrenius et al. 2003); nonetheless, isolated cells undergo spontaneous oscillation of Ca ++ cytosolic concentrations (Uhlén & Fritz 2010). Differently from other cell types, neuronal Ca ++ concentrations are not only intrinsically regulated, but also modified by extracellular ion concentrations, contributing in this way by generating specific circuit behaviors (Gonzalez-Islas & Wenner 2006; Meister et al. 1991; Tritsch et al. 2007). Cortical development is also regulated in several aspects by Ca ++ pathway, which has been shown to act endogenously (Figure i-21)(Uhlén et al. 2015 ; Corlew et al. 2004).

Figure i- 21 .

 21 Figure i-21. Progressive emergence of spontaneous Ca ++ activity in differentiating cortical neurons.

  Robo1 and DCC and allows TC axonal elongation[START_REF] Castillo-Paterna | DCC functions as an accelerator of thalamocortical axonal growth downstream of spontaneous thalamic activity[END_REF][START_REF] Mire | Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth[END_REF]).This process is intrinsically controlled by thalamic neurons: the upregulatation of the Ca ++ dependent pathways results in NF-kB transcription upregulation, which will in turn induce Robo1 and DCC transcription. The rapid translation of attractive and repulsive signals regulating growth cone guidance through Ca ++ signaling, also depend on cAMP and cGMP translation. Many signals can influence axonal growth and guidance; different signals have attractive or repulsive functions.

  mainly occupied by functional columns organized into clusters which respond directly to single whisker stimulation, as indicated by pioneering studies on single whisker deletions (Van der Loos & Woolsey 1973; Belford & Killackey 1980; Durham & Woolsey 1984; Erzurumlu & Gaspar 2012a).This structure is referred to as barrel field, and the region of the somatosensory cortex in which the barrel field is located is named barrel cortex. The organization of these barrels requires sensory activity in the early post-natal days in mice. Careful injury to specific whiskers results in the reduction of the cortical area innervated by sensory information coming from that particular whiskers and a consequent increase of the space dedicated to neightbouring whiskers[START_REF] Van Der Loos | Somatosensory cortex: structural alterations following early injury to sense organs[END_REF]. This critical period covers a period of about 5 days (Figure1-22)(P0 to P5)[START_REF] Durham | Effects of neonatal whisker lesions on mouse central trigeminal pathways[END_REF] Erzurumlu & Gaspar 2012a). The process of development of this circuit involves topographic innervation between the thalamus and the neocortex and maturation of organized synapses in an activity-dependent manner.

Figure i- 22 .

 22 Figure i-22. Progressive definition of S1 depends on thalamic innervation.

Figure i- 23 .

 23 Figure i-23. Attractive and repulsive cues lead thalamo-cortical and cortico-thalamic elongation.

  Figure i-24. Barrel cortex stellate and septa innervation.

  and post-mitotic cortical cells, we generated conditional knock out mutant mice for the COUP-TFI gene specifically for the cortex. The generation of COUP-TFI fl/fl Emx1-Cre was performed as described in Armentano et al. 2007. COUP-TFI floxneo13 ES cells have the 3 rd exon of COUP-TFI surrounded by fox sequences at the 5' and 3'. The flox sequences are Cis oriented, in order to allow excision of the region between them from the Cre recombinase activity. After the 2 nd flox sequence the neomycin 3 rd flox sequence. To obtain COUP-TFI flox mice, COUP-TFI flox neo13 ES cell clones were electroporated with Cre recombinase and COUP-TFI flox clones were selected by PCR. COUP-TFI flox clones were injected in blastocysts in C57Bl/6J and chimeric mice were mated with C57Bl/6J to obtained germline transmission.During COUP-TFI flox mice generation the neomycin was removed in ES cells after the electroporation of Cre recombinase. The allele Emx1-IRES-Cre was inserted via knock-in transgenesis in mice in order to obtain a transgenic line in which Cre recombinase combination happens specifically in cortical cells since E10.5[START_REF] Gorski | Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1expressing lineage[END_REF]. As homozygous COUP-TFI flox mice are viable and fertile they were directly mated with Emx1-IRES-Cre mice, in order to remove COUP-TFI specifically in all cortical cells. COUP-TFI fl/fl;Emx1-IRES-Cre mice were considered as Conditional knock outs (EmxCKO), while COUP-TFI +/+, fl/fl and fl/+ withouth the Emx1-Cre allele were considered as controls.EmxCKO mice are also fertile and viable and present a specific COUP-TFI loss in cortical cells. In order to properly assess the genotype of these mice two differents Polymerase Chain Reactions (PCR) were performed, in order to test the presence of COUP-TFI flox allele and the Emx1-Cre allele. The reaction for the COUP-TFI allele was performed using three primers designed to discern the presence of a wildtype COUP-TFI allele, a COUP-TFI flox allele and a COUP-TFI null allele in which the third exon was actually removed from the genome. The forward primer ARM 531 (ctgctgtaggaatcctgtctc) anneals upstream to the third COUP-TFI exon, the EX351 (aatcctcctcggtgagagtgg) anneals to a sequence inside the 3rd COUP-TFI exon and the ARM402

  CCRmR and CCRmL primers at a concentration of 0.1µM, which anneal into the sequence of the β-actin. While the Cre Fw and Rev amplicon is 400bp the CCRmR (atgtggatggagaggagtcg) and CCRmL amplicon (caaccgagaccttcctgttc) is 250bp, therefore it is possible to use the β-actin amplicon as an internal control and the Cre amplicon as a confirmation of the presence of the Cre allele. It is impossible to understand whether or not the Cre allele is present in homozygosis or heterozygosis. The DNA concentration used is the same as the one indicated for COUP-TFI allele genotyping as also the program used for the reaction.2.b. COUP-TFI fl/fl Nex-Cre.COUP-TFI fl/fl Nex-Cre mice were genotyped using the same protocol used for COUP-TFI fl/fl Emx1-Cre ones as COUP-TFI fl allele was shared between the two mice. The Nex-Cre allele was also genotyped using the same primers used for the Emx1-Cre allele, as they recognize the open reading frame of the Cre-recombinase[START_REF] Goebbels | Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice[END_REF]).

  DNA and RNA were quantified on a Nanodrop spectrophotometer, which converts the absorbance at 260nm of the DNA through the Lambert-Beer law [A=ε λ lC] after measuring a blank sample. The concentration was calculated on the average of three different dilutions in the reading range of the Nanodrop instrument.

RT and 3

 3 times in ice cold Equilibration buffer A (HEPES pH7.4 20mM, EDTA 1 mM, NaCl 150 mM, Triton 0.8%, SDS 0.1% PMSF 10 mg/ml in H 2 O mq) and left overnight in agitation at +4°C. This process took place in low bind 15ml Falcon tubes spun at 1.5g at +4°C at RT Tissues used for the ChIP were collected pooling cortices from 8 wt P0 pups of the same littermate. Cortices from both hemisphere were dissected in ice cold HBSS and removed from meninges and hippocampus, once collected samples were washed twice in 10ml DMEM for 5' at RT collecting cortices by spinning at 1500g for 2' at +4°C.After suspending the tissue in 10 ml of DMEM it was crosslinked adding 270.5 µl of 37% formaldehyde for 10' at RT. The reaction was stopped adding 1.1 ml of glycine 1.25 M and incubating for 5'. Crosslinked samples were washed 3 times with 10ml ice cold cell wash buffer (HEPES pH 7.4 20 mM, NaCl 150 mM, Glycine 0.125 M, PMSF 10mg/ml in H 2 O mq) and suspended in 6ml of Lysis buffer (HEPES pH 7.4 20 mM, NaCl 150 mM, Glycine 0.125 M, SDS 1% PMSF 10mg/ml in H 2 O mq). The solution was moved in a glass potter to mechanically disrupt cell membranes obtaining cell nuclei which were collected by centrifugation at 2.2kg for 5' at +4°C with a slow rate of acceleration and deceleration. After estimating the volume of nuclei obtained it was suspended in Sonication Buffer (HEPES pH 7.4 20 mM, NaCl 150 mM, Glycine 0.125 M, SDS 0.4 % PMSF 10mg/ml in H 2 O mq) to a final volume of 1.2 ml of which a 10µl was taken and used to count the amount of nuclei obtained on a microscope counting dish. After confirming having collected around 9*10 6 nuclei, samples were split in 4 tubes of 300µl volumes each used to sonicate nuclei on an immersion sonicator.The sonication was carried for 6 times 5'' at 10 µm amplitude. Tubes were centrifuged at 14kg for 10' and move on a new tube, in order to discard undissolved clusters of cellular debris. This process was repeated a second time pooling together the 4 tubes. 5µl of sample were collected to verify on an agarose gel that the sonication process generated DNA fragments of about 500bp.The collected samples were yet divided in 4 samples to which 50 µl of Slurry was added and incubated in rotation at +4°C for 1h to perform a pre-clearing, removing DNA fragments that aspecifically binds the Protein-A resin. Samples were cleared off the resin centrifuging at 800g for 2' at +4°C and moving the surnatant into new tubes, to which 1.2ml of SDS dilution buffer ( HEPES pH 7.4 20 mM, NaCl 150 mM, Triton 100 1%, PMSF 10mg/ml in H 2 O mq). This process was repeated twice. Precleared samples were added with a set of different antibodies: in sample I, I added COUP-TFI antibody, in sample II the Egr1 antibody, in sample X the GFP antibody, in sample N no antibody was added. All antibodies were used at 3μg. A new sample was obtained using 300µl of Sonication buffer alone, called mock (M) which does acts as a negative control, not containing any antibody and DNA. Samples with antibodies were incubated overnight at +4°C in gentle rotation to bind antibodies to target proteins.

  tracing injections were performed either injecting CTB-conjugated fluorophores (Invitrogen, 488nm C34775 and 555nm C34776) or DiI diluted in DMSO. In both cases the injection were performed in live animals anaesthetized and immobilized on a Digital Lab Standard Stereotaxic Instrument (Stoelting 51900) applying eye hydrating gel droplets. After appropriate shaving and disinfection of the head the skin was opened at the dorsal base of the skull to visualize the lambda (λ) point at the junction between parietal and occipital bones; once the needle loaded on the Nanoinjector (Nanoject II Auto-Nanoliter Injector, Drummond Scientific Company 3-000-204) was

  platform, normalizing the intensity range and the size of image to be comparable between the different conditions. The results show a line chart with mean and s.e.m. of fluorescence signals normalized from 0 to 1. Pearson colocalization coefficients were also calculated using a custom Matlab program. Pearson coefficient is obtained dividing the covariance of two variables for the product of the two standard deviations. This coefficient ranges from -1 to 1 where -1 indicates absolute complementarity between the two analyzed signals and 1 indicates complete overlapping of the two analyzed signals.Morphological assessment of Stellate and Pyramidal morphology was performed manually analyzing GFP expressing cells in a z-stack in ImageJ, subdividing each individual cell for with the corresponding coordinates. Stellate and Pyramidal morphology was quantified over the total amount of GFP expressing cells. Each subgroup of cells was also analyzed for the expression of proteins using the coordinates and verifying manually the presence of other proteins signals in the same location and comparing the results with the ortagonal view(ZEN). Quantification of single cells count and co-localization evaluation where made on 3 animals minimum on images representing the same anatomic area. Post Hoc analysis on variance within two independent groups was performed using T-test analysis on Excel.25. pCIG::Egr1::IRES::GFP pCDK5::Egr1::IRES::GFP plasmid construction.The Egr1 ORF was amplified using available cDNA synthetized in the lab starting from RNA extracted from P7 wildtype cortices using the four combinations of the two following forward and two reverse primers: Egr1 5' UTR-FW (gagagatcccagcgcgcag) Egr1 5' CDS-FW (ccaacatcagttctccagctcgc) Egr1 3' UTR-rev (gaaggatacacaccacatatcc) Egr1 3' CDS-rev (ccaataccatgcaaatcaagtcc) therefore amplify four possible amplicon containing: Egr1 ORF only (1.8kb), Egr1 ORF ant 5'UTR (2kb), Egr1 ORF and 3'UTR (2.8kb), Egr1 ORF with both 5' and 3'UTR (3kb).Amplification were verified on an agarose gel and directly purified by gel extraction kit (Quiagen), following the manufacturers instruction. Amplicons extracted were cloned by ligation in a TOPO TA plasmid (Invitrogen) following the manufacturers instruction. Ligation mix was directly electroporated in E.coli cells, amplified in Ampicillin LB and extracted by miniprep kit (Quiagen).To verify the ligation samples ligation which contained the Egr1 5'UTR were digested with SmaI and SpeI as SmaI would cut at 234bp in the 5'UTR, while SpeI would cut at about 1kb from the 5' edge of the injection site, allowing to verify the presence and orientation of the insert. Plasmid which did not contained Egr15'UTR were verified by EcoRI digestion, which cuts at the two edges of the insertion site of the TOPO-TA plasmid, allowing to verify the insertion. All digestion mix were performed at 10µl using 2µl NEB4 buffer, 1µl of BSA, 1µl of the enzyme, 1µl of purified plasmid, in H 2 O mq and was carried at +37°C for 2h. Plasmid which contained the insert was then sequenced from the sp6 promoter, in close proximity of the TOPO-TA cloning site. Verified plasmids were amplified into midipreps (Quiagen).Egr1::TOPO::TA plasmids obtained were used to obtain Egr1 sequences to clone in pCIG::IRES::GFP and cDK5::IRES::GFP plasmids. pCIG::IRES::GFP plasmid have a CMV promoter followed by a multiple cloning site (MCS), an IRES sequence which allows the expression of a polycistronic RNA containing two independent translation sites for ribosomes and a GFP ORF. The MCS of this plasmid has an EcoRI unique cutting site which, therefore I extracted Egr1 sequences from the TOPO-TA plasmids digesting with EcoRI and linearized pCIG::IRES::GFP plasmids using the same enzyme. Digestions were carried out in 50µl volume of H 2 O mq which contained 5µl of NEB4 buffer, 3µl of BSA, 3µl of EcoRI and 10-15µg of plasmid. Egr1 digestion were verified on a gel and extracted from the gel with the Quiagen gel extraction kit. pCIG::IRES::GFP linearization were verified on a gel and linearized plasmid (6kb) was extracted from the gel and then dephosphorylated. The dephosphorilation was performed in 50 µl of total volume in H 2 O mq which contained all the extracted linearized plasmid, 5µl NEB3 buffer, 1µl of Calf-Intestine-Phosphatase (CIP, Biolabs), incubated for 1h at +37°C, then 1µl of CIP was added and incubated for an additional hour at +37°C. The reaction was extracted with Phenol-Chloroform twice and precipitated in 1/10 Sodium Acetate 3N, three volume of ethanol 96% and 1µl of glycogen overnight at -20°C in 10 μl of H 2 O mq. All four extracted Egr1 inserts were ligated with pCIG::IRES::GFP linearized plasmid at a stoichiometric ration of 1:2 and 1:5.

  was prepared containing the digested plasmid without the insert. The reaction was let overnight at 16°C. It was then precipitated as previously explained and re-suspended in 10μl of H 2 O mq. 2μl of the ligation were then electroporated in XL1Blue bacterial cells and amplified in minipreps. To verify the ligation plasmids were digested using SmaI, for plasmid ligated with Egr15'UTR and Egr15'3'UTR, which would either give a 3kb+6kb bands in case of in frame ligation, a 9kb band in case of ligation in the wrong frame and a 6kb band in case of no ligation. Plasmid ligated with Egr1CDS were tested with PstI and SmaI which would give 2kb+6kb bands in case of in frame ligation, a 8kb band in case of wrong frame ligation and a 6kb band in case of no ligation. Finally plasmids ligated with Egr3'UTR were digested with EcoRI which would either give 2kb + 6kb bands in case of ligation and 6kb band in case of no ligation, without discerning for frame orientation. Digestions were carried out as previously described. Plasmids with verified ligation were sequenced using the reverse Egr1 primers and verified plasmids were then amplified in Endofree-Maxiprep kits (Quiagen). As only Egr1 CDS and Egr15'3'UTR inserts were verified to have ligated I verified wether these sequences were able to express Egr1 when electroporated in mice neurons performing in utero electroporations with these plasmid. I could then verify that both Egr1 CDS and Egr15'3'UTR sequences inserted in pCIG-IRES-GFP plasmid can be expressed in mice neurons once electroporated.I also cloned Egr1 sequences in cDK5::IRES::GFP which contained a promoter region from the cDK5 gene, which is specifically expressed in post-mitotic cells, which allows expressing a gene of interest after the end of proliferation. I extracted Egr15'3'UTR and Egr1CDS from TOPO-TA-Egr15'3'UTR and TOPO-TA::Egr1 CDS by digesting with EcoRI as previously indicated. I linearized cDK5-IRES-GFP plasmids with MluI enzyme in 50µl volume of H 2 O mq (5µl NEB3, 3µl BSA, 10-15µg plasmid) at 37°C for 3hours. Linearized plasmid were verified on an agarose gel and extracted with Quiagen gel extraction kit. Both the linearized plasmid and the Egr1 fragments were incubated with Klenow fragments to perform fill in on the endings. Fill in reaction was carried in 30µl of volume in H 2 O mq containing all of the plasmids and fragments obtained, 3µl NEB2 buffer, 1,2dNTP 25mM mix, 1µl of Klenow fragment. The reaction was performed for 20' at RT and then samples were extracted in PCA and precipitated in 10 µl of H 2 O mq, for the Egr1 fragments, and 43µl for the linearized plasmid. Then the linearized plasmid was also dephosphorilated as previously indicated and precipitated in 10µl of H 2 O mq. Ligation reaction with Egr15'3'UTR (3kb) Egr15'UTR (2kb) with linearized cDK5::IRES::GFP (7kb) was performed at stoichiometric ration of 1:2 and 1:5 in 20 µl of H 2 O mq total volume with the following mix:

  was prepared containing the digested plasmid without the insert. The reaction was let overnight at 16°C. It was then precipitated as previously explained and re-suspended in 10μl of H 2 O mq. 2μl of the ligation were then electroporated in XL1Blue bacterial cells and amplified in minipreps. To verify the ligation I performed PCR reaction with Egr1 5' CDS.FW + Egr1

  of results interpretation (Figure m-1). Time lapse images acquired were stacked using ImageJ which was further aligned with the "align images in stack" plugin, to correct eventual slice drift during the acquisition. Images were analyzed in a custom Matlab program to acquire information on single cells Ca ++ activity. At first I selected manually outlines for single regions of interests (ROI), defining soma of each single cell in the image. ROI outlines are used singularly to create masks over the entire image stack and the average intensity of all pixels inside the ROI outline are registered in a vector numbered with the ROI selected (Figure m-2.a). At the same time ROI outline center coordinates are registered. To compare the position of different ROI compared to their laminar and columnar distribution I manually determined the position of the Pia line. I used Pia line straight line coordinates as a new x axis Cartesian system, from which ROI coordinates were obtained, calculating their radial and distance from the Pia line and between coordinates and calculating the tangential distance between all ROI (Figure m-2.b). The result of this process is a matrix in which each value indicates the tangential distance calculated between two ROI coordinates over the parallel axis of the Pia line.

Figure m- 1 .

 1 Figure m-1. Schematic representation of workflow of Calcium imaging analysis.

Figure m- 2 .

 2 Figure m-2. Sample of ROI and Pia selection.

Figure m- 3 .

 3 Figure m-3. Bleach correction, standardization and filtering of active cells signals.

Figure m- 4 .

 4 Figure m-4. Detection of burst events and correlation clustergram creation.

  Figure m-5. Sample of k-means clustering of cell pair using correlation and tangential distance parameters.

Part 1 .

 1 (Data published in Alfano & Magrinelli et al. 2014) 1.1. Post-mitotic inactivation of COUP-TFI.As previously discussed, COUP-TFI is expressed in both progenitors and post-mitotic cells of the developing cerebral cortex. The importance of this transcription factor was already demonstrated in cortical development and areal organization, although with no clear distinction between the roles played in progenitors and/or in post-mitotic cells. These studies made use of different lossand gain-of-function mouse models, such as the COUP-TFI constitutive KO, the COUP-TFI fl/flEmx1-Cre

Figure 1 - 1 .

 11 Figure 1-1. Specific post-mitotic Cre expression and COUP-TFI deletion.

Figure 1 - 2 .

 12 Figure 1-2. NexCKO mice have non-significant effects on cortical proliferation.

  -3.a). To understand the specific function of COUP-TFI expression in post-mitotic cells, I compared the expression of area specific markers in control, EmxCKO and NexCKO brains. First, I performed whole mount in situ hybridizations to analyze the overall tangential expression of known areal markers in P7 control, NexCKO and EmxCKO cortices (Figure 1-3.b). The markers used in these experiments were Bhlhb5, Cad6 and Cad8. Bhlhb5 is a transcription factor involved in the organization of barrels in the S1 (Joshi et al. 2008a) and mainly expressed in S1, V2 and A1 areas, but not V1 at P7. In both EmxCKO and NexCKO cortices, Bhlhb5-expressing areas are strongly reduced in size and shifted caudally, similarly to what was already observed in the EmxCKO cortex[START_REF] Armentano | COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas[END_REF]. Cad6 is an adhesion protein expressed in sensory domains. In whole mounts of control brains at P7 it clearly labels sensory areas (S1, S2, V1, V2), and its expression is strongly reduced in size and caudalized in EmxCKO and NexCKO brains. On the contrary, the adhesion protein Cad8 is strongly expressed in Frontal/Motor areas and in V2 cortex surrounding the V1 area in control brains. In good accordance with previous data, Cad8 expression is expanded from rostral to caudal regions in both mutant cortices.

Figure 1 - 3 .

 13 Figure 1-3. Arealization markers and COUP-TFI expression in control, EmxCKO and NexCKO sagittal sections.

EmxCKO

  and NexCKO cortices the strong Ror-β expression domain observed in control S1, is limited to a small portion of the caudal cortex, while the remaining cortical regions show just a faint Ror-β expression in layer 5a that recalls the normal pattern in control motor domains. Finally, the insulin growth factor binding protein 4 (Igfbp4) is expressed all along the rostro-caudal axis of P7 cortices, with the exception of the caudal V1 area receiving geniculocortical axons (S.-J. Chou et al. 2013), Igfbp4 expression is expanded caudally in accordance with the reduction and caudalization of V1 in both EmxCKO and NexCKO brains (Figure 1-3.c).

Figure 1 - 4 .

 14 Figure 1-4. ISH of laminar and areal cortical markers in control, EmxCKO and NexCKO sagittal sections.

  they are not or poorly co-expressed with Ctip2. The amount and distribution of Tbr1 and Fog2 cells in NexCKO and EmxCKO is not significantly different from control cortices, suggesting normal generation of layer 6 neurons. Together, these data indicate that COUP-TFI expression in postmitotic cells is mainly required in the specification of layer 5 Ctip2+ neurons.Foxp2 (Forkhead Box P2) is expressed in layer 6 and 5b of control cortices and its expression is radially expanded in the EmxCKO compared to both control and NexCKO brains, as well as the amount of neurons co-expressing Foxp2 and Ctip2. In NexCKO brains, although the rate of Foxp2+ cells is fairly similar to control brains, the amount of neurons co-expressing Foxp2 and Ctip2 is now increased in NexCKO cortices, most probably due to the increase of Ctip2+ cells. This significant difference between EmxCKO and NexCKO organization suggests that the development of specific neuronal populations is more dependent on COUP-TFI expression in progenitors.Next, I analyzed the laminar identity of upper layer neurons, which have important roles in sensory area functioning. Bhlhb5 protein expression in controls is expressed from layer 5 to layer 2/3 and it spans from somatosensory areas to visual areas. Cux1 is organized similarly and is expressed solely in upper layers, where it directs callosal neuron maturation in an activity dependent manner (Figure1-7)[START_REF] Cubelos | Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex[END_REF][START_REF] Rodríguez-Tornos | Cux1 Enables Interhemispheric Connections of Layer II/III Neurons by Regulating Kv1-Dependent Firing[END_REF]. Both Cux1 and Bhlhb5 are absent or faintly expressed in EmxCKO and NexCKO mS1 cortices and are detectable only in a small domain of the occipital cortex, in accordance to the reduction and shift of sensory domains to caudal regions (Figure1-7).In conclusion, the laminar characterization of EmxCKO and NexCKO cortices indicates an acquisition of motor-like features in somatosensory regions, confirming a crucial role for COUP-TFI in the laminar specification of sensory areas at post-mitotic level.

Figure 1 - 5 .

 15 Figure 1-5. Lower layer organization in motor areas of control, EmxCKO and NexCKO brains.

  EmxCKO: (#) P ≤ 0.05; (##) P ≤ 0.01; (###) P ≤ 0.005. 1. All data were collected from n=3 animals for each genotype and were analyzed by ANOVA (post-hoc) analysis performed by two-tailed t-test. Error bars represent s.e.m.

Figure 1 - 6 .

 16 Figure 1-6. Lower layer organization in S1 in control, EmxCKO and NexCKO.

Figure 1 - 7 .

 17 Figure 1-7. Upper layer organization in control, EmxCKO and NexCKO.

Figure 1 - 8 .

 18 Figure 1-8. Thalamic innervation in control, EmxCKO and NexCKO.

Figure 1 - 9 .

 19 Figure 1-9. Thalamic innervation of the caudal cortex and subplate organization.

Figure 1 -

 1 Figure 1-10. Microarray analysis of control and NexCKO brains.

  NexCKO brains were not significantly different compared to controls, they were strongly reduced in EmxCKO brains (Fold Change NexCKO COUP-TFI=0.972 + 0.127. EmxCKO COUP-TFI=0.228 + 0.166. ANOVA Pvalue< 0.003). This suggests that, in normal conditions, COUP-TFI expression in mitotic cortical cells is negatively regulated by COUP-TFI expression in post-mitotic cells. Thus, COUP-TFI regulates neocortical arealization not only by promoting caudally-associated genes in post-mitotic cells, but also by regulating its own expression and putative rostral patterning genes directly (at mitotic level) and indirectly via a feedback from post-mitotic neurons (Zembrzycki et al. 2007) (Figure 1-12).

Figure 1 -

 1 Figure 1-11. Molecular analysis of gene expression in COUP-TFI mutants.

Figure 1 .

 1 Figure 1.12. Proposed model of COUP-TFI arealization control.

(

  which possess only the CAGGS-lox-stop-lox-hCOUP-TFI and the Nex-Cre alleles) by immunostaining P0 controls and iz/hCOUP-TFI brains with the COUP-TFI and c-myc antibodies. In these brains, endogenous COUP-TFI expression strongly overlaps with the domain of ectopic COUP-TFI in the cortex, as also indicated by the Pearson coefficient (P=0.614)(Figure 1-13). As expected, iz/hCOUP-TFI lack hCOUP-TFI expression (detected by c-Myc antibodies) in cortical progenitor, but express hCOUP-TFI in post-mitotic cortical cells (Figure 1-13.b). To further confirm maintenance of hCOUP-TFI expression in post-mitotic cells of COUP-TFI null mice, KO;iz/hCOUP-TFI cortices were ananlyzed at E13,5, when progenitor cells are more abundant. Co-staining with c-Myc;Tuj1 and c-Myc;Tbr2 showed that hCOUP-TFI was expressed in post-mitotic (Tuj1+) but not in intermediate progenitor (Tbr2) cells (Figure 1-14). These experiments were confirmed for the radial gradient of expression and the Pearson coefficient of radial co-expression. As expected, I observed that KO;iz/hCOUP-TFI cortices express c-Myc tagged COUP-TFI in the domain of Tuj1 only, supported by the radial profile of expression and the Pearson coefficient (P=0.630), but failed to express c-Myc in Tbr2+ cells (P=-0.111). These data further confirm the specific post-mitotic expression of hCOUP-TFI in the KO;iz/hCOUP-TFI mice (Figure 1-13.a). Since ectopic expression of hCOUP-TFI is regulated by the CAG (CMV early enhancer/chicken ßactin promoter) promoter, it does not show the typical COUP-TFI gradient (high caudal to low rostral) and thus ectopic post-mitotic COUP-TFI expression is strongly expressed in both rostral and caudal post-mitotic cortical cells (Figure 1-14.c). This was revealed by IF on sagittal cryosections of P0 control, KO, and KO;iz/hCOUP-TFI cortices (Figure 1-13.c). Thus, despite the limited life span of KO;iz/hCOUP-TFI mice, they provide a solid genetic model, complementary to the NexCKO one, to test whether COUP-TFI expression in post-mitotic cells is sufficient to impose a sensory identity in the developing neocortex.

Figure 1 -

 1 Figure 1-13. COUP-TFI is re-expressed in cortical post-mitotic cells in KO;iz/hCOUP-TFI mice.

Figure 1 -

 1 Figure 1-14. Post-mitotic COUP-TFI expression and areal organization in KO;iz/hCOUP-TFI.

Figure 1 -Figure 1 -

 11 Figure 1-15. Patterning genes expression in control, NexCKO and KO;iz/hCOUP-TFI.

Figure 1 -

 1 Figure 1-17. Areal and laminar organization in control, NexCKO, COUP-TFI KO, and KO;iz/hCOUP-TFI.

Figure 1 -

 1 Figure 1-18. Detailed immune labeling of Bhlhb5, Lmo4 and COUP-TFI in control, COUP-TFI KO, and KO;iz/hCOUP-TFI.

Figure 1 -

 1 Figure 1-19. Thalamic innervation of caudal cortices in KO;iz/hCOUP-TFI.

  -16.a). Additionally, while Lmo4 protein expression negatively correlates with COUP-TFI expression, Bhlhb5 strongly co-localizes with COUP-TFI expression. These data strongly indicate that COUP-TFI post-mitotic expression in rostral regions is sufficient to induce sensory identity not only in mutant parietal cortices, that acquired a motor identity in the absence of COUP-TFI, but also in rostral motor regions where now COUP-TFI is strongly and ectopically induced (Figure 1-16.c). To corroborate these data, I have also examined motor and sensory thalamo-cortical connections by performing retrograde labelling experiments on P0 wt and KO;iz/hCOUP-TFI brains and injecting DiI (red crystals) in rostral regions and DiD (far red crystals; digitally replaced with green) in parietal regions of the neocortex (Figure 1-20). In wt mice, the VPM nucleus projects uniquely to the S1, whereas rostral motor regions do not receive VPM innervation and are strongly innervated by the VL and Po thalamic nuclei. The VL is found more rostrally (Figure 1-20.a), instead the Po and the VPM are palced in medio-caudal regions of the thalamus (Figure 1-20.b). As expected, in wt brains DiI retrolabeled the VL, and the Po in rostral regions, while DiD retrolabeled cells of the VPM in somatosensory cortex. Both the DiI and DiD are visible in the internal capsule (ic) through which thalamic cells project their axons en route versus the cortex. On the other hand, in KO;iz/hCOUP-TFI brains, nor the DiI or the DiD retrolabel VL cells, while both of them retrolabeled VPM cells and, in part the Po. These results show that COUP-TFI overexpression in rostral postmitotic neurons is able not only to reprogram these cells to a sensory identity, but also to redirect talamo-cortical connectivity. This confirms previous molecular data and further demonstrates that COUP-TFI post-mitotic expression is sufficient to induce sensory identity in neocortical young neurons during development.

Figure 1 -

 1 Figure 1-20. Thalamic innervation of parietal and frontal cortices in KO;iz/hCOUP-TFI.

Figure 2 - 1 .

 21 Figure 2-1. Rostro-caudal regionalized differences in calcium frequency and synchronicity is affected in young EmxCKO pups.

Figure 2 - 2 .

 22 Figure 2-2. MEA recording of control and EmxCKO dissociated somatosensory neurons.

  -4.b) suggesting a delay in the network maturation of EmxCKO neurons. Considering the IEI distribution of control neurons at 6 DIV (Figure 2-4.c), it was fitted by a double Gaussian with mean values of 1.5 s and 13.5 s respectively. On average 80% of IEI values belonged to the Gaussian centered on 1.5 s (narrowest in the figure), while 20% of IEIs belonged to the Gaussian centered on 13.5 s. Considering the IEI distribution of EmxCKO neurons after DIV 6 it was fitted by a double Gaussian as well. The average IEI values for each of the two Gaussian distributions were respectively 0.76 s and 10.7. While 60% of IEI values belonged to the narrowest Gaussian centered on 0.76 s, the broadest Gaussian, centered on 10.7 s accounted for the remaining 40% of IEIs (Figure 2-4.c). We than concluded that at early developmental stage the contribution of longer IEI values is almost doubled in EmxCKO mice, confirming a tendency of EmxCKO neurons to fire at lower frequency.

Figure 2 - 3 .Figure 2 - 4 .

 2324 Figure 2-3. Spontaneous recording of dissociated control and EmxCKO cortical neruons at DIV6 and DIV9.

  control and EmxCKO brains (Figure 2-5). At P7 5HT is localized mostly in L4 of S1, where it is also organized in the typical clusters of the barrel field, and V1, and it is only present in few quantity in the rostral area M2 (Figure 2-5.a). While in V1 of EmxCKO 5HT is localized similarly to the control S1 area, in S1 it is still present in upper layers but less concentrated and without a clear organization in clusters (Figure 2-5.a). At P16 TC axons terminals can be visualized with the expression of Vglut2 (Figure 2-5.b).In control S1 area it is clearly expressed in L4 clusters and in L5a, accordingly to the typical pattern of core thalamic nuclei innervation. A similar pattern is observed in V1 and in M2 it is possible to observe a strong labeling in L3b, the layer in M2 which still receives core thalamic innervations. In EmxCKO S1 area, Vglut2 is localized in less defined pattern, as it is both found in L4 and L3b and over all the L5. In V1 it is expressed in a pattern more similar to that of the S1 control brains, accordingly to what was observed at P7 (Figure2-5.a). EmxCKO M2 Vglut2 stayining was instead comparable to that observed in control M2. Finally at P21, in control brains Vglut2 expression recapitulates the one observed at P16, while in EmxCKO brains S1 displays a pattern of Vglut2 expression more similar to the pattern of matrix thalamic nuclei innervation, labeling L2 and L5, as in M2 (Figure 2-15.b). In EmxCKO V1 area Vglut2 is instead localized in L4 (Figure 2-15.c). These data already suggest a dynamic modification of thalamic innervation pattern between P7 and P21 in EmxCKO brains, especially regarding the S1 area, which was more directly studied with axonal tracing techniques.

Figure 2 - 5 .

 25 Figure 2-5. Laminar distribution of thalamic axons in control and EmxCKO M2, S1 and V1 cortices at P7, P16 and P21.

Figure 2 - 6 .

 26 Figure 2-6. DiI injection in S1 cortex at P7 of control and EmxCKO brains.

Figure 2 - 7 .

 27 Figure 2-7. DiI injection in VPM at P7 of control and EmxCKO brains.

Figure 2 - 8 .

 28 Figure 2-8. DiI injection in S1 cortex at P16 of control and EmxCKO brains.

Figure 2 - 9 .

 29 Figure 2-9. DiI injection in VPM at P16 of control and EmxCKO brains.

Figure 2 -

 2 Figure 2-10. DiI injection in S1 cortex at P21 of control and EmxCKO brains.

Figure 2 -

 2 Figure 2-11. DiI injection in VPM at P7 of control and EmxCKO brains.

Figure 2 -

 2 Figure 2-12. Summary scheme of TC connections at P7, P16 and P21 in control and EmxCKO brains.

2. 5 .

 5 COUP-TFI mutant S1 cortices are initially activated by VPM stimulation but fail to respond to external stimuli at P21.To directly assess thalamo-cortical functionality, I next stimulated the VPM and recorded activity in the S1 cortex in slice preparations in collaboration with M. Mantegazza, Institute de Pharmacologie Moleculeire et Cellulaire, Valbonne, France. To this purpose, I prepared acute brain slices cut at a 55° angle, a plane suitable for maintaining the whole thalamo-cortical tract intact during recording[START_REF] Agmon | Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex[END_REF]. I performed field-recording on control and EmxCKO brain slices by applying single stimulations of 0.1ms at the level of the VPM and recording neocortical response at the level of the S1. To this aim, glass microelectrodes were inserted into L6 and L4 and a stimulating electrode was inserted in the VPM. I performed these experiments on the same set of samples used for the DiI injections, analyzing P8, P16 and P21 control and EmxCKO brains, to directly observe the functionality of the thalamo-cortical circuit between VPM and mS1 cortex in EmxCKO mice.By stimulating the VPM in P8 control cortical slices, L6 neurons in the S1 cortex normally responds with a direct negative peak of -0.17 (± 0.04) mV with a time delay from the stimulation of 5.99 (± 0.26) ms (Figure2-13.a and c). Even if the mutant mS1 cortex responded to VPM stimulations, the electrical profile recorded in L6 of EmxCKOs was remarkably different from that of controls (Figure 2-13.b). Nonetheless the amplitude of the first negative peak as well as the delay from the stimulation and the response was not significantly different between the two conditions in L6 (EmxCKO peak amplitude= -0.18 ± 0.04 mV. T test P value control/EmxCKO = 0.862. EmxCKO peak delay = 6.62 ± 0.20 ms. T test P value control/EmxCKO = 0.156) (Figure 2-13.c). The time delay from the stimulation depends from the amount of steps a stimulation has to go through before inducing the first neagative peak. It is therefore an indirect measure that can indicate how many neurons-neuron synapses separate the stimulated ones to the recurded. In both control andEmxCKO the delay observed in L6 neurons was in the range of 6ms it can correspond to a bi-or trisynaptic circuit, which fits the model of the SP relay of thalamic inputs in the cortex[START_REF] Luhmann | Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex[END_REF].At P16, control L6 showed a more consistent negative peak profile with a delay compatible with a model of mono-synaptic response, whereas EmxCKO brains displayed a significantly weaker negative peak with a delay still similar to that of a di-synaptic circuit (Figure2-13) (peak amplitude: control= -0.85 ± 0.21 mV. EmxCKO= -0.10 ± 0.01 mV. T test P value control/EmxCKO = 0.003. peak delay: control= 2.59 ± 0.33 s. EmxCKO= 4.16 ± 0.95 s. T test P value control/EmxCKO = 0.078).Control P21 brains recordings in L6 after VPM stimulation showed a less important negative peak, since at this stage VPM axons directly innervate L4 and L5a and the relay of subplate cells is no more present (Figure2-13.a)[START_REF] Harris | The neocortical circuit: themes and variations[END_REF][START_REF] Luhmann | Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex[END_REF][START_REF] Hoerder-Suabedissen | Development, evolution and pathology of neocortical subplate neurons[END_REF]. Some of the recordings also displayed a quick positive peak, which could be due to the antidromic stimulation of cortico-thalamic cells, meaning a negative peak which is induced by a direct current diffusion traveling from CT axons back the soma of the cells in L6 (Figure2-13.a). However, no real electrical responses were recorded in L6 of EmxCKO brains, apart from some artifacts (Figure 2-13.b and c).

Figure 2 -

 2 Figure 2-13. Field recording in S1 L6 upon VPM stimulation of control and EmxCKO thalamocortical slices.

  (a) Central panel displays field recording traces in L6 for control brains at P21 showing no direct negative peak after the stimulation artifact. (b) Central Panel displays EmxCKO P21 recordings in L6 where no negative peak was observed after the stimulation artifact. (c) Quantification of control and EmxCKO P8, P16, P21 peak delay and peak amplitude parameters. Ctx -cortex, S1 -primary somatosensory areas, Hipphippocampus, dLGN -dorso-lateral-geniculate nucleus, nRT -nucleus reticulatum, VPM, ventro-postero medial nucleus, VL -ventro-lateral nucleus, Po -Posterior nucleus, L -Layer. **: P value < 0.01. ***: P value < 0.005. Error bars display s.e.m. I also analyzed the response of L4 cells to VPM stimulation in control and EmxCKO brains. The profile of response in L4 at P8 was evidently different between control and EmxCKO brains (Figure 2-14.a and b). In control slices, we can observe a clear negative peak at a delay that corresponds to a bi-or tri-synaptic model, whereas in EmxCKO the cortical the peak amplitude of the first negative peak was not significantly similar to that of control brains the mean peak delay lower (Figure 2-14.b and c, Peak delay P8: control= 7.55 ± 0.35 ms. EmxCKO= 5.39 ± 0.88 ms. t-test control/EmxCKO Pvalue = 0.045. Amplitude peak P8: control= -0.22 ± 0.03 mV. EmxCKO= -0.16 ± 0.60 mV. t-test control/EmxCKO. Pvalue= 0.393). Interestingly, in EmxCKO brains the recording in L4 showed a response which involved an abnormal positive peak before or after the expected negative peak, which may indicate a different localization of the negative peak stimulation and therefore of the circuit (Figure 2-14.b). Also, the distribution of the peak delay in EmxCKO brains indicates that the samples analyzed can be separated in three subgroups: a sub group displayed a delay of responses similar to a monosynaptic circuit, a second subgroup displayed a pattern reminiscent of a di-synaptic circuit ,and finally a third subgroup displayed delays similar to a tri-synaptic circuit (Figure 2-14.). This indicates a possible range of variability in the organization of the thalamocortical circuit between VPM and parietal cortex in the absence of COUP-TFI function, which differs from controls, which has to ber further investigated. At P16, L4 recordings confirmed that control brains reinforced the circuit previously observed at P8, displaying bigger negative peak amplitude at a delay compatible with a mono-synaptic response (Figure 2-14.a and c). On the contrary, EmxCKO L4 had a significantly weaker negative peak with a delay compatible with a mono or di-synaptic circuit, suggesting that the connections between TC axons and cortical neurons failed to strengthen in the time window between P8 and P16 (Figure 2-14.b and c Peak delay P16: control= 3.29 ± 0.42 ms. EmxCKO= 3.51 ± 1.08 ms. t-test control/EmxCKO Pvalue = 0.835. Amplitude peak P16: control= -0.54 ± 0.12 mV. EmxCKO= -0.02 ± 0.01 mV. t-test control/ EmxCKO. Pvalue= 0.001). At P16 the relay of SP neurons between TC axons and L4 neuron from is likely to be terminated as stable synapses between TC and L4 neurons has alredy been established (Hoerder-Suabedissen & Molnár 2015). In accordance with DiI labeling, recordings in P21 control brains in L4 confirmed a strengthening of the thalamo-cortical circuit since they clearly showed a negative peak delay compatible with mono or di-synaptic circuits (Figure 2-14.a and c). EmxCKO brains showed instead no negative peak after the stimulation artifact, indicating a complete loss of the thalamo-cortical circuit between VPM and mS1 at P21 (Figure 2-14.b and c).

Figure 2 -

 2 Figure 2-14. Field recording in S1 L4 upon VPM stimulation of control and EmxCKO thalamocortical slices.

Figure 2 -

 2 Figure 2-15. Ion channels, regulators and glutamate receptors distribution in wt cortex.

  -16.c); therefore its downregulation at P0 could be upper layers specific (Figure 2-16.c).These data suggest that several components modulating electrical activity are affected in the absence of COUP-TFI. All these channels and receptors are important for triggering intracellular response to synaptic stimulation and inducing hyperpolarization of cell membranes after depolarization. Precise expression of these regulators could therefore be crucial in determining how neurons respond to stimulation. Moreover, potassium channels are a key component for regulating spontaneous activity in neurons and determining the correct membrane potential.

Figure 2 -

 2 Figure 2-16. rtq-PCR relative quantification of ion channels, regulators and glutamate receptors at P0 and P7 in control and EmxCKO cortices.

  (1 conserved DR site at around 40 kb from the 5'UTR) (Figure 2-17). To verify direct binding of COUP-TFI on these evolutionary conserved sites, I performed chromatin immune precipitation (ChIP) experiments on genomic extracts from whole cortices at P0 by using a specific COUP-TFI antibody allowing the enrichment of immunoprecipitated fractions in which COUP-TFI bound to its target sequences. Then, after reversing the crosslinking between COUP-TFI and its specific genomic targets, I queried the samples for the expected target sequences by rtq-PCR. These experiments confirmed me direct binding of COUP-TFI on the following loci: Hcn1, Cacna2d3 and Kcng3 (Figure 2-17).

Figure 2 -

 2 Figure 2-17. COUP-TFI directly binds regulatory sequences of a set of ion channels, regulators and glutamate receptors.

  -18.a). At P14, Arc expression increases in L6, L4, L3/2 in a medio-caudal high to rostro-lateral low gradient (Figure 2-18.b'''). In S1 Arc expression in L4 is specific in septa (black arrowhead) rather than in barrel cells (empty arrowhead) (Figure 2-18.b''').

Figure 2 -

 2 Figure 2-18. Areal and laminar distribution of the immediate early gene Arc in wt brains.

Figure 2 -Figure

 2 Figure 2-19. Laminar distribution of cFos in control and EmxCKO cortices.

Figure 2 -

 2 Figure 2-20. Laminar distribution of the immediate early gene Egr1 at P7, P16 and P21 in control and EmxCKO cortices.

  COUP-TFI in the neocortex, I queried the ECR browser for COUP-TFI binding sequences on the Egr1 genetic locus and found one evolutionarily conserved DR1 sequence located at around 6kb from the 5'UTR (Figure 2-21.b). To confirm its direct interaction, ChIP experiments on P0 control cortices with our COUP-TFI antibody were performed. The evolutionary conserved DR1 sequence failed to display any significant enrichment in the chromatin precipitated with COUP-TFI when compared to chromatin precipitated with a non-specific antibody (Figure 2-21.b). The sequence containing a Sp1 binding site, located in close proximity to the Egr1 5'UTR (Pipaón et al. 1999) was instead significantly enriched in chromatin fractions immuno-precipitated with the COUP-TFI antibody (Figure 2-21.d).These data indicate that the immediate early gene Egr1 is expressed in a regionalized pattern consistent with the initial activation of L4 cells by core-type thalamic projections, such the ones originating from the VPM. Its altered expression in COUP-TFI mutants (lack of Egr1 in mS1 and overepression in V1) is also consistent with affected thalamo-cortical connections in mS1 and misrouted TC axons in V1. Thus, COUP-TFI by controlling Egr1 expression might be directly involved in pre-pattern the barrel cortex to properly receive thalamic inputs during peri-and postnatal stages of development.

Figure 2 -

 2 Figure 2-21. Comparison of Egr1 and thalamo-cortical axons laminar distribution at P7, P16 and P21 in control and EmxCKO cortices.

Figure 2 -

 2 Figure 2-22. IUE of pCIG::Cre::GFP at E14 in wildtype cortices.

Figure 2 -

 2 Figure 2-23. IUE of pCIG::Cre::GFP at E14 in COUP-TFI fl/fl cortices.

  by COUP-TFI, may mediate the acquisition of stellate morphology in L4 neurons. To directly test this hypothesis, I aimed to rescue stellate cell morphology in COUP-TFI-deficient cells by co-electroporating a pCIG::Cre::IRES::GFP plasmid with a cDK5::Egr1::IRES::GFP plasmid in COUP-TFI fl/fl brains at E14 (Figure 2-24). In this way, electroporated cells become COUP-TFI negative and simultaneously expressed Egr1. By restoring Egr1 expression in COUP-TFI inactivated cells, the stellate cell morphology was remarkably rescued (Figure 2-24 and 2-25.b). Indeed, the percentage of electroporated cells with a stellate morphology in COUP-TFI fl/fl brains was similar to that of wt brains electroporated with the GFP expressing vectors pCIG::Cre::GFP ( Stellate cells/GFP cells in Cre::Egr1 COUP-TFI fl/fl brains = 43.21 ± 6.02%, in wt controls = 36.77 ± 3.57%. t-test Pvalue= 0.333). I also investigated whether Egr1 expression in fluences how electroporated cells distributes inside or outside the cluster of Vglut2 TC axons, and found that the percentage of stellate and pyramidal cells localized within the Vglut2 clusters is not significantly different (Figure 2-25.c. Stellate cells in Vglut2/GFP cells = 97.50 ± 2.50%, Pyramidal cells in Vglut2/GFP cells = 79.27 ± 8.06. t-test Pvalue= 0.128). Furthermore, I evaluated the rate of Egr1 expression in stellate and pyramidal cells in co-electroporated brains. Stellate cells were all expressing Egr1, while some of the GFP cells with pyramidal morphology failed to express Egr1, suggesting that co-expression of Cre end Egr1 was not 100% efficient (Figure 2-25.b. Egr1 expression in stellate cells/GFP cells = 100 ± 0.00%, in pyramidal cells/GFP cells= 81.79 ± 6.42%. t-test Pvalue= 0.018).These data support the hypothesis that Egr1 expression in L4 mediates the acquisition of the stellate morphology and that COUP-TFI influences this process by cell autonomously regulating Egr1 during the establishment of TC topographic connectivity.

Figure 2 -

 2 Figure 2-24. IUE of pCIG::Cre::GFP and cDk5::Egr1::GFP at E14 in COUP-TFI fl/fl cortices.

  Egr1 in my experiments. Even if the simultaneous absence of COUP-TFI and inactivation of Egr1 showed a rescue of L4 stellate morphology, it does not directly tell us whether Egr1 can induce the stellate morphology without VPM inputs. COUP-TFI fl/fl neurons electroporated with Cre fail to acquire the stellate morphology, but we still don't know whether these abnormal neurons also fail to connect with VPM axons specifically. The very same information is still lacking for COUP-TFI fl/fl L4 neurons electroporated with Egr1 and Cre expressing plasmids. Therefore it is still unclear from these experiments whether Egr1 function is necessary to maintain VPM axon synapses, and consequently lead to the acquisition of the stellate morphology, or whether VPM axons fail to synapse electroporated cells, in which case Egr1 could participate in inducing the stellate morphology independently from VPM inputs.

  conclusion, my work has demonstrated a high level of plasticity during the process of neocortical area organization within post-mitotic cortical cells. The function of COUP-TFI is necessary and sufficient in early post-mitotic neurons to control sensory area organization. This differs from what was previously reported, since it shows that progenitors are not the only source for impinging positional identity of their progenies, and indicates how the transcriptional code present in early differentiating neurons can modify cortical identity independently from the information originating from progenitor cells. In addition, my work has shown that COUP-TFI influences sensory area identity by properly differentiating L4 cells, thus allowing thalamic inputs to synapse to L4 neurons and form a barrel cortex in S1. I propose that one of its direct functions during this process is to regulate spontaneous activity of newborn neurons before the arrival of thalamic sensory inputs, as observed by Ca ++ imaging and MEA experiments. My data also show that COUP-TFI directly controls the transcription of glutamate receptors, ion channels and regulators of endogenous activity, which might be important for the establishment of stable synapses between the VPM and S1. This was demonstrated by analyzing the dynamics of the VPM and S1 connections between in control and EmxCKO mutant brains with multiple approaches, such as DiI labeling and field recording in thalamo-cortical slices. Finally, COUP-TFI might directly control Egr1, which normally responds to evoked activity and contributes in shaping L4 stellate morphology, an important cell population in L4 required during barrel organization. However, several experiments are needed in my opinion to finalize these data. It is first necessary to directly characterize the electrophysiological properties of neurons acutely inactivated of COUP-TFI by whole cell patch clamp, in order to confirm their altered electrical properties. Ideally, by reactivating COUP-TFI or better Egr1 in these electroporated cells, newly formed stellate neurons should acquire back their normal electrical properties. Second, it is important to directly prove that re-expression of Egr1 in COUP-TFI-deficient L4 cells and consequent acquisition of a stellate morphology is sufficient to stabilize synapses between VPM and S1 neurons and maintain TC connectivity after P21, a time in which EmxCKO mice loose thalamic innervation. Thus, one needs to perform thalamo-cortical tracing experiments from the VPM in COUP-TFI fl/fl mice electroporated at E14 with the Cre and Egr1 expressing plasmids, and with mice electroporated with Cre expressing plasmid only.

  Emx2 and COUP-TFI expression, Sp8 reduction leads also to COUP-TFI and Emx2 increase. Since these regulators are strong caudalizing factors, it is still not clear whether Sp8 induces rostral identity directly or indirectly, i.e. by repressing COUP-TFI and Emx2 expression through Fgf8. Overexpressing a Sp8/engrailed fusion protein in E11.5 cortices suggested a directly active role for Sp8 in defining neocortical boundaries, rather than promoting rostral identity

[START_REF] Borello | Sp8 and COUP-TF1 Reciprocally Regulate Patterning and Fgf Signaling in Cortical Progenitors[END_REF]

, since engrailed-mediated transcriptional repression induced a generalized reduction of neocortical areas and of neocortical territories.

. The organization of the neocortex. 6.a. The radial organization of the neocortex.

  . Using both complete mouse knock-outs and shRNA approaches, the Nieto

	Similar results were obtained by knocking down Kv1. Firing and proper contralateral innervation
	were rescued by re-expressing Kv1 or reactivating Cux1 postnatally (Rodríguez-Tornos et al. 2016).
	These findings constitute a milestone in our understanding of how activity-dependent processes
	regulate transcriptionally-controlled differentiation programs involved in circuit assembly within
	the neocortex.
	6The mature neocortex is a structure organized radially into six layer of glutamatergic neurons and
	tangentially into different functional domains that process sensory or motor information to
	elaborate and implement motor plans (Alfano & Studer 2013; O'Leary et al. 2013; Rash & Grove
	2006). This six-layered radial organization resides in the most rostral region of the CNS
	(Rubenstein et al. 1998; Kaas 2011), and is not shared by any other regions of the brain,
	representing a landmark of all mammalian species (Figure i-10). Indeed, this six layer organization
	characterize each functional area of the mammalian cerebral cortex (Supèr & Uylings 2001; Aboitiz
	et al. 2003; Puelles 2011). Each of these layers is populated by subtypes of neurons that share
	similar structures but distinct functions (Rash & Grove 2006; Harris & Mrsic-Flogel 2013; Harris &
	Shepherd 2015) in terms of connectivity, morphology, molecular code and electrophysiological
	functions, all of which determined by a specific genetic program implemented during
	corticogenesis.
	As mentioned above, cortical layers are populated by excitatory (glutamatergic) projection
	neurons (PN) and inhibitory (GABAergic) interneurons (IN) (Figure i-11). The first constitute 80% of
	all cortical neurons, they are born directly from cortical progenitors in the pallium and project
	group has demonstrated that Cux1 and 2 are required for dendrite branching and spine axons both locally and to distant targets within and outside the neocortex (Williams & Price 1995;
	organization of upper layer neurons (Cubelos et al. 2010). Cux1, in particular, plays an important
	role in CPN maturation, as it controls their topographic innervation to the contralateral S1 cortex,
	after midline crossing and axon elongation. Interestingly, Cux1 directly modulates expression of
	the voltage-dependent potassium channel Kv1, which provides callosal neurons with a distinct
	firing pattern. Loss of Cux1 function results in a decrease of Kv1 expression, aberrant firing
	responses, and selective loss of contralateral innervation.

  Slides stored at -80°C were let dry 1h at RT and moved into autoclaved shanks for all the reactions. Brains collected where processed to remove meninges and fixed in PFA 4% overnight at +4°C.

	9. In situ hybridization. 10. Whole Mount Hybridization. The revelation was performed in B3 buffer (Tris HCl 100mM, MgCl 2 50mM, NaCl 100mM, Tween
	Epitope 0.1% in H 2 O milli-q) in which the brains were incubated twice for 20' at RT prior incubation with Working Working Incubation Host Source
	concentration in cryosections NBIT-BCIP (SIGMA) tween 0.2%.The reaction was carried out until the desired moment and concentration in vibratome sections conditions animal
	BrdU monoclonal At first slices were permeabilzed in RIPA buffer (NaCl 150 mM, 0.1%SDS, EDTA 1mM, Tris pH 8 50 1:100 / O.N. +4°C mouse SIGMA After fixation and washing in PBS, brains were progressively dehydrated in methanol in stopepd washing 3 times with PBS-tween 0.1% for 10'.
	mM, NP-40 1% and Na deoxycholate 0.5% in H 2 0 mq) 2 times for 10' at RT and then post-fixed for intermediate steps repeated twice for 15' at +4°C:
	cMyc polyclonal 15' at RT in PFA 4%. Samples were washed 3 times in PBS for 10' at RT and incubated 15' at RT in 1:250 / O.N. +4°C rabbit Abcam Aspecific signal can develop during the reaction, which can decrease the signal/noise ratio. A
	cMyc monoclonal Epitope triathanolammine buffer (Triethanolamine 100 mM, Acetic Acid 0.2% and Anhydride acetic 0.25% 1:100 / O.N. +4°C mouse Santa Cruz Working Working Incubation Incubation Host Source PBS-Tween 0.1% 75% 50% 25% 0% destaining process was used when necessary which removes superficial precipitates. All the steps
	COUP-TFI polyclonal concentration in 1:1000 in H 2 0 mq) to acetylate nucleic acids in the sample. Samples were washed again 3 times in PBS for 1:1000 O.N. +4°C rabbit concentration in conditions in conditions in animal Methanol 25% 50% 75% 100% are performed at RT for 1h in gentle rocking and consisted in equilibrating brains in methanol 50% Thermo Fischer cryosections vibratome sections cryosection vibratome sections 10' at RT and then pre-hybridized for at least 1h at 70°C, in order to block aspecific binding of RNA in PBS-Tween 0.1%. Samples were then washed three times in methanol pure and equilibrated in
	COUP-TFI monoclonal molecules, providing a buffer similar to that will be used for incubating the RNA probe, but which 1:1000 1:1000 O.N. +4°C mouse Perseus Proteomics Brains were stored at -20°C in pure methanol and then progressively rehydrated to PBS-Tween B.A.B.B. (Benzyl alcohol 33%, Benzoil benzoate 67%) 50%/methanol 50% and then washed in
	Ctip2 polyclonal Rabbit FC 488 only contains aspecific RNA molecules which also do not incorporate digoxigenin (Formamide 50%, 1:300 1:500 O.N. +4°C rat Abcam 1:300 1:500 2h RT O.N. +4°C goat Life Technologies 0.1% (PBSt) before use.
	Cux1 polyclonal Rabbit FC 594	1:300	1:1000	1:500	1:1000	2h RT	O.N. +4°C	rabbit O.N. +4°C	A. Nepveu gift goat	Life
										Technologies
	Egr1 polyclonal		1:300		1:500		O.N. +4°C	rabbit	Santa Cruz
	Rabbit FC 555	1:300		1:500		2h RT		O.N. +4°C	goat	Life
	Fog2 polyclonal		1:100		/		O.N. +4°C	rabbit	Santa Cruz	Technologies
	Foxp2 polyclonal Rat FC 488	1:300	1:500	1:500	1:1000	2h RT	O.N. +4°C	rabbit O.N. +4°C	Abcam goat	Life
										Technologies
	GFP polyclonal Rat FC 594	1:300	/	1:500	1:1000	2h RT	O.N. +4°C	chicken O.N. +4°C	Abcam goat	Life
	Technologies RNA probe excess was washed incubating samples in Post-hybridization buffer (Formamide 50%,
	Ki67 monoclonal		1:25		/		O.N. +4°C	mouse	Dakocytomation
	Rat FC 633 SSC 5X, Tween-20 0.1% in H 2 0 mq) 2 times at +70°C for 1h. Slides were washed in B1 buffer( 1:300 1:500 2h RT O.N. +4°C goat Life
	Ror-β polyclonal Maleic acid 100mM, NaCl 150 mM, Tween-20 0.1% in H 2 0 mq) 3 times at RT for 10' and blocked in 1:100 1:200 O.N. +4°C rabbit Abcam Technologies
	Satb2 monoclonal Mouse FC 488 B2 ( B1, 10% Sheep Serum) for at last 1h at RT, to block aspecific antibody binding sites. Antibodies 1:20 / O.N. RT mouse Abcam 1:300 1:500 2h RT O.N. +4°C goat Life Technologies
	Serotonin polyclonal Mouse FC 594 1:300 for digoxigenin coupled with alkaline phosphatase enzymes where diluted 1/2000 in B2 and 1:500 1:1000 O.N. +4°C rabbit Calbiochem 1:500 2h RT O.N. +4°C goat Life
	Tbr1 polyclonal incubated over slides overnight at +4°C using 200 µl per slice. The excess of antibody was washed 1:1000 / O.N. +4°C rabbit Hevner's lab Technologies The excess of probes is washed with preheated solution X (Formamide 50%, SSC 2X, SDS 1% in H 2 O
	Tbr2 polyclonal Mouse FC 555 with B1 3 times for 10' at RT before preparing samples for the revelation reaction. Slides were 1:1000 / O.N. +4°C rat 1:300 1:500 2h RT O.N. +4°C goat Life mq) at 4 times 45' at +70°C. Brains were equilibrated in TST (NaCl 0.5M, Tris HCl pH7.5 0.01M, Technologies Chemicon incubated 30' at RT in B3 (Tris HCl pH 9.5 100 mM, MgCl 2 50 mM, NaCl 100 mM, Tween-20 0.1% in Tween 0.1% in H 2 O milli-q). To further eliminate probe excess brains were treated with RNAase A
	Vglut2 polyclonal Chicken FC 488 H 2 O mq). Once samples are equilibrated NBT-BCIP, Tween-20 0.1% was used to perform the 1:500 1:1000 O.N. +4°C guinea pig Millipore / 1:500 / O.N. +4°C goat Life Technologies 10µg/ml in TST twice for 30' at RT, then samples were washed in TST at 10' at RT and in Solution X
	revelation reaction in which the alkaline phosphatase linked to antibodies process the NBT-BCIP to twice for 30' at +65°C. Brains were equilibrated in B1 buffer (Maleic acid 100mM, NaCl 150mM in
	precipitate. The reaction was stopped at the desired point by washing 3 times in PBS, rinsed in H 2 O H 2 O milli-q, pH 7.5) and washed at RT twice 5', once 10' and the 1h. The blocking was done in B2
	mq. After briefly drying the slides they were mounted with Acquatex resin and stored at RT (B1 buffer 20% sheep serum) 1h at RT and then incubated overnight with 1/2000 antibody α-Dig-
	U-AP (Roche) at +4%C in B1 2% sheep serum. The excess of antibody was washed with B1 buffer at
	RT first 5', then 5 times for 1h and then for two days at +4°C.

SSC 5X, Denhardts 5X, Salmon Sperm 500µg/ml, Yeast RNA 250µg/ml in H 2 0 mq). RNA probes were diluted in the same hybridization buffer to a concentration of 1µg/ml and distributed on slides 200 µl each and homogeneously distributed covering the slide with a glass coverslip. The hybridization was carried out at +70°C overnight in a wet chamber (overnight solution: Formamide 50%, SSC 5%, Tween-20 0.1% in H 2 0 mq).

All steps were done in multiweel plate (Falcon) in mild agitation. At first brains were trated with H 2 O 2 6% in PBSt for 1h at RT, allows the destruction of most of the remaining phosphatase in the sample which may give aspecific signals. After washing samples 2 times PBSt they were treated with Proteinase K (20µg/ml) in PBSt for 55' at room temperature. After briefly rinsing with PBSt, brains were post-fixed with P.F.A. 4%, glutharaldheyde 0.2% in PBSt 20' in ice. Samples were washed twice in PBSt and washed in Hybridization buffer (Formamide 50%, SSC 5X, SDS 1%, tRNA 500µg/ml, acetylated BSA 200 µg/ml, heparin 50µg/ml, Tween 2%, Yeast RNA 50µg/ml, in H 2 O milli-q) for 10' at RT, then pre-hybridized 1h at +70°C in hybridization buffer. Hybridization buffer was used to dilute RNA probes up to a concentration of 200ng/ml and samples were incubated overnight at +70°C. B.A.B.B. for several days until the reaching the desired level of signal/noise ration. Brains were then re-equilibrated in PBSt and stored at +4°C in PFA 0.1%/PBSt. 11. DNA and RNA quantification.

  The RNA probes synthesis reaction was carried using 1µg of linearized DNA in a 20µl total volume in H 2 O mq solution containing 2µl of transcription buffer 10x (Roche), 2µl of Dig labeling Mix (Roche), 1µl of RNAase inhibitor (Roche) and 2µl of the proper enzyme.The reaction ran for 2h at +37°C, after which 2µl of DNAase (Roche) were added to digest the linearized DNA in the solution, this reaction ran for 30' at +37°C and was stopped adding 2µl of EDTA 25mM. Probes were either precipitated at -80°C for 30' or overnight at -20°C adding 100µl H 2 O mq, 10µl LiCl 4M, 300µl Ethanol 100%. The solution was centrifuged 15' at 20000g at +4°C, the pellet was washed in 100µl ethanol 70% repeating the same centrifugation. After removing ethanol and air drying the pellet, this was dissolved in 40 µl of H 2 O mq. The quality of the probes was controlled on gel electrophoresis before DNAse reaction and after the precipitation.

	Probes	Linearization Enzyme	RNA polymerase
	Bhlhb5 A.S.	HindIII	T3
	Cad6 A.S.	SmaI	T3
	Cad8 A.S.	SpeI	T7
	Cre-recombinase A.S.	EcoRI	T3
	Ctgf A.S.	NotI	Sp6
	Emx2 A.S.	EcoRI	Sp6
	Id2 A.S.	XbaI	T7
	Igfbp4 A.S.	SalI	T7
	Lmo4 A.S.	BamHI	Sp6
	Pax6 A.S.	BamHI	T3
	Ror-β A.S.	XhoI	T3
	Sp8 A.S.	NheI	T7

  To enrich antibodies bound DNA fragments 50 µl of Slurry were added to each sample and incubated for 3h in gentle agitation at +4°C. Then samples were washed 4 times in ice cold Washing buffer A (HEPES pH 7.4 20 mM, NaCl 150 mM, Triton 100 1%, SDS 0.1% PMSF 10mg/ml in H 2 O mq) and 4 times in ice cold Washing buffer B (Tris pH 8.0 20 mM, EDTA 1 mM, LiCl 250 mM, NP-40 0.5%, Na-Deoxycholate 0.5%, PMSF 10mg/ml in H 2 O mq). Washing were carried out in agitation for 5', after spinning at 800g for 2' at +4°C the surnatant was discarded to add new washing buffer. At the first washing of the N sample, the surnatant was kept and used as a stock for Input (P) samples. After the last wash with Buffer B another wash was done with TE buffer (Tris HCl pH 8.0 20mM, EDTA 1mM, PMSF 10 mg/ml in H 2 O mq), during the second washing all samples were transferred in a new tube. After having washed the resin antibodies were denatured in order to detach DNA fragments form the resin by adding 300 µl of Elution buffer (NaHCO 3 50mM, 1% SDS in H 2 O mq) and incubated in gentle rotation 1h at RT, an Input sample (P) was created mixing 300 µl of Elution buffer and 10 µl of the Input stock previously collected.

Multi Electrode Array neuronal culture recording.

	20.
		-1.8	B +1.5	-3.0	B -1.8	B +1.5	-3.5
	Caudo-medial	/	/	/	/	/	/
	Caudo-lateral	/	/	/	/	/	/

  Immunofluorescence on vibratome sections experiments were taken using ZEISS Imager.Z1 microscope. Images for Whole Mount I.S.H. where acquired using Leica ZM16 microscope. DiI, CTB and in utero electroporation experiments were acquired with a Zeiss 710 confocal microscope with 34-channel QUASAR detector and 2 GaAsP high sensitivity detectors. Ca ++ imaging experiments time lapse were acquires on a Spinning Disk

microscope (Olympus / Andor technology).

  . A subset of VPM neurons located in the most caudal parto of the VPM also

	innervates S1 reaching both L4 and L5 (red). (b) At P16 in wildtype brains VPM neurons innervate V1 L4
	clusters (blue). In EmxCKO brains VPM neurons are likely to innervate V1 L4 (rose), as they did at P7 and as
	they do at P21. A subset of VPM neurons also innervate S1 reaching both L4 (red). (c) At P21 in wildtype
	brains VPM neurons innervate V1 L4 clusters (blue). In EmxCKO brains VPM neurons still innervate V1 L4
	(rose), but they fail to innervate the S1 (red).

  Field recording on L6 of S1 during stimulation of VPM in thalamo-cortical slices on control and EmxCKO EmxCKO. Pvalue= 0.86). (a) Central panel displays field recording traces in L6 for control brains at P16 showing direct negative peak after the stimulation artifact at a delay compatible with the monosynaptic model. (b) Central panel displays EmxCKO P16 recordings in L6 where direct negative peaks are found later delays compared to control recordings and peak amplitudes are significantly lower (Peak delay P16: control= 2.58 ± 0.34 ms. EmxCKO= 4.16 ± 0.95 ms. t-test control/EmxCKO Pvalue = 0.078. Amplitude peak P16: control= -0.85 ± 0.21 mV. EmxCKO= -0.102 ± 0.02 mV. t-test control v EmxCKO. Pvalue= 0.004).

	brains at P8, P16, P21. (a) Upper panel displays field recording traces in L6 for control brains at P8 showing
	a direct negative peak after the artifact of stimulation (red shade) at a delay consistent with di-or
	monosynaptic stimulation, in accordance with subplate direct stimulation model. (b) Upper panel displays
	EmxCKO P8 recordings in L6 display similar amplitude of the negative peak to control, but shorter and with
	more variable delay (Peak delay P8: control= 5.99 ± 0.27 ms. EmxCKO= 6.62 ± 0.20 ms. t-test
	control/EmxCKO Pvalue = 0.156. Amplitude peak P8: control= -0.17 ± 0.04 mV. EmxCKO= -0.18 ± 0.04 mV. t-
	test control/

Glutamate receptors and ion channels are differentially expressed in the EmxCKO cortex.

  Field recording on L4 of S1 during stimulation of VPM in thalamo-cortical slices on control and EmxCKO Central panel displays field recording traces in L4 for control brains at P16 showing direct negative peak after the stimulation artifact at a delay compatible with the mono-or disynaptic model. (b) Central Panel displays EmxCKO P16 recordings in L4 where direct negative peaks are found at similar delays compared to control recordings, but peak amplitudes are significantly lower (Peak delay P16: control= 3.29 ± 0.42 ms. For my analysis, I selected candidates with a strong expression in the cortex, and particularly in layers 4, 5, 6 that are involved in the thalamo-cortical circuitry between P0 and P7. Their patterns of expression are available on Genepaint (http://www.genepaint.org/Frameset.html) and on the Allen brain atlas (http://www.brain-map.org/).

	2.6. The impairments observed in endogenous activity and thalamo-cortical circuitry in COUP-TFI-
	brains at P8, P16, P21. (a) Upper panel displays field recording traces in L4 for control brains at P8 showing deficient cortices prompted me to search for possible alterations in expression levels of regulators
	a direct negative peak after the artifact of stimulation (red shade) at a delay consistent with di-trisynaptic
	stimulation, in accordance with subplate-L4 relay model. (b) Upper panel displays EmxCKO P8 recordings in of electrical activity, such as ion channels, intracellular calcium-mediators, glutamate receptors
	L4 display similar amplitude of negative peak to control, but shorter and more variable delay (Peak delay and/or immediate early genes. To this purpose, I analyzed the expression of several types of ion
	P8: control= 7.55 ± 0.35 ms. EmxCKO= 5.39 ± 0.88 ms. t-test control/ EmxCKO Pvalue = 0.045. Amplitude channels, channel regulators, which interact with ion channels modifying their functionality and
	peak P8: control= -0.22 ± 0.03 mV. EmxCKO= -0.16 ± 0.60 mV. t-test control/EmxCKO. Pvalue= 0.39). (a) localization, and glutamate receptors during different stages of late cortical development (Figure
	2-15).
	EmxCKO= 3.51 ± 1.08 ms. t-test control v EmxCKO Pvalue = 0.835. Amplitude peak P16: control= -0.54 ±
	(b) Central Panel displays EmxCKO P21 recordings in L4 where no negative peak was observed after the
	stimulation artifact (Peak delay P21: control= 4.28 ± 2.78 ms. Amplitude peak P21: control= -0.23 ± 0.02
	mV. EmxCKO= -0.00 ± 0.00 mV). (c) Quantification of control and EmxCKO P8, P16, P21 peak delay and peak
	amplitude parameters. Ctx -cortex, S1 -primary somatosensory areas, Hipp -hippocampus, dLGN -dorso-
	lateral-geniculate nucleus, nRT -nucleus reticulatum, VPM, ventro-postero medial nucleus, VL -ventro-
	lateral nucleus, Po -Posterior nucleus, L -Layer. *: P value < 0.05. Error bars display s.e.m.

0.12 mV. EmxCKO= -0.02 ± 0.01 mV. t-test control/EmxCKO. Pvalue= 0.001). (a) Central panel displays field recording traces in L4 for control brains at P21 showing direct negative peaks after the stimulation artifact at a delay compatible with the mono-or disynaptic model, in accordance with the results observed at P16.

Within the voltage-dependent calcium channel family, I selected Cacna1a, Cacna1b, Cacna1h expressed in SP and L4 neurons at E18.5 and are upregulated in almost all layers at P4. Cacna1g is expressed scarcely at E18.5 and more abundantly in upper layers at P4, whereas Cacna2d1 is expressed mainly in upper layers, and Cacna2d3 in SP, L6 and L5 neurons at P4 and in upper layers

  EmxCKO cortex RNA extracts for cFos and Egr1. cFos expression in control and EmxCKO brains is comparable both at P0 and P7 (cFos P0: control= 1 ± 0.07 f.c., EmxCKO= 0.96 ± 0.00 f.c. t.test Pvalue= 0.717. P7: control= 1 ± 0.14 f.c., EmxCKO= 0.75 ± 0.09 f.c. t.test Pvalue= 0.112). Egr1 expression at P0 is significantly downregulated in EmxCKO at P0, not at P7. The decrease of Egr1 expression in L4 observed by immunofluorescence may indeed be compensated by Egr1 expression in other cortical layers

	Immunofluorescence with Egr1 antibody on control (left panel) and EmxCKO (central panel) P7, P16, P21
	brains. Right panel displays quantification of Egr1+/DAPI percentage in control (blue circle) and EmxCKO
	(red circle) (a) At P7 Egr1 expression in control brains (left panel) is strongly expressed in L4 of S1 (a'') and a
	in the subplate with a lower density. In M2 (a') Egr1 is expressed in rarely in upper layers in and in V1 (a''')
	is strongly expressed in L4,2/3. EmxCKO expression (central panel) is significantly downregulated in upper
	layer in all areas and specifically in L4 of S1. (b) At P16 Egr1 expression increases in all layers, more
	abundantly in L6 and L4 of control brains in S1 (b''), V1 (b''') and in L3/2 in M2 (b'). In EmxCKO brains Egr1
	expression is upregulated as well in L6, but is consistently and significantly downregulated in L4 of S1. In V1,
	Egr1 expression in L4 is comparable to control brains. (c) At P21 the pattern of expression in control and
	EmxCKO brains in M2 (c'), S1 (c'') and V1 (c''') recapitulates the one observed at P16. (d) rtq-PCR at P0 and
	P7 on control and (Egr1 P0: control= 1 ± 0.08 f.c., EmxCKO= 0.59 ± 0.02 f.c. t.test Pvalue= 0.027. P7: control= 1 ± 0.12 f.c.,
	EmxCKO= 0.88 ± 0.04 f.c. t.test Pvalue= 0.075). Abbreviations: M2 -secondary motor area, S1 -primary
	somatosensory area, V1 -primary visual area, L -Layer. Scale bars: (a, b, c) 400 µm. *: P value < 0.05. Error
	bars display s.e.m. All panels of this figure are representative images of at least three independent
	experiments for each genotype.

Results

Magrinelli Elia -Ph.D. Thesis

Insulin-like growth factor-binding protein 4  IL -Intralaminar  IN -Inhibitory Interneuron  IPC -Intermediate Progenitor cell  IRES -Internal ribosome entry site  ish -in situ hybridization  IT -Intrathelencephalic Neuron  IT;CPN -IT Commissural Projection Neuron  IUE -In utero Electroporation  IZ -Intermediate Zone  KCN -Potassium Channel  KO -Knock Out  L -Layer  L4py -Layer 4 Pyramidal nneuron  L4s -Layer 4 spiny-stellate neuron  L4sp -Layer 4 star-pyramidal neuron  LDVL -laterodorsal ventrolateral thalamus  LGE -Lateral Ganglionic Eminence  LGN -Lateral Geniculate Nuclei  Lhx2 -Lim homeodomain 2  Lmo4 -LIM Domain Only 4  LP -Lateral Pallium  LPM -latero posterior, mediorostral nucleus  LTP -Long Term Potentiation  M1-Primary Motor Area  M2 -Secondary Motor Area  MAPK -Mitogen-Activated Protein Kinase  MEA -Multi Electrode Array  MGE -Medial Ganglionic Eminence  MGv -Medio-Ventral Geniculate nuclei  Moxd1 -Monooxygenase, DBH-Like 1  mS1 -presumptive somatosensory areas in EmxCKO and NexCKO  MZ -Marginal Zone  NE -Neuro Epithelium  Ngn2 -Neurogenin 2  nRT -Nucleus reticulatum  Nurr1 -Nuclear Receptor Related 1  OSVZ -Outer Radial Glia Cell  Pax6 -Paired box gene 6  Pf -Parafascicular nuecleus  PFC -Prefrontal Cortical Domains  PN -Excitatotyr Projection Neuron  PoM -Postero-Medial Nucleus  PP -Preplate  Prdm8 -PR Domain containing Protein 8  PrV -Trigeminal Nucleus Principalis  PV -Parvalbumin  RA -Retinoic Acid  ReG -Retina Ganglionic Cell  RGC -Radial Glia Cell  ROI -Region of Interest  Ror-β -RAR-related orphan receptor beta  rtq-PCR -real time Polymerase Chain Reaction  S.D. -Standard Deviation  s.e.m. -Standard error of the mean

Abbreviations

Discussion

Conclusions and perspectives