Le cortex cérébral des mammifères est divisé en plusieurs domaines tangentiels appelés des aires fonctionnelles, qui sont députées à l'élaboration des afférences motrice et sensorielles, la sélection et la mise en oeuvre des plans moteurs, et d'autres fonctions cognitives supérieures. Chaque aire fonctionnelle est constituée par six couches de neurones de projections (PN) glutaminergiques avec différentes morphologies, connectivité et codes moléculaires.

Plusieurs facteurs de transcription spécifiant différentes sous-classes de neurones, comme les neurones calleux (CPN, qui ciblent l'hémisphère controlatéral du néocortex) et les neurones de projection sous cérébraux (SCPN, par exemple les neurones corticopontines et corticospinaux) ont été identifiés à ce jour. Fezf2 et Ctip2 induisent la spécification des neurones sous-cérébraux, alors que Satb2 induit l'identité calleuse, essentiellement en réprimant la transcription de Ctip2 par le recrutement du complexe NURD au locus de Ctip2. Cependant, très peu est connu sur les mécanismes précisant leurs caractéristiques de manière temporelle et régionale. Dans cette étude, je montre que la population de cellules co-exprimant des marqueurs moléculaires des neurones calleux et sous-cérébraux, comme Satb2 et Ctip2, sont d'abord spécifié dans les couches V et VI du néocortex rostro-médiale chez la souris au stades périnataux, et augmente progressivement entre P0 et P21 dans l'aire somatosensorielle. Cependant, les mécanismes qui permettent la co-expression de ces deux facteurs, ainsi que la fonction et la connectivité des cellules co-exprimant satb2/Ctip2 sont encore inconnus. J'ai trouvé qu'en absence de COUP-TFI, un facteur de transcription modulant la spécification des aires fonctionnelles et des sous-types des cellules néocorticales, le nombre de cellules co-exprimant Satb2 et Ctip2 augmente fortement dans la couche V de l'aire somatosensorielle primaire éventuelle. J'ai démontré que l'expression ectopique et prématurée de LMO4 dans la lignée transgénique mutante pour COUP-TFI, de-réprime Ctip2 dans les cellules Satb2 positives en perturbant l'assemblage du complexe NURD au locus de Ctip2. En plus, par l'utilisation d'une lignée transgénique exprimant la GFP dans les neurones de la couche V et de colorants vitaux, j'ai analysé la morphologie, la connectivité et l'activité électrophysiologique de cette classe hybride de neurones à des stades de développement postnataux. Ensemble, mes résultats ont montré un nouveau mécanisme moléculaire spécifiant une nouvelle sous-population neuronale dans la couche V du cerveau.

En conclusion, mon étude a montré que la co-expression de marqueurs neuronaux calleux et sous-cérébraux ne caractérise pas seulement les premières phases de spécification neuronale, mais définit également une sous-population neuronale avec des caractéristiques régionales particulières et un calendrier de développement spécifique dans le néocortex cérébral.

I dedicate this thesis to my family and my fiancé, Ali

for their support and endless love.

I love you all.

I am gratefully thankful to my fiancé Ali that was hand by hand with me during all my studies, and was always there for me in all hard and good situations, I am thankful to all your love, care and support. Without you, these 4 years would not have been possible.

I especially thank my mom and dad. My hard-working parents have sacrificed their lives for my sisters, brother and myself and provided unconditional love and care. I love them so much, and I would not have made it this far without their love, support and encouragement.

Abstract

The mammalian cerebral cortex is subdivided into several tangential domains called functional areas, which are deputed to the elaboration of motor and sensory inputs, the selection and implementation of motor plans, and many other higher cognitive functions. Each functional area is constituted by six layers of projection glutamatergic neurons (PNs) with different morphologies, connectivity and molecular codes. Several transcription factors specifying different subclasses of neurons, such as callosal neurons (CPN, which target the contralateral neocortical hemisphere) and subcerebral projection neurons (SCPN, e.g. corticopontine and corticospinal neurons) have been identified so far. Fezf2 and Ctip2 promote the specification of subcerebral PNs, whereas Satb2 promotes callosal identity, mainly by repressing Ctip2 transcription through the recruitment of the NURD complex to the Ctip2 locus. However, little is known about the mechanisms specifying their features in a timeand areal-specific manner.

In this study I show that a population of cells co-expressing molecular markers of CPN and SCPN neurons, such as Satb2 and Ctip2, becomes first specified in layers V and VI of rostro-medial mouse neocortex at perinatal stages and progressively increases between P0 and P21 in somatosensory areas. However, the mechanisms allowing co-expression of these two factors, as well as function and connectivity of Satb2/Ctip2 co-expressing cells are still unknown. I found that in neocortices lacking COUP-TFI, a transcription factor modulating neocortical areal and cell-type specification, the number of Satb2/Ctip2 co-expressing cells increases abnormally in layer V of the prospective primary somatosensory area. I demonstrated that LMO4 ectopic and premature expression of LMO4 in COUP-TFI mutant transgenic line derepresses Ctip2 in Satb2 positive cells by disturbing the assembly of the NURD complex at Ctip2 locus. Moreover, by the use of a transgenic line expressing GFP in layer V neurons and of vital dyes, I analysed morphology, connectivity and electrophysiological activity of this hybrid class of neurons at postnatal stages of development. Together, my results have unravelled a novel molecular process specifying a new PN sub-population within layer V.

In conclusion, my study demonstrates that the co-expression of CPN and SCPN neuronal markers does not only characterize early phases of neuronal specification, but also defines neuronal subpopulation with different areal-specific features and developmental timing in the mammalian neocortex. postmitotic projection neurons into one of three broad subtype identities. Figure 13-Satb2 protein interacts with both the Ctip2 promoter upstream region and histone deacetylase complex and controls chromatin remodeling. ........ Figure 14

Table of Contents

Introduction

List of figures

I. The mammalian neocortex: Origin and function

Early in embryonic development, the neural tube forms three major brain vesicles: the forebrain positioned at the anterior end, the midbrain and the hindbrain (Figure 1) [START_REF] Rubenstein | Regionalization of the prosencephalic neural plate[END_REF].

The dorsolateral forebrain later evaginates to form the telencephalon and the diencephalon (thalamus/hypothalamus). Subsequently, the telencephalon is subdivided into ventral telencephalon, which gives rise to the striatum and the basal ganglia, and dorsal telencephalon, from which arises the cerebral cortex [START_REF] Rubenstein | Regionalization of the prosencephalic neural plate[END_REF].

The cerebral cortex is the largest and the most complex structure of the mammalian brain, and more than any other brain structure, it has undergone the most dramatic changes during vertebrate evolution [START_REF] Aboitiz | Genetic and developmental homology in amniote brains. Toward conciliating radical views of brain evolution[END_REF][START_REF] Aboitiz | The evolutionary origin of the mammalian isocortex: towards an integrated developmental and functional approach[END_REF][START_REF] Super | The early differentiation of the neocortex: a hypothesis on neocortical evolution[END_REF]. The cerebral cortex is subdivided into the neocortex, which is the largest region and unique to the mammalian forebrain, the archicortex (hippocampus) and the paleocortex (olfactory piriform cortex) [START_REF] Aboitiz | Genetic and developmental homology in amniote brains. Toward conciliating radical views of brain evolution[END_REF][START_REF] Aboitiz | The evolutionary origin of the mammalian isocortex: towards an integrated developmental and functional approach[END_REF][START_REF] Super | 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].

The neocortex has acquired an increasing relevance during evolution, and is deputed to the elaboration of sensory and motor inputs. This structure represents the prevalent centre for the elaboration of peripheral inputs, and the seat of higher functions, such as memory, language and voluntary movements, and in humans, where it constitutes more than 80% of the total brain volume, it is responsible for thoughts and consciousness [2-4, 6, 7].

II. The neocortex: General organization A. Tangential organization

In its tangential dimension, the neocortex is organized into "areas", defined by Brodmann as the "organs of the brain" [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF], which represent a partition of distinct neocortical functions among several tangential regions. In adult, the borders of these areas are sharply defined by differences in cytoarchitecture and chemoarchitecture, input and output connections, and pattern of gene expression [START_REF] O'leary | Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex[END_REF][START_REF] Rash | Area and layer patterning in the developing cerebral cortex[END_REF][START_REF] Sur | Patterning and plasticity of the cerebral cortex[END_REF].

The basic plan of the mammalian neocortex comprises four primary areas; three of these primary areas are sensory: the primary somatosensory (S1), visual (V1) G). BG, basal ganglia; CiC, cingulate cortex; CoC, corpus callosum; LV, lateral ventricle (modified from [START_REF] Geng | Pathogenesis of holoprosencephaly[END_REF]) .

and auditory (A1), which process sensory information arriving respectively from the whole body, the eye (retina) and the inner ear (cochlea). The fourth primary area is motor (M1), which mainly controls voluntary movements [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF].

Areal patterning will be presented in details in Chapter IV.

B. Radial organization

The laminar organization of the neocortex is an important feature that differentiates it from other cortical structures. Each area is radially organized into six major "layers", each containing a heterogeneous population of neurons, distinguished by their morphology, connectivity and function from those of other layers [START_REF] O'leary | Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex[END_REF][START_REF] Rash | Area and layer patterning in the developing cerebral cortex[END_REF][START_REF] Sur | Patterning and plasticity of the cerebral cortex[END_REF]. Each layer is generated according to an inside-out sequence. Early born neurons reside in deeper layers, while late born neurons reside in superficial layers [START_REF] Rakic | Neuronal migration and contact guidance in the primate telencephalon[END_REF]. Lower layer neurons (VI and V) project their axons to subcortical targets (striatum, thalamus, tectum, pons and spinal cord), while upper layer neurons (IV-II) establish cortico-cortical connections, or receive sensory and motor inputs from the thalamus [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]. Cellular organization of the neocortex, different types of progenitors, radial migration and different types of projection neurons will be discussed in chapters III and V.

C. The functional relationship between cortical areas and the thalamus

One of the principal functions of the thalamus, originating from the diencephalon, is to receive sensory and motor afferents from peripheral systems, and to relay them to the main elaboration centre: the neocortex [START_REF] Aboitiz | The evolutionary origin of the mammalian isocortex: towards an integrated developmental and functional approach[END_REF]. The dorsal thalamus has four principal thalamic nuclei that are functionally and topographically connected to the four primary cortical areas. This relationship between a primary cortical area and a nucleus in the dorsal thalamus is critical for both adult function and developmental differentiation of the areas [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF].

The S1 receive thalamocortical afferents (TCA) from the ventral postero-medial nucleus (VPM), the V1 from the dorsolateral geniculate nucleus (dLGN) and the A1 from the ventral medial geniculate nucleus (MGv). TCAs will reach the fourth layer of the targeted sensory areas [START_REF] Molnar | Mechanisms controlling the guidance of thalamocortical axons through the embryonic forebrain[END_REF][START_REF] Clasca | Unveiling the diversity of thalamocortical neuron subtypes[END_REF][START_REF] Grant | Development of the corticothalamic projections[END_REF], while the motor area, which is connected to the ventrolateral nucleus (VL), receives TCAs in lower layer III [START_REF] Strick | Synaptic termination of afferents from the ventrolateral nucleus of the thalamus in the cat motor cortex. A light and electron microscopy study[END_REF][START_REF] Jones | Lamination and differential distribution of thalamic afferents within the sensory-motor cortex of the squirrel monkey[END_REF][START_REF] Donoghue | The motor cortex of the rat: cytoarchitecture and microstimulation mapping[END_REF]. In the somatosensory area, TCA innervation of layer IV granular cells gives rise to the cortical barrels, which are constituted by columns of thalamocortical afferents surrounded by layer IV stellate (granule) cells. In turn targeted areas send corticothalamic afferents (CTAs) from layers V and VI to the respective thalamic nuclei [START_REF] Clasca | Unveiling the diversity of thalamocortical neuron subtypes[END_REF][START_REF] Grant | Development of the corticothalamic projections[END_REF] (Figure 2).

III. Neocortical neurogenesis and radial migration A. Cellular organization of the neocortex

A prominent feature of the neocortex is its complex but well-organized cellular architecture. It contains an immense number of neurons, produced through extensive progenitor cell divisions during embryogenesis. These neurons are not randomly dispersed, but spatially organized into horizontal layers, essential for neocortical function. The formation of this laminar structure requires exquisite control of neuronal migration from the birthplace to the final destination in the cortex.

The main constituents of the neocortex are excitatory, inhibitory neurons and glial cells. The glutamatergic excitatory neurons represent the vast majority (70-80%) of neocortical circuit neurons and are responsible for generating the cortical output, while the γ-aminobutyric (GABA)-ergic inhibitory interneurons account for about 20% of cortical neurons, and provide a rich variety of inhibitions that shape the output of functional circuits [START_REF] Tan | Neocortical neurogenesis and neuronal migration[END_REF]. Excitatory and inhibitory neurons convey and modulate motor and sensory inputs, favoring their integration and elaboration and, ultimately the implementation of motor plans. Thus, proper neocortical function critically depends on the production and positioning of a correct number of excitatory and inhibitory neurons, which largely occurs during embryonic stages.

In contrast, glial cells mainly constituted by oligodendrocytes, astrocytes and microglia are involved in a variety of functions including cell feeding, axon myelination, structural support and radial migration of neocortical projection neurons [START_REF] Tan | Neocortical neurogenesis and neuronal migration[END_REF][START_REF] Halassa | Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior[END_REF][START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF]. Moreover, oligodendrocyte precursor cells in the mammalian brain form synapses with neurons, suggesting an even greater degree of complexity in the interactions between neurons and oligodendroglia [START_REF] Lin | Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus[END_REF]. Glutamatergic and glial cells are born from common precursors (through neurogenic and gliogenic processes), a-Visual input from the retina (blue line) is relayed through the dorsal lateral geniculate nucleus (dLGN) to reach the visual cortex (V1, blue area in b). Somatosensory information from the whiskers (red line) reaches the ventrobasal complex (VB) through the brainstem, and is relayed to the barrel field of the primary somatosensory cortex (S1, red area in b). Acoustic information (purple line) arrives at the medial geniculate nucleus through numerous relays, and is then relayed to the primary auditory cortex (A1, purple area in b). b-The sensory and motor cortical areas of the mouse brain. Orange dashed line indicates plane of section in part c. c-Forebrain section, showing S1 and VB and the pathways that link them. The red line indicates the thalamocortical fibre running from VB to layer IV of S1 (red shading). The blue line indicates thalamocortical projections to the anterior segment of V1 (blue shading). Neurons in layer VI of the same area project to VB. Layer V extends projections to the cerebral peduncle (CP). White brackets in layer IV represent septa of barrels. CPU, caudate putamen; GP, globus pallidus; IC, internal capsule; M1, primary motor area; M2, secondary motor area; S2, secondary somatosensory cortex; TRN, thalamic reticular nucleus; vLGN, ventral lateral geniculate nucleus; WM, white matter (taken from [START_REF] Lopez-Bendito | Thalamocortical development: how are we going to get there?[END_REF]).

while excitatory and inhibitory neurons arise from different developmental lineages, and their progenitors are fully segregated in space [START_REF] Tan | Neocortical neurogenesis and neuronal migration[END_REF][START_REF] Halassa | Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior[END_REF][START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF]. Excitatory neurons are generated in the proliferative zone of the dorsal telencephalon and then migrate radially to constitute the future neocortex. Instead, inhibitory interneurons are produced in the proliferative zone of the ventral telencephalon and migrate tangentially to reach the neocortex [START_REF] Tan | Neocortical neurogenesis and neuronal migration[END_REF].

In this section, I will present only glutamatergic cell specification and migration.

B. Progenitors diversity and corticogenesis

Early in development, the telencephalic wall is composed of neuroepithelial cells (Figures 3 and4). Most neuroepithelial progenitor cells undergo symmetric division; two neuroepithelial cells are produced at each division, expanding the population of founder cells. These neuroepithelial cells are multipotent, capable of generating radial glial cells (RGCs) and the first group of neurons in the neocortex [START_REF] Tan | Neocortical neurogenesis and neuronal migration[END_REF][START_REF] Haubensak | Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis[END_REF][START_REF] Williams | Evidence for multiple precursor cell types in the embryonic rat cerebral cortex[END_REF]. Around embryonic day E9.5-E10.5 in the mouse, some neuroepithelial cells begin to differentiate into RGCs establishing the ventricular zone (VZ) [START_REF] Haubensak | Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis[END_REF]. RGCs are characterized by their radial bipolar morphology. They expand across the entire thickness of the developing neocortex with a long basal radial process pointing to the pial surface, and a short apical ventricular endfoot reaching the VZ surface, with their soma located in the VZ [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]. RGCs divide symmetrically to self renew, and at later stages they undergo, alternatively, symmetric and asymmetric divisions to either self renew or generate neurons, respectively [START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Miyata | Asymmetric inheritance of radial glial fibers by cortical neurons[END_REF]. RGC symmetric divisions give rise also to other two classes of progenitors: the intermediate progenitor cells (IPC) and the outer radial glia (or outer subventricular zone OSVZ progenitor cell), which reside between the upper VZ and the subventricular zone (SVZ) [START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Miyata | Asymmetric inheritance of radial glial fibers by cortical neurons[END_REF][START_REF] Wang | A new subtype of progenitor cell in the mouse embryonic 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][START_REF] Fietz | OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling[END_REF].

Intermediate progenitors have a multipolar morphology, and are not anchored to either the apical or basal surface. They act primarily as transit amplifying cells, undergoing limited proliferative divisions, and more often divide symmetrically to produce two neurons [START_REF] Haubensak | Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis[END_REF][START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF][START_REF] Wu | Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone[END_REF][START_REF] Kowalczyk | Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex[END_REF]. The generation of neocortical neurons through IPCs increases the number of neurons produced by individual RGCs. Hence, it has been This schematic depicts the sequential generation of neocortical projection neuron subtypes and their migration to appropriate layers over the course of mouse embryonic development. a | Radial glia (RG) in the ventricular zone (VZ) begin to produce projection neurons around embryonic day 11.5 (E11.5).

At the same time, RG generate intermediate progenitors (IPs) and outer RG (oRG), which establish the subventricular zone (SVZ) and act as transit-amplifying cells to increase neuronal production. After neurogenesis is complete, neural progenitors transition to a gliogenic mode, generating astrocytes and oligodendrocytes (not shown). Cajal-Retzius (CR) cells primarily migrate into neocortical layer I from non-cortical locations, whereas other projection neurons are born in the neocortical VZ and/or SVZ and migrate along radial glial processes to reach their final laminar destinations. b | Distinct projection neuron subtypes are born in sequential waves over the course of neurogenesis. The peak birth of subplate neurons (SPN) occurs around E11.5, with the peak birth of corticothalamic projection neurons (CThPN) and subcerebral projection neurons (SCPN) occurring at E12.5 and E13.5, respectively. Layer IV granular neurons (GN) are born around E14.5. Some callosal projection neurons (CPN) are born starting at E12.5, and those CPN born concurrently with CThPN and SCPN also migrate to deep layers. Most CPN are born between E14.5 and E16.5, and these late-born CPN migrate to superficial cortical layers. Peak sizes are proportional to the approximate number of neurons of each subtype born on each day. NE, neuroepithelial cell (taken from [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]).

postulated that the abundance of IPCs may contribute to the evolutionary expansion of the neocortex [START_REF] Martinez-Cerdeno | The role of intermediate progenitor cells in the evolutionary expansion of the cerebral cortex[END_REF].

Outer radial glia have unipolar morphology, compared to RGCs, they retain only the basal process and lack the apical process [START_REF] Wang | A new subtype of progenitor cell in the mouse embryonic neocortex[END_REF][START_REF] Hansen | Neurogenic radial glia in the outer subventricular zone of human neocortex[END_REF]. They undergo asymmetrical divisions to self renew and generate neurons [START_REF] Fietz | OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling[END_REF][START_REF] Kelava | Abundant occurrence of basal radial glia in the subventricular zone of embryonic neocortex of a lissencephalic primate, the common marmoset Callithrix jacchus[END_REF]. A very small population of outer radial glia was observed in the developing mouse neocortex [START_REF] Wang | A new subtype of progenitor cell in the mouse embryonic neocortex[END_REF][START_REF] Shitamukai | Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors[END_REF]. A fourth class of progenitors, that either possesses a short or lack the basal process reside in the VZ, they are termed as short neural precursors (SNPs). Most SNPs produce postmitotic neurons directly within the VZ [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] (Figure 4).

Neocortical progenitors begin to produce glutamatergic projection neurons around embryonic day (E) 10. establishing the six layered structure of the mature cortex in an "inside out" manner [START_REF] Marin-Padilla | Dual origin of the mammalian neocortex and evolution of the cortical plate[END_REF][START_REF] Raedler | Autoradiographic study of early neurogenesis in rat neocortex[END_REF][START_REF] Luskin | Studies of the earliest generated cells of the cat's visual cortex: cogeneration of subplate and marginal zones[END_REF][START_REF] Rakic | Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition[END_REF] (Figure 3).

C. Neocortical "glia guided" radial migration

One of the major processes during cortical development is radial cell migration. After exiting the cell cycle, newborn projection neurons leave the VZ and migrate radially to give rise to the cortical plate, while starting their specification. During early stages of mammalian corticogenesis (E11-E13.5), newborn neurons leave the VZ by somal translocation. At this stage neurons inherit from their progenitors a process in contact with the pial surface, and use it as a puller, which progressively translocates the cell body towards the primordial cortical plate [START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF][START_REF] Miyata | Asymmetric production of surface-dividing and nonsurface-dividing cortical progenitor cells[END_REF][START_REF] Nadarajah | Ventricle-directed migration in the developing cerebral cortex[END_REF][START_REF] Nadarajah | Two modes of radial migration in early development of the cerebral cortex[END_REF]. Radial migration becomes more complex at late stages of corticogenesis (from E13.5), when neurons migrate along the glia scaffold generated by RGCs; this type of migration is called "glia guided". Once moved from the VZ to the SVZ, the neurons adopt a multipolar shape, elongating and retracting thin neurites, and detach from the glia continuing their migration both radially and tangentially with respect to the ventricular surface. Then, once they have reached the upper limit of the intermediate zone (IZ), they re-attach to the glia scaffold, adopt a bipolar shape, and enter the cortical plate [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]. Finally, they contact the pial surface with their leading process, leave the glia scaffold, and move towards the pia by somal translocation. This last step of migration is critical for Outer subventricular zone (OSVZ) progenitors likely originate from RGCs through oblique division, which leads to loss of the apical process and ascension of the nucleus toward the cortical plate/intermediate zone (CP/IZ). The minor population of OSVZ progenitors can also generate neurons through asymmetric division outside of the VZ (taken from [START_REF] Tan | Neocortical neurogenesis and neuronal migration[END_REF]).

the inside-out order of layer formation, since it allows the incoming neurons to settle beyond their predecessors [START_REF] Nadarajah | Ventricle-directed migration in the developing cerebral cortex[END_REF][START_REF] Rakic | Mode of cell migration to the superficial layers of fetal monkey neocortex[END_REF] (Figure 5).

D. Progenitor lineage commitment

Neocortical progenitors generate the different subtypes of PN in sequential waves, however the lineages leading to specific neuronal subtypes remain largely unknown.

Two models have been proposed: the first model states that only one single lineage of progenitors exists and generates all subtypes of PNs, and the competence of a given progenitor to generate specific subtypes becomes progressively limited over the course of development [START_REF] Mcconnell | Fates of visual cortical neurons in the ferret after isochronic and heterochronic transplantation[END_REF][START_REF] Mcconnell | Cell cycle dependence of laminar determination in developing neocortex[END_REF][START_REF] Frantz | Restriction of late cerebral cortical progenitors to an upper-layer fate[END_REF][START_REF] Luskin | Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus[END_REF][START_REF] Walsh | Clonally related cortical cells show several migration patterns[END_REF][START_REF] Price | Cell lineage in the rat cerebral cortex: a study using retroviral-mediated gene transfer[END_REF][START_REF] Reid | Systematic widespread clonal organization in cerebral cortex[END_REF]. The alternative model proposes that different independent fate restricted lineages of progenitors generate specific neuronal subtypes. In support to this latter model, a number of subtype specific transcription factors was shown to be expressed in progenitors, suggesting that distinct subsets of progenitors might be committed to generate specific classes of PNs. For instance, Fez family zinc finger 2 (Fezf2, also known as Fezl) is sparsely expressed in the proliferative zone during deep layer neurogenesis, and its postmitotic expression is specific to corticofugal PN [START_REF] Inoue | Fez1 is layer-specifically expressed in the adult mouse neocortex[END_REF][START_REF] Hirata | Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons[END_REF][START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF][START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF][START_REF] Chen | Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex[END_REF]. See more details in chapter V.

Conversely, Cut-like homeobox 1 (Cux1) and Cux2 are expressed in the VZ and SVZ during upper layer neurogenesis, and its post-mitotic expression is specific to CPN, and other superficial layer 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] Zimmer | Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons[END_REF][START_REF] Molyneaux | Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons[END_REF]. In favor of the second model, recent work has demonstrated that a subset of progenitors, positive for Cux2 and present from E10.5 corresponding to the earliest stages of corticogenesis, exclusively produces CPN and other superficial-layer neuron subtypes, and that early born neurons derived from the Cux2 lineage become deep layer CPN [START_REF] Franco | Fate-restricted neural progenitors in the mammalian cerebral cortex[END_REF].

However, another recent paper [START_REF] Guo | Fezf2 expression identifies a multipotent progenitor for neocortical projection neurons, astrocytes, and oligodendrocytes[END_REF] demonstrated that Fezf2-expressing radial glial cells (RGCs) exist throughout cortical development and sequentially generate all major projection neuron subtypes and glia. Moreover, this study showed that the vast majority of Cux2-positive cells in the VZ and SVZ are migrating interneurons derived from the subcortical telencephalon, and Cux2-positive RGCs generate both deep-and upper-layer projection neurons. Overall, this work identified Fezf2-positive radial glial cells as multipotent neocortical progenitors and suggests that determining whether laminar-fate-restricted RGCs exist since early stages of neurogenesis requires further investigation [START_REF] Guo | Fezf2 expression identifies a multipotent progenitor for neocortical projection neurons, astrocytes, and oligodendrocytes[END_REF].

IV. Genetic regulation of neocortical area mapping

As mentioned above, the neocortex has four primary areas; each of them is the cornerstone of clusters of functionally related areas that include scores of higher order areas prominently interconnected. Three of these primary areas are sensory, processing primary information from the whole body (S1), the inner ear/ cochlea (A1)

and the eye/retina (V1), while the fourth primary area is motor (M1), controlling voluntary movements. Each area is innervated by thalamocortical afferents from distinct thalamic nuclei, establishing a topographical relationship between cortical areas and thalamic nuclei, which is crucial both for the development of functional areas and for adult brain functioning (Figure 2).

Neocortical areas form the basis for sensory perception, control our movements and mediate our behavior. Many features must be properly specified during arealization, not only the unique properties that determine an area function and interaction with other brain structures, but also the appropriate area size and positioning. Arealization of the neocortex is controlled by interplay between intrinsic mechanisms (genetic mechanisms that operate within the cortex), and extrinsic mechanisms (TCA innervation and relayed inputs) [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF]. Two hypotheses were proposed in this issue: the "protomap" versus the "protocortex" hypothesis (Figure 6). The protocortex hypothesis states that area pattern in the developing neocortex is extrinsically specified by the innervating thalamocortical afferents, and thus the naïve cortical primordium may be seen as a "tabula rasa" patterned by outside factors [START_REF] Van Der Loos | Somatosensory cortex: structural alterations following early injury to sense organs[END_REF][START_REF] O'leary | Do cortical areas emerge from a protocortex?[END_REF]. In contrast, the protomap hypothesis proposed that neocortical areas are patterned by the interactions among several morphogens and transcription factors expressed by neuronal progenitors in different neocortical and allocortical (external structures to the neocortex) regions [START_REF] Rakic | Specification of cerebral cortical areas[END_REF]. However, the truth lies in the middle, both extrinsic and intrinsic mechanisms are working in combination to regulate specification and development of neocortical areas. During early stages, before innervation by thalamocortical afferents, area identity is broadly established in the neocortex through intrinsic information in progenitors and postmitotic neurons, whereas at later stages, extrinsic input refines and sharpens these boundaries (Figure 7) [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF].

Neocortical areal patterning is controlled by a regulatory hierarchy beginning with morphogens secreted from patterning centers positioned at the boundaries of the dorsal telencephalon. These morphogens or signaling molecules establish, within The "protocortex hypothesis" states that the neocortex is like a tabula rasa until the arrival of TCA from distinct thalamic nuclei: the dorsolateral geniculate (dLG), the ventropostero medial (VPM), the VL. Abbreviations: F/M: frontal-motor areas; S1: primary somatosensory area; V1: primary visual area; A1: primary auditory area; Ncx: neocortex; RGC: radial glia cells (taken from [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]).

cortical progenitors, the differential graded expression of transcription factors that determine the areal identity of the neurons that will give rise to the cortical plate [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF].

A. Morphogens and patterning centers

The early specification of cortical territories is controlled by a complex interaction between different morphogens, including fibroblast growth factors (Fgfs), sonic hedgehog (Shh), retinoic acid (RA), bone morphogenetic proteins (Bmps), vertebrate orthologs of Drosophila wingless (Wnts) and epidermal growth factors (Egfs) [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF]] (Figure 7).

Four telencephalic patterning centers appear to be involved directly or indirectly in cortical patterning. Two of them are directly involved in arealization: the commissural plate (CoP), which expresses Fgfs, and the cortical hem that releases Wnts and Bmps.

The third patterning center is the antihem, producing Egfs (Neuregulin 1 and 3), Tgfα, Fgf7, and the Wnt antagonist secreted frizzled related protein (Sfrp2). The fourth patterning center is located in ventral telencephalon and in the ventral diencephalon (hypothalamus), expressing Shh (Figure 7) [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF].

Anterior neural ridge: Fgfs 1.

The anterior neural ridge (ANR), the anterior junction between neural and non-neural ectoderm, which becomes later through morphogenesis the CoP, is considered as an anterior patterning center. It begins to release Fgf8 at E8-E8.5, and shortly after Fgf15, 17 and 18 [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF][START_REF] Maruoka | Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo[END_REF]. Fgf8 and Fgf17 have been most implicated in arealization. They act by inducing ETS family of transcription factors, establishing the gradients of empty spiracles homeobox (Emx2) and chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) within cortical progenitors [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][START_REF] Fukuchi-Shimogori | Neocortex patterning by the secreted signaling molecule FGF8[END_REF]. Fgf8 and Fgf17 have crucial effects on area patterning, by altering TF levels, most probably through repression of COUP-TFI, Emx2, and other TFs [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][START_REF] Fukuchi-Shimogori | Neocortex patterning by the secreted signaling molecule FGF8[END_REF]. However, there is a big debate between O'Leary and Grove whether Fgf8 controls Emx2 expression directly or indirectly. Other studies showed that Fgf8 controls the size of The initial, tangential gradients of transcription factors (TFs) in the ventricular zone (VZ) are established by signaling molecules/morphogens secreted from telencephalic patterning centers, such as Fgf8 and Fgf17 from anterior neural ridge (ANR), which later becomes the commissural plate (CoP), and Wnts and BMPs from the cortical hem. The antihem is a putative patterning center identified based on its expression of secreted signaling molecules (e.g. Tgfa, Fgf7, Sfrp2, as well as Neuregulin1 and Neuregulin3) with known patterning functions. A fourth telencephalic patterning center is defined by the expression domains of sonic hedgehog (Shh) in ventral telencephalon, but it does not have defined roles in dorsal telencephalic (dTel) patterning. The graded expression of certain TFs, such as Pax6, Emx2, COUP-TFI, and Sp8, imparts positional or area identities to cortical progenitors which is imparted to their neuronal progeny that form the cortical plate (CP). The CP also initially exhibits gradients of gene expression that are gradually converted to distinct patterns with sharp borders. Coincident with this process, distinct cortical layers, and the anatomically and functionally distinct areas seen in the adult (M1, S1, A1, V1), differentiate from the CP. Genes that are differentially expressed across the cortex are often expressed in different patterns in different layers, suggesting that area-specific regulation of such genes is modulated by layer-specific properties, and questions the definition of area identity. Although the initial establishment of the graded gene expression in the embryonic CP is controlled by mechanisms intrinsic to the telencephalon, the more complex differentiation patterns established postnatally might be controlled in part by extrinsic mechanisms, for example, TCA input and the sensory activity that it relays from the periphery to the cortex (Taken from [START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF]).

both dorsal frontal cortex and ventral/orbital frontal cortex, while Fgf17 selectively controls the size of dorsal frontal cortex [START_REF] Cholfin | Patterning of frontal cortex subdivisions by Fgf17[END_REF].

Overexpressing Fgf8 by in utero electroporation causes a caudal expansion of rostromedial areas [START_REF] Fukuchi-Shimogori | Neocortex patterning by the secreted signaling molecule FGF8[END_REF][START_REF] Toyoda | FGF8 acts as a classic diffusible morphogen to pattern the neocortex[END_REF]. In contrary, Fgf8 reduction in hypomorphic mutants causes caudal areas of the neocortex to expand rostrally. A similar phenotype was observed by overexpressing the cytoplasmic domain of Fgf8 receptor Fgfr3C, which reduced rostral Fgf8 expression [START_REF] Garel | Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants[END_REF][START_REF] Cholfin | Patterning of frontal cortex subdivisions by Fgf17[END_REF].

A mutual inhibiting loop between Fgf8 and Bmp/Wnt signaling has been reported in different studies [START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF][START_REF] Grove | The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice[END_REF][START_REF] Aoto | Mouse GLI3 regulates Fgf8 expression and apoptosis in the developing neural tube, face, and limb bud[END_REF][START_REF] Kuschel | A disrupted balance between Bmp/Wnt and Fgf signaling underlies the ventralization of the Gli3 mutant telencephalon[END_REF]. In contrast, Shh, together with Noggin and chordin (two Bmps antagonists secreted by the ANR), favors Fgf8 expression presumably by inhibiting Wnt signaling [START_REF] Aoto | Mouse GLI3 regulates Fgf8 expression and apoptosis in the developing neural tube, face, and limb bud[END_REF][START_REF] Anderson | Chordin and noggin promote organizing centers of forebrain development in the mouse[END_REF][START_REF] Crossley | Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles[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][START_REF] Panman | Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial-mesenchymal signalling[END_REF][START_REF] Piccolo | Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4[END_REF][START_REF] Shimamura | Longitudinal organization of the anterior neural plate and neural tube[END_REF][START_REF] Shimogori | Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex[END_REF][START_REF] Sousa | Sonic hedgehog functions through dynamic changes in temporal competence in the developing forebrain[END_REF][START_REF] Zimmerman | The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4[END_REF]. In turn, Fgf8 maintains Shh expression rostrally in a dose dependent manner [START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF].

Cortical hem: Bmps and Wnts

2.

The cortical hem, which is a neuroepithelium extending from the medial cortex to the dorsal midline, expresses Bmps and Wnts [START_REF] Grove | The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice[END_REF][START_REF] Furuta | Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development[END_REF]. Very recently, important roles for the hem in dorsoventral and areal patterning have been revealed [START_REF] Caronia-Brown | The cortical hem regulates the size and patterning of neocortex[END_REF]. Hem ablation caused a reduction in the size of the dorsomedial cortex, whereas ventrolateral cortex was expanded rostrally. Moreover, hem ablation perturbed regional neocortical patterning. Genetically engineered mice lacking the hem showed an expansion of rostral neocortical domains, while caudal domains were diminished [START_REF] Caronia-Brown | The cortical hem regulates the size and patterning of neocortex[END_REF].

In addition, Lhx2 and Lhx5, two members of the LIM/homeobox protein (Lhx) class of Lim homeodomain proteins, have been shown to control the development of the cortical hem [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][100][101]. Lhx2 is expressed in the cortical VZ in a high posterior-medial to low anterior-lateral gradient. After repression of Bmp2 and Bmp4 in the roof plate, Lhx2 exhibits an abrupt decline in its posterior-medial expression, e.g. in the cortical hem [START_REF] Monuki | Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway[END_REF]. In the absence of Lhx2 function, the neocortex is dramatically reduced in size, and proliferation is prematurely arrested [START_REF] Monuki | Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway[END_REF][100][101]. Other studies in Lhx2 conditional knock out (KO) mice show that Lhx2 specifies cortical identity in a cell autonomous manner [102].

Antihem: Sfrp2, EGFs, Tgfα, Fgf15 and Fgf7 3.

The antihem, located at the ventricular edge of the ventral pallium, is so called due to its opposite position with respect to the hem, and due to the production of Sfrp2, an antagonist of Wnt signaling [103]. However, the anti-hem releases also EGFs (Neuregulin 1 and 3), Transforming Growth Factor α (Tgfα), Fgf15 and Fgf7.

No clear function has been found for the anti-hem in cortical patterning [START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF], but it is essentially absent in small eye (Sey) mutant mice lacking functional Paired box gene 6 (Pax6) protein, and therefore some of the major telencephalic defects observed in these mutants could be due to the absence of the antihem [104].

Ventral telencephalon: Shh 4.

The ventral telencephalon and the hypothalamic region of the ventral diencephalon produce high levels of Shh. It has been shown that Shh is involved in regional patterning of the forebrain [START_REF] Rash | Area and layer patterning in the developing cerebral cortex[END_REF][START_REF] Grove | The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice[END_REF][START_REF] Kuschel | A disrupted balance between Bmp/Wnt and Fgf signaling underlies the ventralization of the Gli3 mutant telencephalon[END_REF][START_REF] Crossley | Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles[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][START_REF] Shimogori | Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex[END_REF]. Gain-and loss-of-functions experiments show that Shh is involved in controlling levels of proliferation of neural progenitor cells along the entire central nervous system (CNS) including the neocortex [105,106]. However, other studies have led to the proposal that Shh is not directly involved in areal patterning [START_REF] Rash | Area and layer patterning in the developing cerebral cortex[END_REF].

In conclusion, most of these signaling molecules act in a time-and dose-dependent manner to regulate proliferation, specification and survival of stem cells and neural progenitors. Some of them can even promote neurogenesis and differentiation. Their antagonizing effects influence, initially, the balance between cellular processes contributing to dorsoventral (DV) and anteroposterior (AP) patterning, and then the specification of neocortical domains [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. However, among the different morphogens, the best described to date to play a clear cut role in cortical arealization belong to the Fgf family [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF] and, very recently, to the Wnt family and the other families of molecules expressed by the hem [START_REF] Caronia-Brown | The cortical hem regulates the size and patterning of neocortex[END_REF].

B. Graded expression of transcription factors

Cortical morphogens and signaling molecules induce a graded expression of transcription factors (TFs) in ventricular zone progenitors. These TFs match the basic criteria required for candidate genes in areal identity specification. They are regulatory genes, expressed in different AP and DV gradients by progenitors in the VZ, or in both the VZ and the SVZ. These characteristics allow for these transcription factors to act in a combinatorial manner across the cortical axes, which is essential to impart area identities. Among these, four are expressed from early stages of development in the cortical primordium: Paired box gene 6 (Pax6) and empty spiracles homeobox (Emx2) are expressed in the VZ in reciprocal rostro-lateral to caudo-medial gradients, whereas trans acting transcription factor 8 (Sp8) and chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) are expressed in reciprocal rostro-medial to caudo-lateral gradients (Figure 8).

In mice, the expression of these TFs begins at E8.0-E8.5 in the ventricular zone, and lasts, apart from Sp8, during the entire period of neurogenesis. Except for COUP-TFI, the expression of all these TFs is exclusively limited to the progenitor cells in the VZ. COUP-TFI is the only TF expressed in both mitotic and postmitotic cells [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF][START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF]

Sp8

1.

Sp8 is a zinc finger transcription factor belonging to the family of Bottonhead (btd) homeotic genes. Its expression begins from E8.0 to E8.5 in the ANR, and from E9.5 in the cortical primordium, in a high antero-medial to low postero-lateral gradient.

Sp8 is a direct transcriptional activator of Fgf8 in the CoP [107]. Overexpression of Fgf8 at E11.5 induces an ectopic expression of Sp8 indicating a reciprocal induction between these two genes [108,109]. In vitro assays showed that Emx2 co-expresses with Sp8 in cortical progenitors but not in the CoP. Emx2 interferes with the ability of Sp8 to bind regulatory elements of Fgf8 to induce its expression, explaining why Sp8 induces Fgf8 expression only in the CoP but not within cortical progenitors [107].

Gain-and loss-of-function analyses by in utero electroporation, and the use of a conditional knock out ablating Sp8 specifically in the telencephalon (Sp8 fl/fl Foxg1-Cre ), Those changes to the expression patterns were identified in the knockout mice; thus, these interactions do not necessarily imply direct control of one TF on another. For instance, Emx2 suppression by Sp8 might be due to an enhancement of Fgf8 expression, which in turn acts negatively on Emx2 expression. +, positive interaction; _, negative interaction. (C) Summary of all reports of loss-of-function or gain-of-function mice mutant for TFs that regulate area patterning. Reducing Emx2 levels in the cortex of the heterozygote mutant mice results in posterior shifts of areas with shrinkage of V1, while overexpression of Emx2 under the control of the nestin promoter shifts areas anteriorly. The small eye mutant without functional Pax6 shows anterior area shifts. Unlike the Emx2 transgenic mice, YAC transgenic mice of Pax6 do not show area changes other than a slight, but significant, reduction in the size of S1 (asterisk). Loss of COUP-TFI in cortical progenitors transforms the fate of primary sensory areas into frontal/motor areas. The analysis of Sp8 conditional knockout mice shows anterior shifts of gene markers (taken from [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF]).

further unraveled the roles of Sp8 in areal patterning [107,109]. Analyses of these mutant mice at E18.5 showed an anterior shift of cortical markers, suggesting that Sp8 specifies frontal/motor area identity. However, the use of the Foxg1-Cre line results in the deletion of Sp8 in the ANR/CoP. Since Sp8 is a direct activator of Fgf8 in the CoP, and is required for its maintenance, and since Fgf8 is involved in frontal/motor area specification it is not clear if Sp8 promote directly or indirectly frontal/motor identity. Moreover, Fgf8 negatively regulates the gradients of COUP-TFI and Emx2, thus it is not clear whether the regionalized marker shifts observed in the conditional knock out of Sp8 are mediated by the decrease of Fgf8 expression in the CoP, or by impairments in normal COUP-TFI and Emx2 gradients [107,109]. However, a new work by Borello et al [110] using a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner, demonstrated a reciprocal cross-regulation between COUP-TFI and Sp8, and proposed that Sp8 Pax6 is a paired box domain TF. Its expression can be detected from E8.5 in the cortical primordium in a high anterior lateral to low posterior medial gradient, which correlates well with the neocortical neurogenic gradient [START_REF] O'leary | Area patterning of the mammalian cortex[END_REF][START_REF] Rakic | Specification of cerebral cortical areas[END_REF]111,112]. Pax6 is involved in controlling the balance between proliferation and neurogenesis. It is also involved in many other events, such as mediating the fate of neuronal precursors in the neocortex, and controlling the progression from apical to basal progenitors and orienting the mitotic spindle, which are at the basis of the neurogenic process [113][114][115]. Small eye (Sey) mutant mice, which lack a functional Pax6 protein, lack eyes and nasal structures, have major lamination defects and a cortex reduced by a third, in which TCA fail to reach the cortex [111,[116][117][118]. Moreover, analysis of arealspecific markers indicated a rostralization of sensory areas proposing Pax6 as a rostralizing agent [111,[116][117][118]. However, Pax6 overexpression using a YAC transgenic approach, reported no changes in areal patterning despite a partial reduction of the somatosensory area that can be explained by an induced proneurogenic and pro-apoptotic effects in response to increased Pax6 levels [119,120].

The analysis of Pax6 conditional knock out (CKO) mice under the control of the cortical-specific Emx1-Cre promoter showed only few changes in arealization markers and no defects in TCA innervation. The only remarkable change was the shrinkage of the somatosensory area, while its position was not changed, suggesting a minor role for Pax6 in arealization [121]. Further analyses are required to explain these discrepancies and better define the role of Pax6 in areal patterning.

Emx2

3.

Emx2 is a homeotic genes belonging to the family of Empty spiracles TFs. It is expressed from E8.5 in the rostro-lateral neural plate, and later in the cortical primordium in a high caudo-medial to low rostro-lateral expression gradient [111,112,122]. Emx2 promotes caudal cortical fate in a dose-dependent manner [111,112,122]. In Emx2 mutants, sensory areas are shifted caudally and the V1 is reduced and mis-positioned at the most caudal portion of the cortex [111]. In contrast to the hypothesized role of Emx2 through down-regulation of Fgf8 expression, different genetic studies supported a direct role of Emx2 in conveying positional information to cortical precursor cells [123][124][125]. Transgenic mice in which Emx2 was overexpressed under the control of the Nestin promoter (Ne-Emx2) showed a rostral expansion of the V1 and a significant reduction of the postero-medial barrel subfield (PMBSF) of S1, which was shifted more rostrally. In these experiments, Emx2 acted in a dose dependent manner in cortical patterning and had no effects on the morphology or the dimension of the neocortex [123]. In conclusion, Emx2 is a crucial factor for V1 development, with a limited action on size and organization of the somatosensory area.

COUP-TFI 4.

COUP-TFI is an orphan nuclear receptor that belongs to the steroid/thyroid hormone receptor superfamily, acting as a strong transcriptional repressor [126][127][128][129][130], but also as an activator [131][132][133]. COUP-TFI is involved in the temporal specification of neuronal precursor cells in the mouse neocortex [134]. COUP-TFI expression starts at E8.5 in anterior regions, and from E9.5, it is expressed in a high caudo-lateral to low rostro-medial gradient in the cortical primordium. In contrast to other TFs involved in cortical arealization, COUP-TFI is the only TF among areal patterning genes, expressed in both mitotic and post-mitotic cells in the neocortex, with expression maintained in adult brains [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF][START_REF] O'leary | Area patterning of the mammalian cortex[END_REF]135,136].

First analyses in COUP-TFI constitutive null mutants, showed major defects in subplate and layer IV specification [137][138][139]. Moreover, TCAs fail to exit the internal capsule, and very few of them reach the subplate and the cortical plate. The cortices of these mutants showed a strong caudalization of sensory areas. However, since COUP-TFI is also expressed in the dorsal thalamus, and could be involved in the maturation of TCAs, these effects could not be assessed as a direct role for COUP-TFI in arealization or as a result of TCA innervation [137][138][139]. To overcome the potential effects of the TCAs, COUP-TFI was selectively ablated from the cortex using the COUP-TFIfl/fl EmxCre mice (Emx CKOs). These mutant mice survive after birth to adulthood and TCAs invade the neocortex. In the absence of cortical COUP-TFI, rostral motor regions show an impressive caudal expansion at the expense of the caudal sensory areas, which are shrunken and shifted toward the occipital cortex [140] (see Chapter VII).

Another study from Tomassy et al,[134] showed that the parietal/somatosensory cortex acquired peculiar frontal/motor laminar organization in COUP-TFI conditional mutants, indicating a tangential (areal) and radial (laminar) control of COUP-TFI in neocortical organization. In these mutant mice, the expression of COUP-TFI interacting protein 2 (Ctip2), a TF involved in the specification of layer V subcerebral projection neurons [134], was considerably expanded in the fifth and sixth layers of the parietal cortex, considered as a "motorized" S1, while characteristic layer IV granule cells were lost [134] (see Chapter VII). Some evidence suggests that COUP-TFI inhibits rostral fate and promotes caudal ones by out-competing with Fgf8 signaling, which positively regulates Mitogen-activated protein kinases (MAPK). COUP-TFI interferes with MAPK signaling: the phosphorylation of the Extracellular signal-Regulated Kinase (ERK) (a main player in the MAPK cascade) is strongly affected in D6/COUP-TFI transgenic mice, which express ectopic high levels of COUP-TFI in the antero-dorsomedial region of the cortex [141,142]. Accordingly, a consistent down-regulation of Ets genes (downstream targets of MAPK pathway) has been observed in these mice. This negative regulation of the Fgf8 signaling can be mediated by Sprouty 1 and 2, which are two inhibitors of the MAPK pathway positively regulated by COUP-TFI [141,142]. Thus, COUP-TFI may interfere with Fgf8 rostralizing function by directly promoting Sprouty 1 and 2 transcription, supporting an active and direct role for COUP-TFI in the balance between rostral and caudal fate specification [141,142].

The roles of COUP-TFI in areal patterning and neocortical projection neurons subtype specification will be presented in details in chapter VII.

Areal patterning is determined by the expression of mitotic and post-mitotic patterning gens such as Bhlhb5, Tbr1, LMO4 and COUP-TFI [143][144][145][146]. However, it is still unclear whether post-mitotic genes just refine area properties specified in progenitors or independently define areal features. A recent study from our group used genetic gain and loss-of function approaches to assess the roles of COUP-TFI at mitotic and post-mitotic levels. By comparing two transgenic lines, where COUP-TFI is ablated in all neocortical cells (Emx CKOs) and only in newborn cortical neurons COUP-TFIfl/fl NexCRE mice (Nex CKOs), and by re-expressing the human ortholog of COUP-TFI (hCOUP-TFI) exclusively in post-mitotic neurons of COUP-TFI KO, we showed that COUP-TFI post-mitotic expression is necessary and sufficient to drive sensory area specification. Moreover, this study demonstrate that ectopic post-mitotic overexpression of COUP-TFI in rostral cortical regions reprograms frontal/motor areal features into sensory ones indicating that areal patterning is a more plastic process than previously expected (Alfano et al, 2014 under revision).

V. Neocortical projection neuron specification and diversity

The organization of the mammalian neocortex into only six histologically distinct layers belies an extraordinary diversity of neuronal subtypes. Individual phenotypic characteristics, such as dendritic morphology, electrophysiological properties and projection patterns have been used in the past to classify projection neurons (PNs).

Nowadays, the commonly used characteristic to group neocortical neurons is by the target of their axons, because "hodology", defined as the path followed by axons to reach their targets [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF], is centrally related to projection neuron function. Moreover, the establishment of appropriate projections requires successful execution of developmental and molecular programs. As previously mentioned, the absence of COUP-TFI function, an important areal patterning gene, results in both areal and laminar alterations in the neocortex. Other genes such as LMO4, Bhlhb5, and Tbr1 are also involved in neocortical arealization and subtype specification of neocortical projection neurons (details will be discussed in chapter VII) [143][144][145][146]. Thus, it is becoming increasingly clear, that specification of PN subtypes and area identity are two related and highly dependent processes.

PNs progressively acquire area and subtype identities, and their development can be followed along three different axes: time, subtype differentiation and area differentiation. Therefore, genetic programs operate in establishing boundaries in ndimensional "identity space" among different PNs subtypes, and different neocortical areas [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]. CPN are interhemispheric commissural pyramidal neurons, whose myelinated axons make up the corpus callosum, the largest white-matter tract in the placental mammalian brain, which connects the two cerebral hemispheres. The corpus callosum is not unique in its ability to connect the two neocortical hemispheres, since the anterior and the hippocampal commissures also cross the forebrain midline, but the corpus callosum is the only fiber tract devoted solely to integrate information from the two cortical hemispheres. Thus CPN play a key role in the high level complexity of cognition and associative behavior [147][148][149]. Classified by hodology, there are three basic classes of cortical projection neuron: associative, commissural and corticofugal. Below are some principal subtypes:

A. Major subtypes of projection neurons within the neocortex

Commissural: Callosal projection neurons. Projection neurons of small to medium pyramidal size that are primarily located in layers II/III, V and VI, and extend an axon across the corpus callosum (CC) (panel a). At least three major types of callosal neuron can be classified. These maintain: single projections to the contralateral cortex (black); dual projections to the contralateral cortex and ipsilateral or contralateral striatum (blue); and dual projections to the contralateral cortex and ipsilateral frontal cortex (green). These never project axons to targets outside the telencephalon. Str, striatum.

Corticofugal (subcortical): Corticothalamic neurons. Projection neurons primarily located in cortical layer VI, with a smaller population in layer V, that project subcortically to different nuclei of the thalamus (Th) (panel b).

Subcerebral projection neurons. Also referred to as type I layer V projection neurons (panel c). These include pyramidal neurons of the largest size, which are located in deep-layer V and extend projections to the brainstem and spinal cord. neuron subtypes. Among them:

Corticotectal neurons (orange) are located in the visual area of the cortex and maintain primary projections to the superior colliculus, with secondary collateral projections to the rostral pons (Po). Corticopontine neurons (pink) maintain primary projections to the pons. Corticospinal motor neurons (purple) are located in the sensorimotor area of the cortex and maintain primary projections to the spinal cord, with secondary collaterals to the striatum, red nucleus, caudal pons and medulla. Many other subtypes of subcerebral projection neuron exist that send axons to different areas of the brainstem or have different combinations of collaterals, but are not depicted here for simplicity.Crb, cerebellum; OB, olfactory bulb; SC, spinal cord (Taken from [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]).

Abnormalities in CPN can lead to cognitive deficits. The absence of CPN in humans is associated with defects in abstract reasoning, problem solving and generalization.

CPN dysgenesis is one of only few reproducibly identified pathologies in autism spectrum disorders (ASD), with reduced corpus callosum relative to the overall brain volume [150][151][152][153][154][155][156].

CPNs are more abundant than CFuPN and comprise the largest class of commissural neurons in placental mammals. CPNs are a heterogeneous population of neocortical neurons, with respect to their birthdate, final laminar destinations and distinct projections patterns. Their cell bodies principally reside in layers II-III (approximately 80% in rodents), layer V (approximately 20% in rodents) and to a lesser extent in layer VI [147][148][149]. In mice, layer VI CPN are born at embryonic day E12.5, layer V CPN are born around E13.5, and superficial layers CPN are born from approximately E15.5 to E17.5 [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]148,157].

CPN axons innervate their target in the contralateral hemisphere in a homotopic manner; thus, the location of a CPN within the cortex defines the target of its axon [START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF]158,159]. In addition to homotopic interhemispheric projections extended by all CPN, subpopulations of CPN can be defined by the variety of longrange dual axonal projections. Subpopulations of CPN send dual projections to contralateral or ipsilateral striatum (intralencephalic corticostriatal PN; CStrPNi). In Projection neurons are broadly classified according to whether they extend axons within one cortical hemisphere (associative projection neurons), across the midline to the contralateral hemisphere (commissural projection neurons) or away from the cortex (corticofugal projection neurons). Importantly, neurons of a given subtype residing in different cortical areas (motor, somatosensory, visual and auditory) project to anatomically and functionally distinct targets.

Most commissural projection neurons cross the midline through the corpus callosum (CC) -these are called callosal projection neurons (CPN) -whereas a smaller population of these neurons cross through the anterior commissure (see the figure ,part a). CPN reside primarily in layer II/III, with fewer residing in layers V and VI, and extend axons to mirror-image locations in the same functional area of the contralateral hemisphere, enabling bilateral integration of information.

Present in all cortical layers, associative projection neurons include short-distance intrahemispheric projection neurons, which extend axons within a single cortical column or to nearby cortical columns (such as layer IV granular neurons) and long-distance intrahemispheric projection neurons, which extend axons to adjacent or distant cortical areas (such as forward and backward projection neurons; see the figure ,part b).

Corticofugal projection neurons include corticothalamic projection neurons (CThPN), which reside in layer VI, and subcerebral projection neurons (SCPN), which reside in layer V (see the figure ,part c). CThPN extend axons to specific thalamic nuclei: motor cortex (M1) CThPN establish connections with the ventral lateral (VL) and ventral anterior nuclei, somatosensory cortex (S1) CThPN with the ventral posterior (VP) nucleus and visual cortex (V1) CThPN with the lateral geniculate nucleus (LGN). SCPN extend axons to different primary targets in the brainstem and spinal cord (SC). In general: M1 SCPN project to the SC (corticospinal motor neurons) and brainstem motor nuclei (cortico-brainstem motor neurons); S1 SCPN project to the trigeminal principal sensory nucleus and dorsal column medullary nuclei (corticobulbar projection neurons); and visual cortex SCPN project to the optic tectum (OT) (corticotectal projection neurons). Neurons that send projections to multiple targets (see the figure, part d) can sometimes be classified into more than one of these categories. Examples include CPN with frontal projections, which extend axons to the contralateral hemisphere and to the ipsilateral frontal cortex; SCPN with backward projections, which extend axons to subcerebral targets and to the ipsilateral caudal cortex; and intratelencephalic corticostriatal projection neurons, which extend projections to the contralateral hemisphere and to the ipsilateral striatum (Str). Other neurons that project to multiple targets, such as pyramidal corticostriatal projection neurons, can be classified into only one category. A1, primary auditory cortex; Crb, cerebellum; OB, olfactory bulb; Th, thalamus (taken from [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]). Ipsilaterally, superficial layer CPN send collaterals to pyramidal neurons in layers II-III, V and VI. Thus, in addition to their role in integrating two homotopic regions of the neocortical hemispheres, CPN are also responsible for functional association and integration among different neuronal types in ipsilateral and contralateral hemispheres [163].

CPN establish exuberant projections early in development, with maximum number of dual projections at postnatal day 8 (P8) [160,164,165]. These dual projections are progressively refined until P21, through activity-dependent mechanisms [164,165]. Finally, CPN arose relatively recently in evolution, since they first appeared in placental mammals. Their broad laminar distribution not only reflects a broad time window of generation, but also suggests preferential expansion of this neuronal population throughout evolution [147,148]. Indeed, a large portion of CPN with known heterotopic long-range dual-projecting axons reside in deep neocortical layers, suggesting that deep-layer CPN might have been co-opted from existing populations of CFuPN, during evolution, to project not only to subcerebral targets but also to cortical ones to connect and integrate the two neocortical hemispheres [148].

Corticofugal Projection neurons (CFuPNs) 2.

In contrast to CPN, CFuPNs send their axons away from the cortex. CFuPNs can be further subdivided into subplate and corticothalamic neurons, which form together the subcortical PNs and project to the thalamus, and subcerebral projection neurons, which extend their primary axons to targets in the midbrain, hindbrain and spinal cord [148,166]. These distinct subtypes of cortical projection neurons are born in a tightly orchestrated sequence and reside in distinct laminar locations in the neocortex [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]167] through reciprocal connections formed by TCA and CTA, representing a highly integrated processing unit that dynamically regulates thalamic transmission of peripheral information for cortical processing [172]. CThPNs reside in layer VI with a smaller population in layer V. TCA and CTA contribute to form the internal capsule (IC), a large axonal highway, which also comprises output subcerebral axons proceeding toward the cerebral peduncle and the pyramidal tract [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]173,174].

Layer VI CThPN innervate thalamic nuclei depending on their tangential identity, projecting to the first order thalamic nuclei from which they receive sensory input. Thus, CThPN from the V1 project to the dLGN, while CThPN from S1 innervate the VPM, CThPN from A1 project to the MGv and CThPN from the M1 project to the VL [175][176][177][178]. These CTA form numerous glutamatergic synapses on the distal dendrites of the relay cells, modulating their activity and gating pathways that transmit peripheral information. Layer VI CTAs send also collaterals to the reticular thalamic nucleus, generating an inhibitory circuit modifying the activity of relay cells [172,179,180]. In addition, collaterals from layer V corticobulbar and corticospinal motor neurons send inputs to higher order thalamic nuclei (pulvinar group, mediodorsal thalamic group and lateral posterior nucleus), through glutamatergic synapses on matrix cells. Higher order thalamic nuclei, in turn, project excitatory fibers to the upper and lower layers of the corresponding cortical areas distributing cortico-cortical information, and integrating different cortical areas into a global synchronized network [172,181].

c) Subcerebral projection neurons (SCPNs)

SCPNs are born around E13. 5 [157]; SCPNs migrate to the deeper layer V and its production ceases after E14.5 [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]166]. Even if all SCPNs share a common laminar position and are born within the same developmental time frame, they are quite diverse and include several unique subtypes.

Cortico-Spinal Motor Neurons (CSMN) are large pyramidal neurons that reside in sensory and motor areas and extend their primary axons to the spinal cord, with some secondary collaterals to the striatum, red nucleus, caudal pons and medulla.

Instead, Corticopontine PNs extend a primary axon to hindbrain targets in the pons and medulla. Finally, Corticotectal PNs reside mainly in the visual cortex and extend their primary axon to the superior colliculus in the midbrain [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF][START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]166]. All SCPN normally extend a primary axon through the internal capsule and the pyramidal tract to the spinal cord (inappropriate connections are later pruned) such that subcerebral PNs in the sensorimotor cortex project to the caudal pons and spinal cord, while those in the visual cortex project to the rostral pons and superior colliculus [182][183][184]. Since SCPN have a common pattern of initial development, it is not surprising that many transcription factors regulating their early specification and differentiation are coexpressed in their distinct subtypes. The most well studied subtype of SCPNs is the CSMN, whose molecular, physiological and morphological features have been extensively characterized [185,186].

CSMNs are of great interest because they control voluntary movements in humans;

their degeneration is a key factor in motor neuron degenerative diseases, including amyotrophic lateral sclerosis (ALS). CSMN injury contributes centrally to the loss of motor function following spinal cord injury [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]149]. Among the different subcerebral axonal tracts, the corticospinal tract is one of the longest longitudinal projections in the vertebrate central nervous system, and the major output from the motor cortex connecting the cerebral cortex to the spinal cord [185,186].

CSMNs extend their axons ipsilaterally via the internal capsule, descending to the cerebral peduncle, through the midbrain and hindbrain, until they reach the most caudal part of the hindbrain, where most of them, cross the midline dorsally toward the contralateral side, and form the pyramidal decussation. They further project in a region containing the dorsal funiculus in rodents, and innervate neurons located in the spinal gray matter. Some of them do not decussate in the medulla, but continue downward in the ventral funiculus and decussate just prior the spinal gray matter. The complete trajectory reaches a maximum level of gray matter spinal cord innervation at P14 in mice. Then, this connectivity is gradually refined in the following weeks. Once fully developed, it allows the execution of precise voluntary movements [187][188][189][190][191].

Layer V neocortical PN, callosal or subcerebral, send collaterals either ipsilaterally or contralaterally to the striatum; therefore, they are considered as corticostriatal projection neurons.

Corticostriatal projection neurons (CStrPN) 3.

Corticostriatal PN project from the neocortex to ipsilateral or contralateral striata, and are the cortical efferents of the corticobasal ganglia circuitry. Connectivity between cortex and striatum is directional; cortex connects mono-synaptically to the striatum, while the striatum communicates only indirectly to the cortex, via polysynaptic downstream circuits.

Corticostriatal projections are crucial components of forebrain circuitries, and are widely involved in motivated behavior, cognitive and motor functions, which include for instance, action selection, motor control, sequence learning and habit formation [192][193][194]. CStrPN are clinically important since their injury and degeneration are implicated in the pathophysiology of several neurological disorders. They are the cortical population that predominantly degenerates in the Huntington's disease. Their impairment contributes to multiple forms of cerebral palsy [195][196][197][198][199][200][201][202].

CStrPNs are formed by two distinct classes of neurons: The intratelencephalic IT-type CStrPNs (CStrPNi) project to targets within the telencephalon. They have the unique attribute of being both corticofugal because they project to the striata bilaterally, and callosal because their axons cross the midline. They possess dual callosal and corticofugal anatomic and molecular characteristics, suggesting that they might be evolutionary "hybrids" with both callosal and corticofugal features [203]. They are located in layer Va of the neocortex. In mice, they are born between E12.5 and E14.5; they can be distinguished from pure callosal around P3-P4 when they first invade the contralateral striatum [203]. In addition to CStrPNi, there is another population of subcerebral projection neurons (corticospinal and related corticobrainstem) that sends axon collaterals to the ipsilateral striatum, the co called pyramidal tract-corticostriatal projection neurons (PT-CStrPN). PT-CStrPN are only restricted to layer Vb [193,194,203]. Finally, it is important to recall that CStrPN are either IT or PT but not both (Figure 11).

PT-type and IT-type are neurochemically, morphologically and electrophysiologically distinct pyramidal neuron types [204]. For example, PT-type neurons are larger than IT-type in the rat cortex. These two neuronal populations also differ in their dendritic arborization. While PT-type have a prominent apical dendrite, that ascends and branches profusely in layer I of the cortex, IT-type are more slender and their arborization in layer I is sparser [193].

Moreover, PT-type and IT-type neurons have different electrophysiological activity, they convey distinct signals to the striatum. For instance, PT-type neurons in primates are three to four times more rapid than [205][206][207][208]. In addition, in the motor cortex, PT-type fire during movement, while IT-type fire in relation to movement planning [206,208,209]. There is growing evidence, that imbalance between the two classes of CStrPN, the intratelencephalic (IT) versus pyramidal tract (PT), is an etiological factor of neurodevelopmental, neuropsychiatric and motor disorders, including autism, amyotrophic lateral sclerosis, obsessive-compulsive disorder, schizophrenia, Huntington's and Parkinson's diseases and major depression (Reviewed in [194]).

B. Neocortical projection neuron specification

Corticofugal fate determination 1. 81 (Er81), insulin-like growth factor binding protein 4 (Igfbp4), cysteine rich transmembrane BMP regulator 1 (Crim1) and many others [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF] as CSMN specific genes. Later, the roles of some of these genes were investigated, and it was revealed their crucial involvement in SCPN specification. Diverse studies have shown that the specification and differentiation of SCPN are directed by a combinatorial code of TFs including Fezf2, Ctip2 and SRY-box-containing gene 5 (Sox5).

SCPNs and

(

1) Fezf2

Fezf2 is a zinc finger TF, crucial for SCPN specification; it is expressed by a subset of progenitors during deep layer PN generation and is expressed at high levels by all post-mitotic subcerebral PNs throughout adulthood [START_REF] Inoue | Fez1 is layer-specifically expressed in the adult mouse neocortex[END_REF][START_REF] Hirata | Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons[END_REF][START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF][START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF][START_REF] Chen | Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex[END_REF]210]. In the absence of Fezf2 function, in null mutant mice, the entire population of layer V pyramidal subcerebral PN is absent. Moreover, expression of SCPN specific genes is lost, and there are no projection neurons from the cerebral cortex to either the spinal cord or brainstem [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF][START_REF] Chen | Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex[END_REF]. Importantly, without Fezf2, neocortical progenitors still produce similar numbers of layer V neurons [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF]210], however, using a Fezf2 mutant with a human placental alkaline phosphatase inserted at the Fezf2 locus, the authors demonstrated that these neurons adopt CPN-like properties, by extending axons across the midline via the anterior commissure, and expressing the CPN-specific molecular marker Special AT-rich sequence-binding protein 2 (Satb2) [211], a critical factor in the specification of CPN identity [150,212]. In addition, more neurons in layer V display electrophysiological characteristics typical of CPNs [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF]210]. In contrast, superficial-layer pyramidal neurons are born correctly and appear normal [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF]210]. Therefore, Fezf2 does not affect the ability of progenitors to generate layer V glutamatergic neurons, it likely acts in directing the specification, and defining characteristics of SCPN. Moreover, Fezf2 appears to repress Satb2 expression, directly or indirectly, repressing thereby callosal identity. Furthermore, misexpression of Fezf2 by in utero electroporation causes layer II/III CPN to redirect their axons towards a broad set of subcerebral targets, including brainstem, spinal cord but also thalamus [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF][START_REF] Chen | Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex[END_REF]211,213]. These data further support the involvement of Fezf2 not only in the specification of SCPNs, but also in determining general CFuPN identity (as it will be discussed below). A second set of genes controls later aspects of SCPN development, possibly acting downstream of Fezf2. The most studied and, probably, one of the most important members is Ctip2.

(2) Ctip2

Ctip2 (also known as Bcl11b) is a zinc finger transcription factor that acts as a transcriptional repressor [214,215]; it begins to be expressed at high level in all postmitotic SCPNs, once they reach the cortical plate [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. Ctip2 is a major downstream effector of Fezf2, in regulating the extension of axons toward subcortical targets Ctip2 is a crucial regulator of subcerebral axon extension and collaterals refinement [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]211]. In Ctip2 mutant mice, SCPN are still born and migrate to layer V, however they exhibit defects in fasciculation, outgrowth and pathfinding. SCPN axons fail to reach the spinal cord, as they are misrouted in the forebrain; they rarely reach the pons and never reach the pyramidal decussation [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. Moreover, reduced Ctip2 expression in heterozygous mice results in a defective pruning of transient projections to the spinal cord from SCPN residing in the somatosensory cortex, further demonstrating different roles for this TF at different levels of expression [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF].

Interestingly, Ctip2 also controls proper differentiation of striatal medium

b)

Corticothalamic PN fate specification:

(1) Fezf2, Ctip2 and Sox5

Similar to layer V SCPN, the fate of layer VI CThPN is at least in part specified by the expression of Fezf2, Ctip2 and Sox5. Fezf2 and Ctip2 are expressed at lower levels by layer VI CThPNs and subplate neurons [START_REF] Inoue | Fez1 is layer-specifically expressed in the adult mouse neocortex[END_REF][START_REF] Hirata | Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons[END_REF][START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF][START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF][START_REF] Chen | Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex[END_REF]210]. In Fezf2 null mice,

CThPN and subplate neurons appear to be disorganized, and a number of specific CThPN genes including protein phosphatase 1, regulatory subunit 1B (Ppp1r1b), transducing like enhancer of split 4 (Tle4) and Forkhead box P2 (Foxp2), fail to be expressed [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF]210]. This strongly indicates that low level of Fezf2 expression by

CThPN and subplate neurons is necessary for precise differentiation of these populations, and that the level of Fezf2 protein is directly linked to its function, and (

1) Satb2

Special AT-Rich Sequence-Binding Protein 2 (Satb2) was the first identified key regulator of CPN specification. Satb2 is a DNA binding TF, showed to bind AT-rich DNA sequences [226][227][228]. Satb2 is expressed by a subset of neurons in all cortical layers, with a prominent expression in layers II-V, but not in SVZ progenitors [150,212,226]. It is expressed at high levels by CPNs, and probably also by associative neurons throughout cortical layers [150,212].

Two independent laboratories inactivated Satb2 in the mouse and characterized its cortical phenotype [150,212]. In Satb2 null mice, almost no axons cross the corpus callosum, even though establishment of the midline appears normal.

Instead, some neurons expressing Satb2 in normal conditions project towards the Interestingly, Ski null mice mimic the phenotype shown by Satb2-null mice. In the absence of Ski, CPNs redirect their axons to subcerebral targets similarly to Satb2 null mice. In the absence of Ski, upper layer Satb2-positive neurons ectopically express Ctip2, and other deep layer-specific genes, such as Clim1, Ldb2 and Cdh13, but not Fezf2, suggesting that Ski-deficient CPNs acquire some but not all SCPN characteristics.

In addition, the expression of CPN specific genes (such as including Lmo4, Cdh10 and Ptn) was also up-regulated upon loss of Ski, revealing further changes in the regulation of CPN genetic program. Finally, ChIP, Co-IP, antibody based proximity ligation assay (PLA), and luciferase reporter transfection essays demonstrated that, altough Satb2 directly binds to MAR sequences in the Ctip2 locus and recruits MTA2 independently of Ski, this latter factor is essential for attracting HDAC1, thereby allowing the NURD complex to form properly, and enabling the Satb2-containing protein assembly to act as an inhibitory complex [234] (Figure 14).

(

2) Genes identifying distinct CPN subpopulations

A study from Molyneaux and colleagues [START_REF] Molyneaux | Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons[END_REF] defined a set of genes that identify and molecularly categorized distinct populations of CPN during embryonic and post-natal development. On the one hand, they showed that genes highly expressed at early (

3) Genes involved in upper layer specification: Brn and Cux

The POU domain TFs Brn1 (Pou3f3) and Brn2 (Pou3f2) expressed in superficial layers promote the birth of upper layer neurons. In Brn1 and Brn2 double mutants, superficial layer pyramidal neurons are not generated [235,236].

Moreover, TF Cut-like homeobox 1 and 2, Cux 1 and Cux2 are expressed in SVZ cells and in their progeny in layers II-IV [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] Zimmer | Dynamics of Cux2 expression suggests that an early pool of SVZ precursors is fated to become upper cortical layer neurons[END_REF]. The analysis of Cux2 knockout

VI. Neocortical projection neuron: Callosal versus Subcerebral axon guidance control

Neocortical neurons form connections through specific and predetermined trajectories in the brain in response to several guidance cues present in the environment.

These guidance factors determine the direction of neocortical axons to different cortical or subcerebral targets. Neurons express specific combinations of receptors for secreted ligands, mediating either a chemoattractive or chemorepulsive response.

A. Callosal projection neurons

A wide diversity of developmental processes regulates the midline crossing of callosal As aforementioned, Satb2 and Ctip2 have been shown to orchestrate important and mutually exclusive genetic programs to establish corticocortical versus corticofugal connections. Whereas Satb2 is indispensable for the formation of the corpus callosum, Ctip2 is required for the fasciculation and pathfinding of subcerebral projection neurons [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]226]. In normal development, Satb2 overrides the Ctip2 driven molecular pathway in order to establish interhemispheric projections instead of corticofugal ones [150,212].

VII. Trancriptional regulators involved in arealization and cortical subtype specification

Post-mitotic regulators transform continuous gradients of positional information inherited from progenitors into sharp areal boundaries, instruct the formation of motor and sensory maps and direct projection neurons to acquire areal-specific phenotypic characteristics [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]. To date, only few of such controls have been identified: the Basic-loop-helix domain containing, class B5 (Bhlhb5), Lim domain only 4 (LMO4), Tbr1, and possibly COUP-TFI.

A. Basic-loop-helix domain containing, class B5 (Bhlhb5)

Bhlhb5 starts to be expressed in the cortical plate at E12.5, and is maintained in postmitotic neurons all along corticogenesis. From E15 onwards, Bhlhb5 is expressed in a high-caudomedial to low-rostrolateral gradient. After birth, its expression becomes progressively restricted to layers II-V of the primary sensory areas (somatosensory, visual and auditory).

Absence of Bhlhb5 does not produce a severe areal shift, but rather induces faint changes in the morphology of sensory areas. The V1 is slightly increased and the boundaries of the S1 barrels become faintly discernible. Moreover, the thalamocortical inputs appear to be more diffuse, and some area-specific genes such as LMO4 are aberrantly expressed. This suggests that Bhlhb5 refines the shape and boundaries of somatosensory and caudal areas [144].

Notably, the sensorimotor region, which has a crucial function in the control of voluntary movements in rodents and contains the highest percentage of CSMN resulted mispecified in Bhlhb5 mutants [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]144]. Bhlhb5 is strongly expressed in

B. Tbr1

Tbr1, which has been discussed above as a critical control over CThPN subtype identity, contributes also to area identity acquisition and is expressed mostly in rostral areas of the cortex. Tbr1 mutants lack TCA innervation to the CP and, although they die at birth, regional and laminar markers indicated a rostral shift of their caudal areas, suggesting a role for Tbr1 in arealization [143]. Moreover, Fgf17, ets variant 1 (Etv1) and Sprouty1, which are downstream targets of Fgf8, are increased in Tbr1 mutants at E12.5, suggesting that normally, Tbr1 negatively regulates Fgf8 signaling [143]. Since Tbr1 is expressed in newborn neurons and not in progenitors [221,282],

this negative regulation could be a post-mitotic feedback exerted on mitotic compartment [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]. Finally, Tbr1 has been shown to have a direct transcriptional control on the expression of the frontal marker autism susceptibility candidate 2 (Auts2)

further confirming the role of Tbr1 in frontal area development [283]. Interestingly, mutations in the Auts2 locus were found in cases of Autism and mental retardation [283,284].

C. LIM domain only 4 (LMO4)

Given the important and various roles of LMO4 and its central role in my work, a more detailed description for LMO4 structure and different aspects of its function will be presented in this section. LMO proteins control transcription both negatively and positively depending on the cell context and their binding partners (Figure 18).

LMO4 is a 165 amino acid protein broadly expressed in human tissues, and playing important roles in several developmental systems, including epithelial, mammary, ear and neural development [297,[307][308][309][310][311][312]. However, it also acts as a mammary oncoprotein and is overexpressed in more than 50% of primary breast tumors [313], squamous cell carcinomas of the oral cavity [314], and primary prostate cancer [315]. Overexpression of LMO4 markedly reduces epithelial cell differentiation, implicating it as an oncogene [313,316].

LMO4 was found to interact with several kinds of proteins. It binds to the ovarian tumor suppressor protein Breast Cancer 1 (BRCA1) and inhibits its transcriptional activity in yeast and mammalian cells [317]. LMO4 is also a binding partner of the basic helix-loop-helix protein (HEN1) [318] involved in hematopoiesis and specifically expressed in the developing nervous system [319]. Furthermore, LMO4 interacts with the peroxisome proliferation activated receptor-γ (PPARγ) required for neuron protection in ischemic injury. It interacts also with deformed epidermal regulatory factor 1 (DEAF1), a DNA binding protein that interacts with regulatory sequences and modulates transcriptional outcome [297]. Finally, LMO4 can also associate with other proteins, including the repulsive guidance molecule A receptor Neogenin in human SH-SY5Y cells, in human NTERA neurons, and in embryonic rat cortical neurons [320], the cAMP response element-binding protein (CREB) complex, the glycoprotein 130 subunit, a common receptor subunit for interleukin 6 (IL-6) type cytokines [321] and the transcription modulator Cbp/p300interacting transactivator (Cited2) [322].

Interestingly, LMO4 was found in vivo to bind to the Estrogen Receptor α (ERα) and MTA1 by establishing a multi-protein complex constituted by LMO4, ERα, MTA1 and histone deacetylases (HDACs) [323]. Similarly to MTA2 (see above), MTA1 is also part of the NuRD complex and functions by recruiting HDACs.

In physiologic conditions LMO4 was identified as an important component of the NuRD complex, which act as a potent repressor of ERα, a ligand-dependent TF controlling a variety of essential physiologic and developmental processes [323].

Another study revealed that LMO4 associates with HDAC2, and enhances granulocyte colony-stimulating factor (GCSF)-induced Stat3 signaling in mice Overall, LMO4 promotes the acquisition of cortical neuronal identities by forming a complex with Ngn2 and subsequently activating Ngn2 target genes [326].

b) LMO4 has a dynamic and region-specific expression in the mouse developing cortex

Onset of cortical LMO4 expression starts at E12.5, where LMO4 is detected in the preplate, and in the striatum [325]. At E15.5, LMO4 expression is detected in the cortical SVZ and in the hippocampus. At P0, LMO4 is mostly expressed in postmitotic neurons of the cortical plate [145,325]. At this stage, the expression of LMO4 is mainly localized in the anterior and posterior cortical regions, corresponding to motor and visual cortices, respectively, but not in the parietal region, which corresponds to the future somatosensory areas. After P0 LMO4 expression progressively increases in layers III/IV of the somatosensory regions, and at P10 LMO4 is expressed from lower to upper layers of the parietal cortex [325]. This dynamic and region-specific expression of LMO4 during critical periods for areal specification and neuronal subtype differentiation suggests that this factor may play important roles in these processes.

D. COUP-TFI

COUP-TFI controls the tangential (areal) and radial (laminar) neocortical specification (Figure 19). Moreover, COUP-TFI has been shown to be involved in different aspects of forebrain development, from neuronal migration to neurogenesis, gliogenesis and the formation of commissural projections.

COUP-TFI is involved in neurogenesis, gliogenesis and cell cycle control 1.

In the developing CNS, subtypes of neurons and glial cells are generated according to a temporal sequence defined by cell-intrinsic mechanisms that function at progenitor level.

A study by Naka et al [334] showed that knocking down COUP-TFI and COUP-TFII in neurospheres (NS) obtained from mice embryonic stem cells (ESC) delays the onset of gliogenesis. The time of neurogenesis was prolonged to the third stage of NS, while in normal conditions gliogenesis starts at the second generation of NS.

Furthermore, the knock-down of COUP-TFI and COUP-TFII in the cerebral cortex leads to an increase of early born neurons and to the production of neurons at the expense of glia cells, suggesting that COUP-TFs impinge on progenitor cell neuropotency.

Analysis of the promoter of Glial fibrillary acidic protein (GFAP), which is an important molecule for glia cell specification in COUP-TFI/II knocked-down cells revealed an altered methylation pattern, indicating that COUP-TFI promote gliogenesis through epigenetic modifications. However, COUP-TFI and II appeared to be necessary but not sufficient to induce gliogenesis, suggesting that they limit the neurogenic temporal window rather than promoting gliogenesis [334].

In this context, another study by our group [142] has investigated the role of COUP-TFI in neurogenesis and laminar fate using a gain-of-function mouse model overexpressing COUP-TFI in the neocortex under the promoter of the mDach1 gene (D6/COUP-TF1 transgenic mice) and COUP-TFI KO mice. This study showed that COUP-TFI influences dorso-ventral patterning of the cortex and promotes cell cycle COUP-TFI influences dorso-ventral patterning of the cortex and promotes cell cycle exit and neural differentiation. In addition, this study demonstrated that COUP-TFI A: Schematic representation of an embryonic mouse brain delineating the prospective frontal/motor (F/M), somatosensory (S), and visual (V) areas. According to the radial unit hypothesis, newborn neurons receive positional information from their progenitors. During neurogenesis, the thickening of a given area or areal region (bottom left represents a cortical section) will increase (black arrows), while an increment of the selfrenewing progenitor pool will lead to area surface expansion (bottom right, green arrows). B: Schematic representation of the hypothetical function of the areal patterning gene COUP-TFI in the specification of layer VI in primary motor (M1) and somatosensory (S1) areas. When COUP-TFI (which is highly expressed in parietal and occipital neocortical regions) is active, it delays the onset of the layer V (red) specification program in S1 area, where neurogenesis proceeds slower than in M1. As a result layer VI (blue) of S1 acquires approximately the same thickness of that of M1, while layer V results thinner in S1 compared to M1. When COUP-TFI is ablated, it fails to repress layer V program in S1, which in turn expands its layer V neuronal pool at the expense of layer VI neurons (taken from [START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]).

regulates the balance between early and late born neurons, and provides evidences In COUP-TFI mutants callosal projections fail to decussate and stop abruptly at the midline. They form aberrantly oriented fibres, known as Probst bundles.

Moreover, hippocampal commissure fails to cross the midline, and shows the presence of ventrally oriented thick ectopic bundles [338]. However, the anterior commissure was thicker [338]. In summary, in the absence of COUP-TFI, all the major forebrain commissures show abnormalities along the antero-posterior and the dorso-ventral axes.

Moreover, COUP-TFI deficient hippocampal neurons have a defect in neurite outgrowth and show an abnormal axonal morphology (REF). These defects could be due to the altered expression of various cytoskeletal molecules involved in axon guidance and neuronal migration in the absence of COUP-TFI function, such as the two microtubule-associated proteins regulating microtubule dynamics, MAP1B and MAP2, the member of the Rho GTPases family Rnd2, and the cyclase-associated protein 1 (CAP1) known to regulate actin dynamics. Overall, this study provides strong evidence that COUP-TFI is intrinsically required for proper axonal outgrowth in the developing forebrain [338].

COUP-TFI promotes radial migration and proper morphology of callosal 3. projection neurons by repressing Rnd2 expression

As mentioned in chapter II, the inside-out pattern of corticogenesis was made possible by the appearance of glia-guided migration [START_REF] Aboitiz | The evolutionary origin of the mammalian isocortex: towards an integrated developmental and functional approach[END_REF]339]. While early born neurons give rise to the preplate and lower layers by somal translocation [START_REF] Miyata | Asymmetric production of surface-dividing and nonsurface-dividing cortical progenitor cells[END_REF][START_REF] Nadarajah | Ventricle-directed migration in the developing cerebral cortex[END_REF], upper layer neurons follow a more complex migration pattern (see Chapter II).

The small Rho-GTPase Rnd2 is involved in the regulation of multipolar

COUP-TFI regulates the balance between frontal/motor and sensory

areas

COUP-TFI role in area patterning was suggested by its high caudolateral to low rostromedial expression gradient in the progenitors of the VZ, and later in their neuronal progeny in the cortical plate [138,342]. As previously mentioned, COUP-TFI roles were studied by our group using genetically engineered mice to inactivate COUP-TFI expression selectively in the cortex [140]. These mice were generated to overcome the complications raised by constitutive loss of COUP-TFI. Indeed, COUP-TFI constitutive knockout mice die after birth, before areas can be defined, and analyses were therefore limited to regional markers and connections between the cortex and the thalamus [138]. Moreover, the interpretations of these analyses were complicated due to the robust expression of COUP-TFI in forebrain structures that are critical for cortical development, such as the dorsal thalamus and the ganglionic eminences [338,342,343]. Emx1-Cre ) animals, instead, were viable and fertile [140]. To assess COUP-TFI role in arealization, the position of the somatosensory areas and thalamocortical axons were analyzed by Serotonin immunostaining, which revealed that each primary sensory area in COUP-TFI CKO mice are strikingly reduced in size and are aberrantly positioned at the occipital edges of the cortex. These primary sensory areas maintained their positions relative to one another along the mediolateral cortical axis, but not along the rostrocaudal axis.

COUP-TFI CKO (COUP-TFI flox/flox

Although S1 was substantially reduced in size and caudally shifted, its barrel-like pattern was still identifiable by serotonin staining, but the barrels were smaller than in control brains (Figure 20). Accordingly, layer IV neurons were caudally shifted as revealed by Nissl staining and molecular markers specific to layer 4 demonstrating that basic mechanisms sculpting barrels in S1 are not completely lost in COUP-TFI CKO, even though S1 is much smaller and ectopically positioned. But, since primary sensory areas are shifted caudally, most of the neocortex, in these mice, shows a lower density of Nissl stained cells in layer IV and a reduced expression of layer IV markers, which is a characteristic of frontal/motor areas. Finally, topographic shift of cortical areas was paralleled by a shift in thalamocortical connectivity.

In addition to this caudal shift of sensory areas, Cadherin 8 (Cadh8), which is a cell adhesion protein labeling the rostral motor domains, showed a remarkable caudal expansion covering almost the entire cortical hemisphere, with the exception of the region corresponding to the shrunken primary sensory areas (Figure 20).

Similarly, frontal markers analyses including Id2, Fezf2 and Tbr1 indicate a caudal expansion of frontal/motor areas in COUP-TFI CKOs. DiI and DiA labeling in frontal and parietal cortices showed that most projections targeted either the ventrolateral thalamic nucleus, which is normally connected only to the frontal/motor area, or the cerebral peduncle, suggesting that corticothalamic and corticofugal projection neurons of the S1 area adopted the identity of motor area PNs. In summary, the molecular, cytoarchitectural and hodological features of frontal areas expanded massively to parietal and caudal regions of the neocortex after ablation of COUP-TFI (Figure 20).

All together, these data indicate a role for COUP-TFI in specifying both layer properties that are unique for different sensory areas, and the positional information that controls development of a precise connectivity network between cortex and dorsal thalamus. Thus COUP-TFI might be involved in repressing frontal/motor area identities along its expression domains in parietal and occipital cortices [140].

Area-specific temporal control of corticospinal motor neuron

differentiation

The adult primary motor cortex contains a large number of CSMNs and has a thick layer V, while the somatosensory area is characterized by a thick layer IV, where the neurons receiving relayed sensory inputs are located [182]. CSMNs are generated at a higher rate in the developing motor cortex than in sensory areas in mice [344]. A recent study from our group showed that COUP-TFI CKO mice, in accordance with In COUP-TFI fl/fl, Emx1-Cre conditional mutant brains, the primary sensory areas are much smaller than in controls and are compressed to ectopic positions at the posterior pole of the cortical hemisphere. The barrelfield of the ectopic S1 retains its characteristic patterning but is substantially reduced in size and caudally shifted, while a reduced V1 is located medial and a reduced A1 lateral to the miniature S1 barrelfield. (c and d) In situ hybridization for Cad8 on whole mounts of P7 wild-type (+/+; Emx1-Cre) and homozygous conditional mutant (COUP-TFIfl/fl; Emx1-Cre) brains uniquely marks the frontal/motor areas (F/M). The F/M areas substantially expand following selective deletion of COUP-TFI from cortex. The reduced ectopic primary sensory areas (V1, S1) can be identified by small domains of diminished cad8 expression in posterior cortex (Taken from [START_REF] O'leary | Genetic regulation of arealization of the neocortex[END_REF]) .

previously described areal impairments, show also a F/M-like laminar organization of the parietal cortex.

Analyses of CSMN markers in the frontal and parietal region of COUP-TF1

CKO, called from now on motorized S1 (mS1), showed that Fezf2 and Ctip2 were dramatically increased in layers VI and V of the mS1 [134]. Ctip2 was strikingly increased in the upper sixth layer of the mS1, and other area specific markers known to be expressed in layer V neurons of the M1, such as Cysteine-Rich Motor Neuron 1 (Crim1), FOXP2 and insulin-like growth factor binding protein 4 (Igfbp4), were expressed by layer V neurons of the mS1 (Figure 21). Thus, in the absence of COUP-TF1 function, the number of neurons expressing high levels of F/M-specific markers in layer V and VI of the motorized S1 is dramatically increased. Correspondingly, fluorogold retrograde labeling in the cerebral peduncle showed that SCPNs, which are confined to layer V in S1, were expanded to the layer VI of mS1 where abnormally high Ctip2 expressing cells were located. Additional retrograde labeling from the spinal cord reveals that these abnormal SCPN of the motorized layer VI are able to successfully send axonal projections to more caudal targets in the cervical spinal cord. Instead, in the mS1 layer V, genuine CSMNs that

VIII. Aim of this work

As previously described, in the absence of COUP-TF1 function, in S1 cortex, layer VI neurons that would normally differentiate into CThPN, prematurely and abnormally differentiate as CSMNs and send their axons to all segmental levels of the spinal cord.

However, COUP-TF1 deficient neurons in layer V, expressing abnormally high levels of Fezf2, Ctip2, FOXP2, Tbr1, Crim1 and Igfbp4 and showing an abnormally increased number of neurons co-expressing corticothalamic and corticospinal markers FOXP2/Ctip2 and Tbr1/Ctip2 are abnormally differentiated. These genuine CSMN neurons reach the cerebral peduncle, but fail to project to the spinal cord, resulting in impaired fine motor skills in COUP-TF1 CKO adult mice [134].

The initial aim of my project was to unravel the molecular mechanisms underling the abnormal differentiation of layer V neurons in COUP-TFI mutants. As a first approach, I checked the expression of the two molecular controls regulating the differentiation of the two main populations of layer V neurons: callosal and subcerebral PNs in the S1 of WT and COUP-TFI mutant cortices. Given the increase in Fezf2 and Ctip2 expression observed in COUP-TFI CKOs, a decrease in Satb2 expression was expected. However, Satb2 expression did not decrease in COUP-TFI mutants, but was even increased in layer Vb, and more surprisingly, a remarkable increase in the number of Satb2/Ctip2 co-expressing neurons was observed in COUP-TFI mutants. I observed that a population expressing these two mutually exclusive markers, normally representing 4% of layer V neurons at P0, increases dramatically in COUP-TFI mutants to represent 18% of layer V neurons and 8% of layer V neurons.

Since Satb2/Ctip2 co-expressing neurons constitute a previously uncharacterized cell population, I aimed to investigate the molecular mechanisms allowing Satb2 and Ctip2 co-expression, and the temporal and areal distribution of these cells. Moreover, by the use of molecular markers, vital dyes, and biochemical and electrophysiological approaches I tried to unravel the specific features of Satb2/Ctip2-positive cells. Thus, overall, my work was aimed to characterize a novel and previously uncharacterized cell population at molecular, hodological, electrophysiological and morphological levels. All the reactions were done in a total volume of 10µl using 1µl (100ng-500ng) of the genomic DNA solution and 9µl of the following master mixes:

I. Mutant Mice and genotyping

II. Histological procedures:

A. Tissue processing Brains were collected from postnatal P0, P7 or P21 mice after perfusion with paraformaldehyde (PFA) 4%, in order to fix the tissue and remove as much blood as possible from the brain to avoid its intrinsic fluorescence and the phosphatases that could interfere with the histologic analysis. After dissection the brains were fixed with PFA 4% at +4°c in gentle rocking, for 2 hours, followed by 3 washes in PBS, 10' each.

B. Immunofluorescence on cryosections

After PFA fixation, brains were gradually equilibrated in PBS sucrose 10%, 20% and 30%. Sucrose traces were removed by washing the brains in Optimal Cutting Temperature (OCT), in which they were embedded and frozen. Brains were then stored at -80°C until cutting.

Samples were cut with a leica cryostat, at a thickness of 20 μm. Slices were collected on polarized slides (Thermo Scientific), and were stored at -80°C until usage.

Before immunofluorescence the slides were incubated in Unmasking Buffer (Sodium citrate 85mM, pH6.0) and boiled twice (first time for 15'' and the second time just brought to the boiling point after changing the buffer), for antigen retrieval.

The slides were cooled in ice 10' and washed 3 times 10' in PBS, before incubating them 1h at RT with the blocking solution (PBS 0.3% Triton and 10% Goat Serum or New Born Calf Serum in case one of the primary antibody is made in goat) to block all the sites that would aspecifically bind antibodies. After blocking, slides were incubated ON at +4°c with primary antibodies diluted in 200 µl/slide of PBS 0.3% Slices were washed again as previously described after primary antibodies, and mounted on slides with Vectashield medium with or without DAPI (clinisciences).

D. RNA digoxigenin probes synthesis

Plasmid containing the open reading frame (ORF) of LMO4 was linearized using a restriction enzyme (BamHI) that cut the plasmid in a single site positioned at the 5' of the ORF.

III. Imaging and Morphological analyses

Images of immunostained cryosections were taken using the LEICA DM6000 microscope, while images of immunofluorescences carried out on thick vibratome sections were taken using the Zeiss 710 confocal microscope. Images from optical and confocal microscopes were than processed using Photoshop and Zen-lite 2012, respectively. Finally, images for Whole Mount I.S.H. where acquired using a Leica Spot microscope. To compare the counting performed on different sections to analyze triple colocalizations, the counting was performed on cortical images with a constant width of 600μm.

IV. Countings

V. Molecular biology methods

A. DNA Extraction from murine tissue

To genotype animals, earmarks of P21 weaned mice were collected and used for the extraction of genomic DNA, while for sacrificed postnatal pups, DNA was extracted from the tip of the tail. Tissue lysis was performed by 2 hours incubation at +58°C, in agitation, in 500µl of lysis buffer (50mM Tris pH 7.5, 100mM EDTA pH8, 100mM

NaCl, 1% SDS, 0.2µg/µl Proteinase K). DNA was extracted with an equal volume of isopropanol added and mixed by inversion. Samples were centrifuged for 5' at 20000 g. DNA pellet was washed with 1ml of Ethanol 70%, followed by a centrifugation for 2' at 20000 g. DNA was air-dried and resuspended in an appropriate volume of H2O (70 µl for earmarks and 500 µl for tails) at 37°C with agitation for 1 hour. DNA was stored at 4°C until usage.

B. pCDK5-LMO4-IRES-GFP plasmid construction

The LMO4 ORF was amplified using available cDNA, previously synthetized in the lab starting from RNA extracted from P0 cortices, and the following primers: MluI- The mix was left 1h at 37°C, then another µl of CIP was added and the mix was left again for 1h at 37°C. Dephosphorylated plasmid was then precipitated as previously described and re-suspended in 10 µl of sterile water. The digested LMO4 ORF was then cloned in the expression vector by ligation in a 1:5 stoichiometric ratio between the plasmid (6kb) and the insert (600 bp), respectively, as follows: As a control, a mix was prepared containing solely the digested plasmid without the insert. The reaction was left ON at 16°C and then precipitated as previously explained and re-suspended in 10µl of sterile water. Two µl of the ligation were then electroporated in XL1Blue bacterial cells and the cells were re-suspended in 1 ml of LB medium and left shaking at 160 rpm for 1h at 37°C. 100µl of electroporated growing cells were then plated on LB-Agar solid medium and left ON at 37°C. The day after single colonies were picked and left growing in 5 ml LB + Ampicilline (10mg/ml dil. 1:10000) at 160 rpm ON at 37°C. These minipreps were then processed using QIAGEN Miniprep Kit (according to the manufacturer's protocol) and the extracted plasmid (pCDK5-LMO4-IRES-GFP) was sequenced to verify the presence and orientation of the insert using the following primer: pCDK5C.fw (5'-AGGACTAAACGCGTCGTGTCC-3'). Positive clones were amplified by maxiprep (in 400 ml LB + Amp) and purified using Endofree Maxiprep Kit of QIAGEN (according to manufacturer's instructions) to avoid the presence of endonucleases in the DNA used for in utero electroporations.

VI. In utero electroporations

In utero electroporations were performed as previously described in [346] with some modifications as follows:

The DNA mix to inject in embryonic brains was prepared as follows: DNA 1mg/ml, 2

l FASTGREEN (Sigma) in a total volume of 20l reached by adding sterile water.

E13.5 pregnant mice were anesthetized with Ketamine/Xylazine. After shaving and cleaning the abdomen with 70% ethanol, a 3-cm midline laparotomy was performed, and the uteri were extroflected. For DNA microinjection, 75-mm glass capillary tubes (Drummond Scientific, Broomall, PA) were used and 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 m external diameter by pinching with forceps. One microliter of DNA solution was injected into the lateral ventricle (Figure 4) using a

Femtojet microinjector (Eppendorf). The dorsal surface of the telencephalon was visible through the uterine wall by illuminating with a fiber-optics light source (Figure 4). Square electric pulses were delivered at a rate of one pulse per second to embryos through the uterus by holding them with forceps-type electrodes CUY650P3

MluI MluI

(NEPAGENE, Japan) (Figure 4), while the uterus was kept wet by dropping saline (prewarmed at 37°C) between the electrodes. Four electric pulses (37V, 50 ms each)

were delivered by using a NEPA21 electroporator (NEPAGENE, Japan). Then, the uterine horns were repositioned in the abdominal cavity. The abdominal wall and skin were sewed up with surgical sutures. Finally, operated mice were peritoneal injected with Gentamicin 10 g/ml and Ketofen 50 g/ml (Sigma) to prevent infections and decrease post-operative pains, respectively.

VII. Co-Immunoprecipitation

Co-Immunoprecipitation was performed as described in [212] with some modifications. Postnatal day one (P1) pups were anesthetized with ketamine/xylazine, perfused with cold PBS and decapitated. Cerebral cortices were dissected and nuclear proteins were purified using the NE-PER kit from Pierce (Thermo scientific) according to the manufacturer's instructions. Nuclear proteins were dialyzed against buffer D (20% glycerol, 20mM HEPES (pH=7.9), 100mM KCl, 0.2mM EDTA, 0.5mM DTT, 0.5mM PMSF, all from Sigma-Aldrich) for 2 hours, changing the buffer after 50 minutes at +4°C, in 3500 Molecular Weight Cut-Off Slyde-A-Lyzer Mini Dialysis Units (Fisher scientific). For pre-clearing, 50μg of the nuclear extracts were incubated with 100 μl Protein A Sepharose 50% bead slurry (sigma aldrish) in 400 μl of total volume with constant, slow rotation for 1 hour at 4 °C, prior to being pelleted by centrifugation at 10,000 g for 10 minutes at 4°C. The precleared nuclear extracts were then immunoprecipitated with 2 μg of anti-Satb2 antibody (mouse monoclonal, abcam) or anti-LMO4 antibody (rat monoclonal, donated by Jane Visvader lab, Australia) or a control antibody (anti-Brdu, mouse monolclonal, sigma) for 2 hours.

Immunocomplexes were captured by adding 100 μl of Protein A Sepharose bead slurry. After one additional hour of rotation at 4 °C, the bound fraction was separated by pulse centrifugation. The beads were washed three times with 1 ml of ice-cold washing buffer: 1x Phosphate Buffered Saline (PBS) containing 0.1% Igepal (sigma).

After the last wash, the beads were re-suspended in 2x loading buffer containing 0.1M

DTT and lithium dodecyl sulfate sample buffer (sigma). Samples were denatured at +99 °C for 10 minutes, centrifuged at 10,000 g for 5 minutes at room temperature, and subjected to SDS 10% polyacrylamide gel-electrophoresis using Biorad reagents and Mini-Protean tetra cell (Biorad) for protein electrophoresis. After the electrotransfer (Trans-Blot ® SD Semi-Dry Transfer Cell, Biorad), proteins were identified by immunoblotting with the following antibodies: anti HDAC1 (rabbit polyclonal, Millipore, 1/500), anti-Ski (rabbit polyclonal, santa cruz biotechnology, 1/100)), anti-Brdu (mouse monoclonal, sigma (1/500)), anti-Satb2 (mouse monoclonal, abcam, 1/80), anti LMO4 (rat monoclonal, donated by Jane Vasvader lab, Australia (1/500)) and anti-β-actin (rabbit polyclonal, abcam, 1/500). Biotinylated secondary antibodies (anti-rabbit biotinylated, Vector (1/500), and anti-mouse biotinylated, Vector (1/500) a,d anti-rat biotinylated, Vector (1/500)) were then added and followed by the ABC kit (Vector laboratories). Immunoblots were visualized using SuperSignal West Pico Chemiluminescent Kit (Thermo scientific) with LAS 3000.

VIII. Chromatin immunoprecipiation

Cortices from P1 controls and COUP-TFI CKO mutant mice (4 pups for each genotype) were dissected in HBSS, washed with DMEM, centrifuged at 500g for 1 min and fixed in PFA 1% for 10'. Cortices were then washed twice in 10 ml chilled Cell Wash buffer (20mM HEPES pH7.4, 150mM NaCl, 125mM Glycine, PMSF 0.2 mg/ml).

Samples were lysed in a lysis buffer (20mM HEPES pH7.4, 1mM EDTA, 150mM

NaCl, 1%SDS, 125mM Glycine, PMSF 0.2 mg/ml).

Tissues were homogenized and membranes were fractured to obtain nuclei using a glass potter connected to an electric drill until homogenate becomes clear.

Homogenized tissues were then resuspended in sonication buffer (20mM HEPES pH7.4, 1mM EDTA, 150mM NaCl, 0.4% SDS, PMSF 0.2mg/ml) in a total volume of 1200 μl, and then divided into 4 aliquots for each genotype and sonicated 6 times for The PCR products were separated on 2% agarose and bands were visualized with SYBR-Safe DNA stain (Invitrogen) and a FUJI 3000 LAS intelligent dark box equipped with a CCD camera.

IX. Retrograde labeling

For retrograde labeling, mice pups were anesthetized by hypothermia at postnatal day 2 (P2). Pups were placed on a stereotaxic apparatus and callosal projecting neurons in the somatosensory cortex were retrogradely labeled via 92 nl injections of Alexa

Fluor 488-conjugated cholera toxin subunit B (1 mg/ml; Invitrogen). Coordinates (in mm) were: AP: _+1.2; and ML: 1.3 from the lambda; DV: _0.2 from the pial surface.

Subcerebral injections, into the pons or spinal cord, were performed under ultrasound guidance (Vevo 660, VisualSonics), via 92 nl injections of Alexa Fluor 555conjugated cholera toxin subunit B (1 mg/ml; Invitrogen). Mice were perfused with 4% formaldehyde and brains were collected at P7.

For retrograde labeling of corticospinal motor neurons (CSMN), corticospinal tracts (CST) of deeply anesthetized postnatal day 2 (P2) pups were visualized using a Vevo 770 ultrasound backscatter microscopy system (Visual Sonics) at cervical vertebral level 1 (C1) to C2. 92 nl injections of Alexa Fluor 555-conjugated cholera toxin subunit B (1 mg/ml; Invitrogen) were injected bilaterally into the CST using a Drummond Nanoject II. Mice were perfused and brains were collected at P7.

For retrograde labeling of corticobrainstem motor neurons (CBMN), descending subcerebral projection tracts of deeply anesthetized P2 pups were visualized using a Vevo 770 ultrasound backscatter microscopy system (Visual Sonics) at the midbrain-hindbrain junction. 92 nl injections of Alexa Fluor 555conjugated cholera toxin subunit B (1 mg/ml; Invitrogen) were deposited bilaterally into the CST using a Drummond Nanoject II. Mice were perfused and brains were collected at P7.

Dual retrograde labeling of CBMN and CPN was performed with Red

Retrobeads or Green IX Retrobeads (Lumafluor Inc) respectively at P2 and P3 as described for single retrograde labeling. Pups were perfused and brains were collected at P7.

X. Electrophysiology on acute slices

Thy1-eYFP-H mice (23-28 days) were anesthetized with isoflurane, and decapitated.

The dissected brain was vibratome cut to prepare 300 μm-thick coronal slices. The slices were recovered in a holding chamber for 1 h at 37°C in oxygenated artificial

XI. Molecular and morphological analysis of recorded cells

Clamped cells were filled with biocytin through the recording pipette and fixed 4 hours in 4% PFA. To visualize labeled cells and recognize their molecular code, acute brain slices were rinsed in PBS, and incubated ON in PBS with 0.3% Triton-X, 3%

BSA and 10% goat serum at +4°C. Then, the slices were incubated in primary antibodies diluted in PBS with 3% goat serum, 3% BSA and 0.3% triton ON at +4°C. Used primary antibodies were anti-Satb2 (1:80) mouse (abcam) and anti Ctip2 (1:500) rabbit (abcam). Slices were washed many times for 1 hour each, and biocitin was detected with Texas Red Avidin D (1:500; Vector Laboratories), whereas Satb2 and Ctip2 primary antibodies were detected by the use of Alexafluor anti-mouse far red 633 (life technologies) and anti-rabbit blue 350 (life technologies) secondary fluorescent antibodies (1:300). Avidin and secondary antibodies were diluted as in the same solution used for primary antibodies ON at +4°C. Immunolabeled slices were mounted on polarized slides (Thermo Scientific) using Vectashield without DAPI (clinisciences). Images were taken using a zeiss 710 confocal microscope, and colocalization of biocytin filled cells with Satb2 and Ctip2 was analyzed using zen lite 2012 software.

XII. Electrophysiological analysis

Electrophysiological analyses were done as in Hattox et al, 2007 [347]:

We plotted the relationship between the amount of injected current and the firing frequency. To calculate the initial slope of the firing current (FI) ratio, we analyzed responses of cells to threshold currents injected to generating action potentials and their responses to two times the threshold current. The difference in the number of action potentials in the two traces was divided by the difference in current levels. To capture the slower phase of spike frequency adaptation, which may represent a biologically separate process from fast adaptation, we defined the adaptation ratio as the ratio between the third interspike interval and the last one. This adaptation ratio was analyzed using responses recorded injected two times the threshold current. To B. Satb2/Ctip2-positive neurons are more represented in the motor/frontal area than in the somatosensory region.

I. Characterization of a neocortical population of double Satb2/Ctip2-expressing neurons in normal and COUP-TFI-deficient cortices

In order to understand whether double Satb2/Ctip2-positive neurons are only restricted to somatosensory cortex, I checked the distribution of Satb2 and Ctip2 proteins in the motor area of P0 cortices, a region normally characterized by high levels of Ctip2 [134] (Figure 2A, C andE). Interestingly, the percentage of double Satb2/Ctip2-positive cells is higher in layer V of M1 than S1 of control brains, representing 8% of counted cells (compared to 4± 0.3% in the S1), whereas in layer VI the number of double positive neurons remains nearly unaltered between M1 and S1 areas (0.4% versus 0.35± 0.01%, respectively) (Figure 1A'', B'' and G, Figure 2 A'', B'' and Figure 3 B). Notably, even if double-positive cells are increased in the motor area of COUP-TFI mutant cortices compared to controls, they represent only 12% and 2% of layer V and VI neurons, respectively, thus a 1.5-fold increase in layer V (compared to a 4.5-fold increase in S1) (Figure 2 C'', D'' andG). These data indicate that the population of Satb2/Ctip2 positive cells is normally higher in M1 than in S1 area at P0. This trend is inverted in the absence of COUP-TFI function, the percentage of Satb2/Ctip2 positive cells in the motor area of COUP-TFI CKO is lower than in the "motorized" somatosensory area [134] (Figure 3D) indicating a difference between the "genuine" motor area and the expanded one (the "motorized" somatosensory area (mS1)). Thus, the number of double Satb2/Ctip2-positive cells varies region specifically between M1 and S1 at P0, suggesting differential expression of genes involved in their specification between motor and somatosensory areas (Figure 3D).

C. Tangential and radial increase of LMO4 expression in COUP-TFI CKO cortices

To identify the gene(s) involved in the specification of these double Satb2/Ctip2expressing cells, which are more represented in motor than S1 regions in control brains, I checked for candidate genes characterized by differential expression between motor and S1 areas, which might abnormally increase in COUP-TFI-deficient S1 cortex. LMO4 turned out to be a good candidate since it is a region-specific gene described to be expressed in rostral/motor and caudal/visual regions of P0 cortices, but absent in the somatosensory region (Figure 4 A) [325]. It is also known to play a role in patterning the somatosensory barrel field [146,325] At P0, upper layer neurons have not completed their migration, whereas lower layer PN are fully settled, even if they are at an intermediate stage of their differentiation in which axons have not fully reached their final targets [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]. At this stage of development, LMO4 is normally expressed at low levels in layer V of the somatosensory area, and at slightly higher levels in layer VI (Figures 4 B and5 A).

In the absence of COUP-TFI, LMO4 expression levels increase in all cortical layers, particularly in layer V and part of upper layers (Bins 3, express LMO4 and no obvious differences could be observed between controls and COUP-TFI mutant mice (Figure 5E andF). Similarly, the amount of Satb2/Ctip2positive cells in controls becomes progressively more comparable to COUP-TFI CKO, even if their number is still higher in the mutants (in layer V, 8.56±1.9% in the controls, versus 17.2±1.48% in the CKO (P=0.01), in layer VI, 24.7±0.9% in the

II. A novel molecular mechanism regulating Ctip2 expression via chromatin remodeling

A. The working hypothesis: LMO4 might interfere with the deacetylation process on the Ctip2 locus Previous reports have shown that Satb2 binds to the Ctip2 upstream promoter region, allowing the assembly of the NURD chromatin remodeling complex on the Ctip2 locus, so that HDAC1 can deacetylate the histones at this locus converting the chromatin from an active to an inactivate state, and therefore inhibiting Ctip2 expression [150,212]. LMO proteins function essentially as transcriptional coregulators, mediating protein-protein interactions of various transcription factors or chromatin remodeling proteins [318][319][320][321][322]. LMO4 is also known to bind to HDACs and to MTA1 to modulate the expression of different genes [323,324]; in particular, it can bind and sequester HDAC2 and allow expression of its target gene [324].

My previous data show a good correlation between the strong increase of LMO4 expression and the increase of Satb2/Ctip2 positive cells in layer V of the mS1. Therefore, I hypothesized that in the absence of COUP-TFI at P0, ectopically expressed LMO4 protein in layer V might bind to HDAC1, and possibly subtracts it from the interaction with Satb2. This would interfere with normal deacetylation of histones at the Ctip2 locus and de-repress Ctip2 expression (Figure 7). This mechanism could explain the increased number of double Satb2/Ctip2-positive cells in COUP-TFI mutant brains (Figure 1) in which I observed a higher number of LMO4-expressing neurons (in layer V, 60±4.4% in COUP-TFI CKO versus 15±2.4% in controls (P<0.001), in layer VI, 32±2.3% in COUP-TFI CKO versus 26±1.57% in controls (P=0.06) and in lower part of upper layers 51±5.3% in COUP-TFI CKO versus 10±2.8% in controls (P<0.001)) (Figure 4).

D. LMO4 de-represses Ctip2 transcription by binding HDAC1

In order to directly assess whether LMO4 can bind to HDAC1 and eventually subtract it from Satb2, as proposed by my hypothesis (Figure 7), I immunoprecipitated nuclear proteins isolated form whole P1 cortices, using antibodies against LMO4 and Satb2. I choosed P1 cortices, because at this stage a drastic difference in the percentage of double labeled Satb2/Ctip2 positive cells exists between COUP-TFI mutants and controls. Immunoblots using HDAC1 antibody showed that in controls Satb2 binds HDAC1, as previously described [150,212]. I also found that LMO4 can interact with HDAC1 in normal conditions (Figure 10A). Interestingly, the interaction between LMO4 and HDAC1 drastically increases, whereas the Satb2/HDAC1 interaction strongly decreases in COUP-TFI mutant cortical extracts (Figure 10A), indicating that excess of LMO4 has subtracted part of the HDAC1 normally interacting with Satb2.

To further investigate whether this interaction affected the deacetylation of the (A) LMO4/HDAC1 interaction increases while Satb2/HDAC1 interaction decreases in COUP-TFI CKO compared to controls. Nuclear extracts from P0 controls and COUP-TFI CKO cortices were immunoprecipitated using either anti-LMO4 or anti-Satb2 or a control (Brdu) antibody. Following immunoprecipiation, bound and free fractions were separated on gels, and analyzed by immunoblotting using specific antibody to HDAC1 for bound fraction and β-actin as a control for free action.

Molecular weight is shown in kDa on the right side.

E. LMO4 binds to Ski: another component of the NuRD complex

Ski is another partner of Satb2, recruiting the histone deacetylases to the Ctip2 locus, and involved in Ctip2 inhibition at P0 [234]. In order to investigate whether LMO4 could bind to Ski, I immunoprecipitated nuclear proteins isolated form P1 cortices with antibodies against LMO4 and Satb2, as described above. Then, I immunoblotted the precipitated complexes with an antibody against Ski and found that, as for HDAC1, Satb2 interacts less with Ski in COUP-TFI CKO cortex extracts (Figure 11 A). On the contrary, the interaction of LMO4 with Ski remains unchanged (Figure 11 A). These data suggest that recruitement of HDAC1 to Ctip2 locus is impaired due to an absence of Ski in this locus.

Since Ski interaction was impaired, I checked whether Ski expression levels and distribution was altered in COUP-TFI CKO brains by comparing them to controls at P7, since my previous analyses showed that the highest percentage of Satb2/Ctip2 positive cells in lower layers is detected at this stage (Figure 6). Although Ski Sun [325]. I first used the LMO4 antibody on coronal sections of these brains and detected no LMO4 expression in the neocortex at P0, while LMO4 is still expressed in the striatum and dorsal thalamus, confirming the specificity of this cortex-specific KO (Figure 12 A and B). To assess whether absence of LMO4 would impinge on the number of the double Satb2/Ctip2-expressing neurons, double immunofluorescence of Satb2 and Ctip2 was performed on P7 LMO4 CKO brains, a stage in which a high number of Satb2/Ctip2-and Lmo4 positive cells can be normally detected in the S1 compared to P0 brains (see Figure 5 and6). In the absence of LMO4 function, the number of Ctip2-positive cells in layers V and VI of S1 was decreased (in layer V, 14.6±2.6% in controls versus 9.4±0.01% in LMO4 CKO, in layer VI, 14.7±2% in controls versus 2.7±0.8% in LMO4 CKO (P=0.01) (Figure 12 C-I), whereas the number of Satb2-positive cells was not increased, but even decreased in some bins indicating that Ctip2 inhibition is not due to an increase in Satb2 expression (Figure 12 C'-H' and J). Interestingly, the number of double Satb2/Ctip2-positive cells was also significantly decreased compared to controls (in layer V, 8.1±1.9% in controls and 4.3±0.35% in LMO4 CKO, in layer VI, 8.17±1.9% in controls and 1±0.3% in LMO4 CKO, P=0.05) (Figure 12 C''-H'' andK). These data further support a key role for LMO4 in the de-repression of Ctip2 expression and therefore in the specification of Satb2/Ctip2 positive cells. However, I noticed that E13.5 Cdk5-GFP electroporated cells did not localize with any Ctip2+ cells (Figure 13 G-I'''), even if they are supposed to be born at E13.5 [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]. This might suggest, that apical progenitors, which are the only recipient of the electroporated plasmid are not producing Ctip2+ cells anymore. This unexpected observation support the evidence that LMO4 overexpression can de-repress Ctip2 expression in Satb2 positive cells. Hence, our data further support that the enhancement of LMO4 expression from P0 to P21 progressively increases the number of Satb2/Ctip2 positive cells in lower layers.

C. LMO4 downregulation rescues Ctip2 radial expansion in COUP-TFI CKO mutant cortices

To reinforce gain-of-function data I tested whether cell-autonomous downregulation of LMO4 could repress Ctip2 expression in layer V. To this purpose, we used a construct expressing an already tested LMO4-speicfic shRNA which was kindly donated to us from HH Chen's lab [330]. We used COUP-TFI CKO brains in which Ctip2 expression is strongly up-regulated in lower layers. We in utero electroporated the shRNA plasmid in E13.5 mutant brains together with a GFP-expressing construct that allows distinguishing transfected cells. The efficacy and specificity of the shRNA construct was tested using an antibody against LMO4 on P0 COUP-TFI CKO brains electroporated either with the shRNA against LMO4 or with a "scrambled" shRNA, used as a control. The LMO4-specific shRNA strongly downregulates LMO4 expression in the mS1 area (Figure 14 A-A Interestingly, while the totality of labeled CSMN expresses Ctip2, they never coexpress Satb2 and Ctip2 (Figure 17 D-E'''). These data suggest that double Satb2/Ctip2-positive neurons project either through the corpus callosum to the contralateral hemisphere, or to the brainstem, or to both.

To distinguish between these different possibilities, we co-injected CTBcoated beads in the pons and in S1 of wt pups at P2 and P3. In collected brains at P7, I found no co-labeling in layer V neurons (Figure 17 Retrograde labeling showed that Satb2/Ctip2 co-expressing neurons are corticobrainstem but not corticospinal PN. However, my data showed that 70% of Satb2/Ctip2 double labeled cells are also Bhlhb5 positive. It has been reported that Bhlhb5 is mainly expressed in CSMN and involved in their specification, whereas LMO4 is involved in CBMN specification and excluded from CSMN [144,145]. In In order to associate to the two sub-populations of Satb2/Ctip2 co-expressing neurons, the analyzed molecular markers, immunofluorescence against LMO4, Bhlhb5, Sox5 and Er81 were performed in retrogradely double labeled callosal and SCPN coronal sections of P7 wt cortices taken in S1. LMO4 was expressed by both callosal and subcerebral PNs (Figure 19 A-C'''). However, Er81 was expressed only by CPNs (Figure 19 D-F'''). On the other hand, Sox5 and Bhlhb5 were expressed exclusively by SCPN, while they were both excluded from layer V callosal neurons (Figure 20). Thus, association of retrograde labeling with subtype specific genes confirmed that the two sub-populations of Satb2/Ctip2 co-expressing neurons express specifically distinct molecular markers, with callosal sub-population expressing LMO4 and Er81, and corticobrainstem cell population expressing Sox5 and Bhlhb5.

C. Morphological analysis confirms differences between single and double Satb2/Ctip2-positive layer V neurons

Morphological diversity has been observed between several subtypes of layer V neurons in different species [347,351,352]. Thus, to further investigate whether double Satb2/Ctip2-expressing neurons could also exhibit morphological differences with respect to other layer V subpopulations (single Ctip2 or Satb2 positive neurons).

I used a transgenic line expressing the reporter gene YFP under the Thy1 promoter [345]. In this line, YFP is expressed mainly in the soma, dendrites and axons of layer Vb cortical neurons from P14 onwards [353]. Immunostaining for Satb2 and Ctip2

was thus performed on P21 YFP-positive coronal sections, and confocal images were taken in the somatosensory area at different magnifications to highlight any morphological features in the soma shape, apical and secondary dendrites of layer V neurons (Figure 21A).

Morphological analysis of the soma shape of layer V YFP-positive neurons reveals differences in soma size, geometrical morphology, distribution of secondary dendrites, and angles between these dendrites. Thanks to these differences and to the immunoreactivity for Satb2 and Ctip2, I classified YFP-positive neurons into three main shapes: double Satb2/Ctip2 co-expressing neurons (shape I), single Ctip2 (shape II) and single Satb2 (shape III) positive neurons (Figure 21 A-G).

A deeper analysis of the soma shape and distribution of dendrites was performed using the Sholl analysis, which is a method of quantitative analysis commonly used to analyze the morphological characteristics of neurons [354] (Figure 22 A). To perform this analysis I used a freely available plug-in based on the ImageJ software that automatically creates a series of concentric circles around the center of a neuronal arbor (dendritic or axonal), and quantifies how many times the arbor intersects the circumference of these circles (Figure 22 We found that at their resting membrane potentials, all cell subtypes fail to fire spontaneously.

However, in response to depolarizing current injections, Satb2/Ctip2 type 2 and single Ctip2 type 1 fire an initial doublet, whereas Satb2/Ctip2 (type 1) fire a quadruplet and single Ctip2 type 2 a triplet (Figure 24 E-H). This initial firing is followed by a particularly long, large-amplitude AHP (After Hyperpolarization) (Figure 24 J), while DAP (Depolarizing afterhyperpolarization) is higher in Satb2/Ctip2 (type 1) compared to the other three sub-populations (Figure 24 J). After this phase of adaptation, the neurons fall into regular trains of action potentials (Figure 24 E'-H

I. Satb2/Ctip2 co-expression: not a transient state but a permanent cell population.

The impressive ability of the neocortex to process information relies on the accurate establishment of neuronal connections. The expression of Satb2 or Ctip2 in cortical neurons triggers mutually exclusive genetic programs leading to their final connectivity [150,212,355]. Ctip2 imprints a corticofugal fate to neocortical projection neurons and is involved in the fasciculation and appropriate targeting of their axons [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. In contrast, Satb2 is a key determinant for callosal projection neurons, since its deletion results in the absence of the corpus callosum, misrouting of callosal axons towards the internal capsule and spinal cord, and ectopic upregulation of Ctip2 in presumptive callosal neurons [150,212].

It was previously stated that the refinement of subtype identity is a progressive mechanism. While mature lower-layer neurons exhibit strikingly specific patterns of gene expression and axonal projection, newly postmitotic neurons often extensively co-express transcription factors that later become restricted to different subtypes and temporarily innervate aspecific targets [150,203,217,229,333,356]. For instance, between E12.4 and E14.5, neurons in the cortical plate co-express high levels of Ctip2 and TBR1/FOG2, which later resolve into SCPN and CThPN respectively. Similarly, at E13.5, lower layer neurons briefly co-express Ctip2 and Satb2, which become over time restricted to SCPN and CPN respectively [150].

On the contrary my data show that the co-expression of two transcription factors, Satb2 and Ctip2, normally restricted to callosal versus subcerebral PNs respectively, does not represent a transient state lasting only the few days needed for post-mitotic refinement. It represents, indeed, a definitive and permanent molecular feature maintained after final differentiation and pruning events (at P21), therefore defining a cell population showing a specific morphology, connectivity and electrical activity.

II.

Satb2/Ctip2 co-expressing neurons: a previously uncharacterized cell population.

It is well reported that Satb2 normally represses Ctip2 expression in the cerebral cortex, leading to a sharp distinction between callosal and sub-cerebral projection neuron subtypes [150,212]. Nevertheless, different reports briefly mentioned the presence of a small number of double Ctip2/Satb2 co-expressing neurons in lower layers [150,212,234,260], in which Satb2 seemed to be expressed at very low levels [212]. It was also stated that a few Satb2-positive neurons failed to downregulate Ctip2 in deep cortical layers [260], however these Satb2 positive cells were never seen projecting to the spinal cord [212]. The authors proposed that these Satb2/Ctip2 positive cells were lacking Ski expression necessary to inhibit Ctip2, and that these Satb2+ /Ctip2+/Ski-cells were most likely non-callosal neurons [234,260].

Beside these minor comments in the above-mentioned papers, to my knowledge, no other studies have thoroughly described this population of double Satb2/Cip2 co-expressing neurons. Differently from what was reported in these previous studies, my data reveal a non-irrelevant population of neurons co-expressing both Satb2 and Ctip2 at high levels in lower layers, and a key role for LMO4 in this process. I provide a molecular mechanism responsible for the de-repression of Ctip2, which allows Satb2 and Ctip2 co-expression in a sub-population of lower layer neurons. Moreover, I characterized major features of this previously uncharacterized cell population, including its connectivity, electrophysiological and morphological characteristics, as well as their specific molecular profile.

III. Ectopic and precocious expression of LMO4 in COUP-TFI CKO.

A. LMO4 allows Ctip2 expression in Satb2-positive cells of COUP-TFI CKO brains

LMO4 is normally highly expressed in frontal and occipital regions of wt cortices at P0, while it is expressed only at low levels in layers V and VI of the parietal cortex (Figure 4 A). This expression increases with time to reach high levels at P7 (Figure 5). In the absence of COUP-TFI function, LMO4 expression is prematurely increased in all cortical layers of the motorized S1 (Figure 4 [START_REF] Molyneaux | Fezl is required for the birth and specification of corticospinal motor neurons[END_REF][START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]213]. Thus, it is very plausible that the strong up-regulation of Ctip2 in lower layers of COUP-TFI mutant brains (Figure 1 C and E) might be due not only to the ectopic expression of LMO4, which primarily accounts for the increase of Satb2/Ctip2 coexpression, but also to the up-regulation of Fezf2 expression in layers V and VI. This would also explain the lack of Ctip2 expression in the upper layers of COUP-TFI CKO although high levels of Lmo4 in the S1 area (Figure 9).

My triple immunofluorescence data revealed that LMO4 is normally expressed in 68±1.7% of double Satb2/Ctip2 at P0, and in 92±0.4% of Satb2/Ctip2positive cells in layer V neurons of COUP-TFI mutants (Figure 9 F and G). This confirms that the presence of LMO4 in the majority of layer V Satb2/Ctip2-positive cells is required in the specification of this neuronal population. However, LMO4 is absent in 32±1.7% of Satb2/Ctip2-positive cells in controls, and in 8±0.4% of them in COUP-TFI CKO. Thus, it is possible that another mechanism, independent from LMO4, is responsible for the specification of this complementary percentage, particularly in controls, whereas this complementary mechanism seems to have a minor effect in COUP-TFI CKO brains.

Contrary to LMO4, Ski expression is required for Ctip2 inhibition by Satb2.

Ski is expressed in 57.7% of Satb2/Ctip2 positive cells in layer V and absent in 42.3%

of them (Figure 11 E). Thus, it is likely that in double-positive neurons where LMO4

is not expressed, absence of Ski fails to inhibit Ctip2 transcription, leading thus to Satb2 and Ctip2 co-localization. In the cells where both, LMO4 and Ski are expressed, presence of Ski is not sufficient to interfere with LMO4 function and derepress Ctip2. In summary, at least two independent epigenetic mechanisms might exist in the Ctip2 transcriptional control, which is dependent on the delicate balance between Ski and LMO4.

B. LMO4 binds to HDAC1 and de-represses Ctip2

In physiologic conditions, LMO4 interacts with several kinds of proteins, and modulate transcriptional activity of target genes. LMO4 was found to bind in vivo to the ERα and recruit the member of the NuRD complex MTA1 and HDACs leading to transcriptional repression of ERα target genes [323]. Moreover, LMO4 can also associate with HDAC2, sequester it and thus enhance the activity of Stat3 in mouse cortical neurons [324].

In this study, I showed that LMO4 binds to HDAC1 and prevents it to interact with Satb2, leading to a decrease in the levels of HDAC1 bound to the Ctip2 locus and to an increased histone "acetylation", which maintains the locus in an active transcriptional state (Figure 10). Thus, LMO4 competes with Satb2 in the transcriptional control of Ctip2 allowing the co-expression of Satb2 and Ctip2 in lower layer neurons. At P0, LMO4 is expressed only in few layer V neurons and this would explain why only 4 ± 0.3% of layer V neurons co-expresses Satb2 and Ctip2 at this stage. However, in COUP-TFI CKO, LMO4 is prematurely and ectopically expressed at high levels in lower layers, allowing a high and efficient de-repression of Ctip2 and giving rise to a higher percentage of double-labeled cells (18±1.8% of layer V and 8±0.5% of layer VI neurons) (Figure 1).

Ski was also identified as a Satb2 partner, which recruits HDAC1 at the Ctip2 locus enabling the formation of the NuRD complex and Ctip2 transcriptional repression. My data revealed that LMO4 not only binds and subtract HDAC1 from

Satb2, but that it also binds to Ski (Figure 11 A). Satb2 interaction with these two molecules decreases in the presence of high levels of LMO4, such as in the case of COUP-TFI CKO brains. Therefore, LMO4 perturbs the formation of the NuRD complex at Ctip2 locus. My data show that Satb2 and LMO4 proteins fail to interact either in controls or in COUP-TFI mutants (Figure 11 A). Since, Satb2 still remains bound to Ctip2 upstream regulatory regions in the absence of interaction with Ski and HDAC1[234], the action of LMO4 might happen before the NuRD complex assembles at the Ctip2 locus, and not on the locus itself, further explaining the decrease of Satb2 interactions with HDAC1 and Ski. Therefore, LMO4 does not only perturb the action of the NuRD complex, but it even disturbs its assembly on the Ctip2 locus.

My co-immunoprecipiation data showed also that, even though LMO4 subtracts HDAC1 from Satb2, LMO4 interaction with Ski does not appreciably change between controls and mutants, as for HDAC1 (Figure 11 A). This may be explained by the strong decrease in Ski expression in upper layers of COUP-TFI mutants (Figure 11 B-B''). Our nuclear extracts, indeed, derive from the whole cortex and not only from lower layers. Thus, although the interaction with LMO4 increases, Ski levels decrease in upper layers of COUP-TFI CKO brains, so that its interaction with LMO4 is apparently unchanged, while that with Satb2 is strongly decreased.

IV. Areal and temporal distribution of Satb2/Ctip2 positive neurons in S1 cortex

Previous reports [325] and this study have shown that LMO4 has a regional and temporal specific expression pattern in the developing neocortex being highly expressed in the motor and visual areas at P0 (Figure 5). I have showed that the distribution of Satb2/Ctip2 co-expressing neurons and that of Lmo4-expressing follow a similar trend (Figure 6). Indeed, the percentage of Satb2/Ctip2 co-expressing neurons in layer V of the motor area represents twice the percentage observed in S1

(Figures 2 and3). Interestingly, both LMO4 expression and the percentage of Satb2/Ctip2-positive neurons progressively increase in layers V and VI of S1 after P0

(Figures 5 and6). My triple immunofluorescence LMO4/Satb2/Ctip2 at P0 and P7

(Figures 9 and 16 A-B''' and I) directly proof that LMO4 is normally expressed in the majority of double Satb2/Ctip2-expressing neurons over time. Moreover, my gainand loss-of-function experiments in which we directly manipulated LMO4 expression in S1 (see below) strongly support this evidence.

LMO4 Gain-and loss-of-function A.

The majority, but not the totality of Satb2/Ctip2 co-expressing neurons 1. decrease in the absence of LMO4 in layer V, unraveling a complementary compensatory mechanism My data show that Ctip2 expression strongly decreases in layers V and VI in S1 of LMO4 CKO cortices (Figure 12 F and I). Even more interestingly, the percentage of double Satb2/Ctip2 co-expressing neurons drastically decreases in layers V (1.9 fold decrease) and VI (8 fold decrease) of LMO4 mutants (Figure 12 F'' and K).

Furthermore, although the total population of Satb2/Ctip2-positive cells showed a 3.5fold decrease, Satb2 expression did not increase and even slightly decreased in some bins of LMO4 CKO cortices (Figure 12 F' andJ). This confirms that the lack of Satb2/Ctip2-positive cells in mutant cortices is due to a higher repression of Ctip2 in Satb2 positive cells rather than to a mis-specification of subcerebral Ctip2-positive PNs.

However, despite the absence of LMO4 (in LMO4 CKO), the number of Satb2/Ctip2 co-expressing neurons shows only 1.9 fold decrease in layer V (Figure 12 F'' andK). This implies, as previously mentioned, the presence of a complementary mechanism, which is independent from LMO4 and also required in specifying the Satb2/Ctip2 co-expressing population. Given this relatively high remaining percentage of Satb2/Ctip2 co-expressing neurons in LMO4 mutants, it would be interesting to verify whether Fezf2 and Ski expression levels, which may underlie complementary mechanisms, are changed.

In contrast to layer V, layer VI Satb2/Ctip2 co-expressing neurons were absent in P0 wt cortices, whereas at P7 they were around around 8.2 ±2.5% of the total layer VI neuronal population (Figure 6). Only 1±0.3% of layer VI neurons Satb2/Ctip2-positive remained in layer VI of LMO4 mutants (Figure 12 F'' and K), suggesting that the complementary mechanism favoring Satb2/Ctip2 co-expression in layer V is less active in layer VI. Therefore, I propose that the specification of layer VI Satb2/Ctip2 co-expressing neurons, which appear at P7 together with the increase in LMO4 expression, is primarily dependent on LMO4.

V.

Two distinct cell populations differing in their axonal projection, molecular code, morphological and electrophysiological properties Since the double Satb2/Ctip2-expressing population was not characterized so far, we decided to further investigate its properties in terms of connectivity, molecular code, morphology and electrophysiology. Our comprehensive studies, which included connectivity, electrophysiological, molecular and preliminary morphological analyses, brought to similar conclusions: Satb2/Ctip2 co-expressing cells do not constitute a temporary population of neurons with hybrid callosal/subcerebral features and can be subdivided at least in two distinct sub-populations. One major subpopulation with subcerebral characteristics, projecting to the pons and mainly expressing Bhlhb5 and Sox5 in addition to LMO4 and another minor sub-population, mainly expressing LMO4 and Er81, but not Bhlhb5 and Sox5, and projecting across the corpus callosum to the contralateral hemisphere.

A. Molecular Characterization

The analysis of regulatory genes expressed in layer V PNs (i.e. LMO4, Bhlhb5, Sox5 and Er81), based on subtype specific expression patterns and functional roles, allowed me to subdivide Satb2/Ctip2-positive cells into two sub-populations. Bhlhb5 is strongly expressed in CSMN of sensorimotor cortex and corticotectal PN of the occipital cortex [144]. LMO4 is highly expressed in CBMN in layer Va of the motor cortex, in CPN, in backward projection neurons and in dual callosal or subcerebral/backward projection neurons, however, it is completely excluded from CSMNs [145]. The transcription factor Sox5 is expressed by the major classes of CFuPNs (subplate, corticothalamic and all subcerebral subtypes) but not by corticocortical CPNs, and it controls the sequential generation of these subtypes by its including Satb2/Ctip2 positive cortico-brainstem neurons (Figure 18). Second, Bhlhb5 confers cortico-spinal identity [144], whereas in the absence of LMO4 function, SCPN in layer Va of the rostral motor cortex project to the spinal cord instead of the brainstem [145]. Thus, present data support previous evidence and suggest a scenario in which LMO4 determines cortico-brainstem fate in the somatosensory cortex by either limiting SCPN axonal pathfinding to the brainstem or favoring the pruning of aspecific connectivity to the spinal cord.

Moreover, layer V PNs can also send their axons and/or collaterals to the striatum [204]. Corticostriatal PNs can be of intratelencephalic (IT) type sending their axons to the contralateral striatum, or pyramidal tract (PT) type sending their axons to the ipsilateral striatum. However, corticostriatal PNs can be either IT or PT but never both [204]. Thus, it is not plausible that Satb2/Ctip2 co-expressing neurons, even though they co-express callosal and subcerebral specific genes, project to both targets.

Whether they do also project to the striatum is still under investigation in the lab.

The choice of Satb2/Ctip2 co-expressing neurons to send axonal projections to the brainstem or to the corpus callosum depends probably on the expression of other neuronal identity modulators. The expression of Er81 versus Sox5 and Bhlhb5 could be a potential molecular mechanism, since Er81 expression seems to be exclusive to LMO4 establishes subtypes projection neurons diversity within the rostral motor cortex. In the absence of LMO4 function, the molecular identity of subcerebral and callosal projection neurons in the rostral motor cortex is affected and projection neurons loose their specific connectivity [145]. My molecular analysis shows that LMO4 is expressed in almost all Satb2/Ctip2 co-expressing neurons at P7. Moreover, immunostaining on retrogradely-labeled callosal and subcerebral projection neurons revealed the presence of LMO4 in both of them. Thus, it seems plausible that, rather than directly contribute to the subdivision of Satb2/Ctip2-positive cells, Lmo4 performs two important tasks: it promotes layer V CPN and CBPN identity at the expenses of CSMN identity, and creates the right conditions for the expression of other regulators (such as Er81, Sox5, and Bhlhb5), which likely contribute to the specification of Satb2/Ctip2 sub-types.

Overall, these data indicate that LMO4, in cooperation with other transcriptional regulators, segregates Satb2/Ctip2 positive neurons into two distinct sub-populations, which might regulate axon guidance molecules to direct their axonal projections toward the corpus callosum or the brainstem.

C. Electrophysiology

Physiologically, two major classes of pyramidal neurons, based on differences in their intrinsic firing properties, have been described in layer V: intrinsically bursting neurons that fire bursts of action potentials in response to depolarizing current injection, and regular spiking cells that fire trains of single action potentials [359,360]. However, analyses of additional parameters, including spike frequency adaptation and afterpolarizations [361][362][363][364], have revealed a rich diversity of layer V pyramidal cells.

Previous findings have shown that layer V neurons in the somatosensory cortex projecting to different targets, have distinct physiological properties. Intrinsic membrane and firing properties of cortico-thalamic and cortico-trigeminal neurons differ from those of callosal and cortico-striatal neurons [347]. Similarly, corticobrainstem and cortico-spinal projection neurons can be distinguished by their specific physiological properties [365][366][367], indicating that cortical neurons with different subcortical targets are electrically distinct from each other.

Our electrophysiological studies suggest that the Satb2/Ctip2 type 2 cells are most likely the callosal sub-population of Satb2/Ctip2 co-expressing neurons (Figure 24). This sub-population has very different firing and membrane properties, most importantly a much larger adaptation, compared to the other three subpopulations: the double Satb2/Ctip2 type 1 cells, and two other two populations expressing only Ctip2 (single Ctip2 type 1, and single Ctip2 type 2). These latter two populations show higher similarities and, since my analysis showed no single Ctip2-positive cells projecting to the corpus callosum, they may all be constituted by SCPN. Thus, the Satb2/Ctip2 type 1 neurons, which show similar electrical features, could correspond to the sub-cerebral projecting sub-population of Satb2/Ctip2-positive cells.

Nonetheless, physiological differences exist also among these 3 sub-populations (Figure 24), which is very plausible since they project to different targets. To ultimately confirm the identity of these four cell populations and distinguish the callosal from the subcerebral sub-population Satb2/Ctip2-positive cells, I will use the molecular factors described above.

The differences in intrinsic firing properties observed in distinct subtypes of cortical projection neurons may reflect differences in the readout of cortical activity that they transmit to their targets. Cortical excitatory neurons provide massive recurrent excitation onto other excitatory neurons, therefore amplifying their feedforward signals [368,369]. In addition to inputs from inhibitory neurons, strong spike frequency adaptation of cortical excitatory neurons, including callosal neurons, may be essential for stabilizing the network and preventing runaway excitation.

Instead, little spike frequency adaptation observed in subcerebral projection neurons, which is also a hallmark of cortico-spinal neurons, have been suggested to be important for maintaining rhythmic firing in these cells [370,371], which is required for such long-range targets (brainstem and spinal cord).

D. Morphology

Layer V pyramidal neuron populations projecting to different targets show distinct morphological features. Differences in the length and thickness of the apical dendrite, the laminar position of their bifurcation points, and width of apical tuft were found between callosal, cortico-thalamic, cortico-trigeminal and cortico-striatal layer V neurons in the somatosensory cortex [347], indicating that morphological differences most probably have an effect on the function of a neuron. Interestingly, morphology and electrophysiological properties are very tightly linked parameters. For instance, intrinsically bursting cells have thick apical dendrites, whereas regular spiking cells have thinner apical dendrites [372]. Moreover, membrane capacitance of a neuron is proportional to its soma area and architecture [373].

My analysis associates morphological and electrophysiological differences of layer V neurons to their molecular code, which is a very novel and relevant analysis that will further help understanding PNs diversity within the cerebral cortex. Our morphological data, although preliminary, showed differences in soma shape between Satb2/Ctip2 co-expressing neurons, and single Ctip2 or Satb2 positive neurons, including soma area and distribution of secondary dendrites, angles between the secondary dendrites, and sphericity of the soma (Figures 21 and22). Moreover, we found some clear differences in apical dendrite morphology (Figure 23) between different populations and sub-populations of neurons within each molecular code, including apical dendrite bifurcation point, thickness, length, and apical tuft. All these data are in phase of elaboration by Morpheme mathematicians, which are creating algorithms to allow automatic detection of morphological differences and correlate them with the observed molecular code. Preliminary analysis subdivided Satb2/Ctip2 co-expressing neurons into two main populations further confirming data obtained from retrograde labeling, molecular markers and electrophysiological analyses.

These morphological traits will help in revealing differences in the electrophysiological activity and function of the these neurons: associating these morphological differences with retrograde labeling, analysis of molecular markers and biocytin reconstructions will further help in grouping the characteristics of these two sub-populations of Satb2/Ctip2 co-expressing neurons.

In conclusion, this work has characterized a previously unknown cell population, coexpressing mutually exclusive callosal versus subcerebral transcriptional regulators, and analysed its molecular, hodological, morphological and electrophysiological properties. Importantly, my study demonstrates that the co-expression of callosal and subcerebral neuronal markers does not only characterize early phases of neuronal specification, as previously described, but also defines distinct mature neuronal subpopulations with different areal-specific features and developmental timing in the mammalian neocortex.

Satb2/Ctip2 co-expressing neurons have a precise regional and temporal distribution in layer V and VI of the somatosensory and motor cortex. At postnatal day 0 (P0), these neurons are just a minor population in the somatosensory cortex, while they are highly represented in the motor cortex. From P0 onwards, Satb2/Ctip2 co-expressing neurons progressively increase in the somatosensory cortex together with the increase of LMO4, a transcriptional regulator that I demonstrated to be strongly involved in the specification of this cell population.

Satb2 inhibits Ctip2 by recruiting the chromatin remodeling NuRD complex to Ctip2 locus and, thus, allowing HDAC1 to deacetylate nearby histones and turn the chromatin to an inactive state. My data demonstrate that LMO4 is a main actor in the development of Satb2/Ctip2 co-expressing neurons and acts by de-repressing Ctip2 expression in Satb2-positive neurons. LMO4 binds and sequesters HDAC1 and Ski, another member of the NuRD complex, perturbing the assembly of this machinery on the Ctip2 locus.

Beside the biochemical approach, I have been able to confirm the key role of LMO4 in specifying layer V Satb2/Ctip2 co-expressing neurons in vivo by manipulating LMO4 expression levels starting from E13.5 old embryos. While downregulation of LMO4 diminishes the number of double Satb2/Ctip2-positive neurons in somatosensory cortex, overexpression of LMO4 derepresses Ctip2 expression in Satb2-positive cells, which normally do not co-express these two factors. In addition, in LMO4 mutant mice, both the percentage of Ctip2 and that of Satb2/Ctip2 co-expressing neurons are decreased, and conversely in COUP-TFI mutant cortices, in which LMO4 is prematurely expressed in S1, the specification of Satb2/Ctip2 co-expressing neurons is anticipated and their number is increased.

Since these double Satb2/Ctip2 co-expressing neurons were not described so far, I used several complementary approaches, including double and triple immunostaining with a battery of molecular markers specific to different layer V PN populations, retrograde labeling combined with molecular marker analysis, and morphological and electrophysiological analyses to characterize their cellular and molecular properties. My data clearly show that this "hybrid" population coexpressing Satb2 and Ctip2 gives rise to two distinct PN sub-populations. One population with callosal properties, projecting through the corpus callosum to the contralateral hemisphere and expressing LMO4 and Er81, and the other with subcerebral properties, projecting to the brainstem and expressing LMO4, Sox5 and Bhlhb5.

Finally, electrophysiological and morphological analyses confirmed the existence of two sub-populations of Satb2/Ctip2 co-expressing neurons with different membrane and firing properties, and distinct morphologies, including differences in soma shape, distribution of secondary dendrites and apical dendrite characteristics.

I. To combine the morphological and electrophysiological properties with molecular markers characterizing the two subpopulations of Satb2/Ctip2 co-expressing neurons

To finalize the characterization of the double Satb2/Ctip2 co-expressing neurons I first need to combine the use of molecular markers that allow me to distinguish the callosal sub-population from the subcerebral one, with the morphological and electrophysiological data. In particular, I need to apply the automatized morphometric algorithm, as soon as it will be available, to subdivide the different morphologies identified among YFP-positive layer V neurons and biocytin-injected recorded neurons in specific classes. Then, I will cross these data with molecular and hodological ones to identify the different sub-types of Satb2/Ctip2 co-expressing neurons present in layer V of the S1.

In addition, applying retrograde labeling on the Thy1-YFP brains, which we have used for the morphological and electrophysiological studies, could be useful to properly integrate our multidisciplinary approach and couple the two different subpopulations with their specific morphologies and electrophysiological properties. In this way, I could repeat the immunostaining of the different molecular markers (LMO4, Bhlhb5, Er81 and Sox5) on YFP brains and directly correlate the morphologically different two sub-populations of Satb2/Ctip2 co-expressing neurons to their specific molecular code.

II.

To directly investigate the role of LMO4 on the subtype diversity and axonal projections of cortico-brainstem projection neurons By retrograde labeling LMO4 CKO brains, or alternatively, LMO4 shRNA in utero electroporated brains, I could evaluate whether LMO4 ablation would affect the connectivity of the 2 identified sub-populations of Satb2/Ctip2 co-expressing neurons.

Similarly, retrograde labeling experiments on LMO4 GFP-overexpressing brains, or brains electroporated with LMO4 shRNA, or GFP-electroporated LMO4 CKO brains at E13.5 would be required to evaluate whether LMO4 really limits axonal projection at the level of the pons. Alternatively, electroporation of a GFP-expressing construct in different cortical regions of the gain-and loss-of-function models would help to follow the trajectories of GFP-positive axon and identify final targets of the two major subclasses of Satb2/Ctip2 co-expressing neurons.

Finally, further chromatin immunoprecipitation and co-immunoprecipitation experiments on nuclear and cytoplasmic targets would be required in order to better understand the whole molecular mechanism of Ctip2 de-repression driven by LMO4.

III. Further molecular and functional characterization of Satb2/Ctip2 co-expressing neurons

FACS sorting of Satb2/Ctip2 co-expressing neurons, and RNA extraction from sorted cells would help to identify specific genes highly expressed by the Satb2/Ctip2-positive cells. This may help in identifying the axon guidance molecules and understanding how they direct the two pathways, callosal versus subcerebral, of the two main sub-populations of Satb2/Ctip2 co-expressing neurons. This would be an interesting additional step in our understanding of how different cortical neuron subpopulations reach their final targets during corticogenesis. Moreover, RNAseq on sorted cells could help to discover new important markers of layer V neurons, which could help in the classification of this neuronal population.

More functional and electrophysiological characterization of the Satb2/Ctip2 co-expressing neurons would be required to understand how this cell population accounts for the overall behaviour and the organization of functional circuits in the mammalian brain. Creating mutant mice with a substantial loss or an over-production of Satb2/Ctip2 positive cells would be an interesting and indispensable approach to this aim. Indeed, although LMO4 and COUP-TFI mutants show, respectively, a decrease and a strong increase in the number of Satb2/Ctip2-posotive cells, their phenotypes involve many different neuronal subtypes impeding a clear functional or behavioural analysis. On the contrary, the intersectional approach would be a powerful instrument, in future, to ablate Satb2 and Ctip2 expression only in cells coexpressing these genes and to analyse the impact of their impaired specification on mice behaviour.

Figure 1 -

 1 Figure 1-Development of the mammalian forebrain. ........................................................ Figure 2-Sensory modalities reach the cerebral cortex through different thalamic nuclei. ..............................................................................................................................................Figure 3-Neocortical projection neurons are generated in an 'inside-out' fashion by diverse progenitor types in the VZ and SVZ. .......................................................... Figure 4-Diverse populations of excitatory neuron progenitor cells in the mouse neocortex. ...................................................................................................................................... Figure 5-Neocortical glia-guided radial migration. ........................................................... Figure 6-Graphical representations of the two main hypotheses on the mechanisms underlying formation of cortical areas. .............................................. Figure 7-Patterning centers and graded transcription factors drive arealization of the neocortex. ........................................................................................................................ Figure 8-Summary of Intrinsic Genetic Mechanisms of Area Patterning and Mutant Phenotypes. ................................................................................................................. Figure 9-Major Subtypes of Projection Neurons Within The Neocortex.................. Figure 10-Projection neuron diversity in the neocortex. ............................................... Figure 11-Long-range axonal projections define two classes of Corticostriatal neurons. ......................................................................................................................................... Figure12-Competing molecular programmes direct differentiation of newly postmitotic projection neurons into one of three broad subtype identities. Figure13-Satb2 protein interacts with both the Ctip2 promoter upstream region and histone deacetylase complex and controls chromatin remodeling. ........ Figure14-Ski associates with Satb2 and represses Ctip2 transcription in cortical neurons. ......................................................................................................................................... Figure 15-Satb2-and Ctip2-dependent establishment of cortical connections. .Figure 16-Key events in corticospinal tract guidance. ..................................................... Figure 17-Schematic representations of the human LMOs and LDB1....................... Figure 18-Schematic representations of LMOs in gene transcriptional regulation. ............................................................................................................................................................ Figure 19-Mechanisms underlying cortical thickening, expansion and lamination. ............................................................................................................................................................ Figure 20-Massive expansion of the Frontal motor area and posterior compression of primary sensory areas in COUP-TFI deficient cortex............. Figure 21-Increased expression of molecular hallmarks of CSMN in corticofugal neurons of S1 cortex in COUP-TFI CKO mice. ..............................................................

  Figure 1-Development of the mammalian forebrain. ........................................................ Figure 2-Sensory modalities reach the cerebral cortex through different thalamic nuclei. ..............................................................................................................................................Figure 3-Neocortical projection neurons are generated in an 'inside-out' fashion by diverse progenitor types in the VZ and SVZ. .......................................................... Figure 4-Diverse populations of excitatory neuron progenitor cells in the mouse neocortex. ...................................................................................................................................... Figure 5-Neocortical glia-guided radial migration. ........................................................... Figure 6-Graphical representations of the two main hypotheses on the mechanisms underlying formation of cortical areas. .............................................. Figure 7-Patterning centers and graded transcription factors drive arealization of the neocortex. ........................................................................................................................ Figure 8-Summary of Intrinsic Genetic Mechanisms of Area Patterning and Mutant Phenotypes. ................................................................................................................. Figure 9-Major Subtypes of Projection Neurons Within The Neocortex.................. Figure 10-Projection neuron diversity in the neocortex. ............................................... Figure 11-Long-range axonal projections define two classes of Corticostriatal neurons. ......................................................................................................................................... Figure12-Competing molecular programmes direct differentiation of newly postmitotic projection neurons into one of three broad subtype identities. Figure13-Satb2 protein interacts with both the Ctip2 promoter upstream region and histone deacetylase complex and controls chromatin remodeling. ........ Figure14-Ski associates with Satb2 and represses Ctip2 transcription in cortical neurons. ......................................................................................................................................... Figure 15-Satb2-and Ctip2-dependent establishment of cortical connections. .Figure 16-Key events in corticospinal tract guidance. ..................................................... Figure 17-Schematic representations of the human LMOs and LDB1....................... Figure 18-Schematic representations of LMOs in gene transcriptional regulation. ............................................................................................................................................................ Figure 19-Mechanisms underlying cortical thickening, expansion and lamination. ............................................................................................................................................................ Figure 20-Massive expansion of the Frontal motor area and posterior compression of primary sensory areas in COUP-TFI deficient cortex............. Figure 21-Increased expression of molecular hallmarks of CSMN in corticofugal neurons of S1 cortex in COUP-TFI CKO mice. ..............................................................

Figure 16 -

 16 Figure 1-Development of the mammalian forebrain. ........................................................ Figure 2-Sensory modalities reach the cerebral cortex through different thalamic nuclei. ..............................................................................................................................................Figure 3-Neocortical projection neurons are generated in an 'inside-out' fashion by diverse progenitor types in the VZ and SVZ. .......................................................... Figure 4-Diverse populations of excitatory neuron progenitor cells in the mouse neocortex. ...................................................................................................................................... Figure 5-Neocortical glia-guided radial migration. ........................................................... Figure 6-Graphical representations of the two main hypotheses on the mechanisms underlying formation of cortical areas. .............................................. Figure 7-Patterning centers and graded transcription factors drive arealization of the neocortex. ........................................................................................................................ Figure 8-Summary of Intrinsic Genetic Mechanisms of Area Patterning and Mutant Phenotypes. ................................................................................................................. Figure 9-Major Subtypes of Projection Neurons Within The Neocortex.................. Figure 10-Projection neuron diversity in the neocortex. ............................................... Figure 11-Long-range axonal projections define two classes of Corticostriatal neurons. ......................................................................................................................................... Figure12-Competing molecular programmes direct differentiation of newly postmitotic projection neurons into one of three broad subtype identities. Figure13-Satb2 protein interacts with both the Ctip2 promoter upstream region and histone deacetylase complex and controls chromatin remodeling. ........ Figure14-Ski associates with Satb2 and represses Ctip2 transcription in cortical neurons. ......................................................................................................................................... Figure 15-Satb2-and Ctip2-dependent establishment of cortical connections. .Figure 16-Key events in corticospinal tract guidance. ..................................................... Figure 17-Schematic representations of the human LMOs and LDB1....................... Figure 18-Schematic representations of LMOs in gene transcriptional regulation. ............................................................................................................................................................ Figure 19-Mechanisms underlying cortical thickening, expansion and lamination. ............................................................................................................................................................ Figure 20-Massive expansion of the Frontal motor area and posterior compression of primary sensory areas in COUP-TFI deficient cortex............. Figure 21-Increased expression of molecular hallmarks of CSMN in corticofugal neurons of S1 cortex in COUP-TFI CKO mice. ..............................................................

Figure 1 -

 1 Figure 1-Development of the mammalian forebrain. (A) At early somite stage (E8.5 for mouse; Carnegie Stage 10 (CS10) for human), the neural ectoderm has been specified into different regions along the anterior-posterior axis and the axial mesoderm is underlying the midline of the neural ectoderm. ANC, anterior notochord; PFB, prospective forebrain (or ANR: Anterior neural ridge); PrCP: prechordal plate; PH, prospective hindbrain; PM, prospective midbrain; PNC, posterior notochord; PSC, prospective spinal chord. (B) Neural tube closure occurs at around the 15-somite stage (E9.0 for mouse; CS11 for human). The forebrain gets further regionalized into telencephalon, diencephalon, and prospective hypothalamus (PH). OV, optic vesicle. (C) Approximately at E10.5 in the mouse or at CS14 in human embryos, the expanding telencephalon bifurcates dorsally to form the two hemispheres and gets patterned into dorsal telencephalon (DT) and ventral telencephalon (VT). (D and F) Lateral views of adult mouse (D) and human brain (F). OB, olfactory bulb. Black dashed lines in D and F indicate the location of coronal sections shown in E and G. (E and G) Coronal sections of adult mouse (E) and human brain (G). BG, basal ganglia; CiC, cingulate cortex; CoC, corpus callosum; LV, lateral ventricle (modified from[START_REF] Geng | Pathogenesis of holoprosencephaly[END_REF]) .

Figure 2 -

 2 Figure 2-Sensory modalities reach the cerebral cortex through different thalamic nuclei.

Figure 3 -

 3 Figure 3-Neocortical projection neurons are generated in an 'inside-out' fashion by diverse progenitor types in the VZ and SVZ.

Figure 4 -

 4 Figure 4-Diverse populations of excitatory neuron progenitor cells in the mouse neocortex.During early brain development, neuroepithelial cells (NECs) are the major neural progenitors in the neocortex. NECs divide symmetrically to generate additional NECs, some of which give rise to the first group of neurons (N) through asymmetric division. As the developing brain epithelium thickens, NECs elongate and transit to radial glial cells (RGCs). RGCs can also divide symmetrically to expand the progenitor pool or asymmetrically to generate neurons either directly or indirectly through intermediate progenitor cells (IPCs), which generate neurons directly through symmetric division in the subventricular zone. It remains unclear how short neural precursors (SNPs) are generated. It is possible that they are generated from RGCs or they may be a distinct population originated directly from NECs. Most SNPs produce postmitotic neurons directly in the ventricular zone (VZ). Outer subventricular zone (OSVZ) progenitors likely originate from RGCs through oblique division, which leads to loss of the apical process and ascension of the nucleus toward the cortical plate/intermediate zone (CP/IZ). The minor population of OSVZ progenitors can also generate neurons through asymmetric division outside of the VZ

Figure 5 -

 5 Figure 5-Neocortical glia-guided radial migration. New neurons generated from radial progenitors undergo a transient multipolar migratory state in the SVZ and IZ prior to adopting a bipolar morphology and radial glial-guided migration to the cortical plate where they differentiate (modified from [73]).

Figure 6 -

 6 Figure 6-Graphical representations of the two main hypotheses on the mechanisms underlying formation of cortical areas.A: The "protomap hypothesis" is mainly based on two models: the cooperative concentration model and the radial unit model. Various overlapping gradients of TFs and morphogens (represented in different colors) act synergistically and in a dose-dependent manner to define distinct neocortical domains. These signals control the specification of neuronal progenitors, which are committed to a given, areal-specific, neurogenetic program. Successively, these neuronal progenitors transfer both areal commitment and positional information to their offspring, giving rise to the peculiar cytoarchitecture and functional features of a given area. B: The "protocortex hypothesis" states that the neocortex is like a tabula rasa until the arrival of TCA from distinct thalamic nuclei: the dorsolateral geniculate (dLG), the ventropostero medial (VPM), the VL. Abbreviations: F/M: frontal-motor areas; S1: primary somatosensory area; V1: primary visual area; A1: primary auditory area; Ncx: neocortex; RGC: radial glia cells (taken from[START_REF] Alfano | Neocortical arealization: evolution, mechanisms, and open questions[END_REF]).

Figure 7 -

 7 Figure 7-Patterning centers and graded transcription factors drive arealization of the neocortex.

Figure 8 -

 8 Figure 8-Summary of Intrinsic Genetic Mechanisms of Area Patterning and Mutant Phenotypes.

  (A) Graded expression of Emx2, Pax6, Coup-TFI, and Sp8 along anterior-posterior and lateral-medial axes. Key TFs for cortical area patterning show distinct graded expression patterns along anterior-posterior (A, P) and lateralmedial (L, M) axes. Emx2 is expressed in a high P-M to low A-L gradient. Pax6 expression pattern is opposite to that of Emx2, with a high A-L to low P-M gradient. Coup-TFI has a high P-L to low A-M gradient. Sp8 is expressed in a high A-M to low P-L gradient. While the expression of Emx2, Pax6, and Coup-TFI is sustained in the VZ, Sp8 expression is quickly downregulated around the onset of cortical neurogenesis. (B) In the anterior signaling center, Fgf8 establishes the low anterior-graded expression of the TFs Emx2 and COUP-TFI by repression, and promotes the high anterior gradient of Sp8 expression. Fgf8 expression is also regulated positively by direct transcriptional activation by Sp8 through its binding to Fgf8 regulatory elements, and indirectly by Emx2, which represses the ability of Sp8 to directly induce Fgf8. The asterisk marking the activation of Fgf8 by Sp8 indicates the only interaction that has been shown to be due to direct binding and transcriptional activation. Putative posterior signaling molecules Bmps and Wnts, expressed in the cortical hem, positively regulate the high caudal gradient of Emx2 expression. Genetic interactions between TFs also participate in the establishment of their graded expression. For example, Emx2 and Pax6 mutually suppress each other's expression, Coup-TFI suppresses Pax6 expression and enhances Emx2 expression, and Sp8 suppresses Emx2 expression.

  promotes rostral and dorsomedial cortical development by repressing COUP-TFI and promoting Fgf signaling in pallial progenitors [110]. Pax6 2.

  Within the broad class of neocortical PNs, many subtypes exist with distinct connectivity, soma laminar location and gene expression patterns. Classified by hodology, there are three basic subtypes of cortical PNs: Commissural callosal PNs (CPN), Associative PNs and corticofugal PNs (CFuPN)

Figure 9 -

 9 Figure 9-Major Subtypes of Projection Neurons Within The Neocortex.

Figure 10 -

 10 Figure 10-Projection neuron diversity in the neocortex.

(

  CThPNs are closely related subtypes of CFuPN, residing in deep layers of the neocortex and are sequentially generated early in corticogenesis. Substantial plasticity exists in the specification of these two subtypes, and in the absence of critical molecular controls, each of them can expand at the expense of the other by Arlotta et al[START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF] have led to the identification of genes controlling SCPN specification, in particular CSMN, by purifying CSMN at distinct stages of development, and comparing their gene expression to two other neuronal populations: callosal and corticotectal PNs. This work identified Ctip2, Fezf2, SRYbox-containing gene 5 (Sox5), Orthodenticle homeobox 1 (Otx1), Ets-Related Protein

Figure 11 -

 11 Figure 11-Long-range axonal projections define two classes of Corticostriatal neurons. a | pyramidal tract (PT) neurons (blue) project to ipsilateral striatum, thalamus, subthalamic nucleus (STN) and many brainstem and spinal cord regions. PT neurons also project to contralateral brainstem and spinal cord. Intratelencephalic (IT) neurons project ipsi-or bilaterally (via corpus callosum) within the cerebral hemispheres to cortex (IT-C-Cort orange), and many IT neurons also project to striatum (IT CStr; red. The ipsilateral striatum (black) is unique in receiving CStr input from both IT and PT neurons. Layer 6 (L6) corticothalamic neurons (CT; green) project subcortically only to thalamus and its reticular nucleus (RTN). b | retrogradely labeled spinally projecting PT neurons and callosaly projecting IT-CStr neurons in mouse motor cortex. IT (green) and PT (orange) neurons are intermingled in L5B, but are not double labeled. c | Single PT neuron's axon is multiprojectional sending branches to many subcortical areas (blue) (taken from [194]).

Figure 12 -

 12 Figure 12-Competing molecular programmes direct differentiation of newly postmitotic projection neurons into one of three broad subtype identities.a | The subtype identities of postmitotic projection neurons are depicted within a theoretical n-dimensional 'subtype space' in which individual subtype identities (as defined by gene expression, morphology, dendritic structure, projection patterns, physiology and other characteristics) occupy distinct coordinates. Boundaries between these identities, which prevent neurons of one subtype from taking on characteristics of another subtype, are established by the action of cross-repressive molecular controls. One boundary exists between neurons specified as subcerebral projection neurons (SCPN) and those specified as corticothalamic projection neurons (CThPN), and another exists between corticofugal projection neurons (CFuPN) (SCPN and/or CThPN) and callosal projection neurons (CPN). Early in corticogenesis, undifferentiated neurons have largely overlapping subtype identities (top). As development proceeds, neurons differentiate and subtypes become more distinct from each other (bottom). b | Known molecular controls form key nodes of an elaborate transcriptional network, which is only beginning to be elucidated (top). Arrows indicate known cases of genetic or transcriptional activation or repression, and further interactions and molecular controls remain to be identified (bottom). 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 well-defined 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-box containing protein 5; TBR1, T-box brain protein 1 (taken from[START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]).

[

  211]. Ctip2 is not expressed in layer V of Fezf2 null mutant mice and it can rescue the axonal phenotype of Fezf2 mutants [211].

  spiny neurons (MSN), and the organization of their patch matrix, which is traversed by CSMN axons. Ctip2 controls also the expression of a set of axons guidance signals by MSN, suggesting an additional non-cell autonomous function for Ctip2 in CSMN axon growth and fasciculation [216]. All these data identified Ctip2 as a crucial regulator of SCPN axon extension and their collaterals refinement. Although Ctip2 activation by Fezf2 is critical for SCPN development, several transcriptional controls over CPN, CThPN and subplate development, including Satb2 [150, 212], Sox5 [166] and COUP-TFI [134], operate at least in part by repressing Ctip2 expression, suggesting that Ctip2 is a critical target for transcriptional regulation during the development of neocortical PNs. TF belonging to the SRY-box-containing gene family. Sox5 is expressed at high levels in layers V, VI and subplate in all CFuPNs, and is is lacking in almost all CPNs [166, 217]. Sox5 controls the sequential generation of CFuPN subtypes, including subplate cells, CThPNs and SCPNs, by repressing high level expression of SCPN genes, such as Fezf2 and Ctip2, until the generation of subplate and CThPN is concluded, and preventing premature generation of SCPN during early stages of corticogenesis [166]. It has been shown that Sox5 directly represses Fezf2 expression by binding to an enhancer element required for Fezf2 expression in the forebrain [218]. To support its role in specifying CFuPN identity, ectopic Sox5 expression in upper layer neurons prevented them from extending axons across the corpus callosum, and stimulated the extension of corticofugal axons [166]. Sox5 loss-of-function causes a striking overlap among the three principal CFuPN subtypes. In Sox5 null cortices, subplate neurons aberrantly develop molecular hallmarks and connectivity of SCPN, while SCPN axons show pathfinding defects, including extensive defasciculation in the midbrain and formation of an accessory subcerebral tract that projects through the external capsule, suggesting that Sox5 is crucial to control SCPN timing of generation, and connectivity [166, 217].(4) Otx1: A gene controlling the refinement and pruning of SCPN Another key TF known to function in the choice of final SCPN targets is Otx1, which is a homeodomain TF, expressed in putative deep-layer progenitors in the VZ, and exhibiting a decreasing level of expression in the VZ during the generation of superficial-layer neurons [63, 219]. As deep-layer PN mature, Otx1 localization shifts from the cytoplasm to the nucleus [219, 220]. Post-natally, Otx1 is expressed by 40-50% of layer V SCPN primarily within the visual cortex, while it is completely absent from CPN [219]. In the absence of Otx1 function, defects in corticotectal PN development are observed. Corticotectal PNs maintain collaterals to the spinal cord and caudal pontine nuclei, which are normally pruned during late stages of their differentiation [219]. This indicates that Otx1 has a later role in SCPN development than Fezf2 and Ctip2, possibly in controlling the refinement and pruning of axonal collaterals.

  exerts different functions in distinct neuronal populations in a dose-dependent manner. Moreover, in the absence of Fezf2 function, the expression of the T-box brain protein 1 (Tbr1), a transcription factor crucial for CThPN development, expands into presumptive layer V, and many of these Tbr1-positive cells project to the thalamus, (whereas others are converted to CPNs as discussed above). Thus, Fezf2 specifies SCPN identity at least in part by repressing CThPN fate. Sox5 regulates the differentiation of the three CFuPN subtypes; it is normally expressed by layer V SCPN, layer VI CThPN and subplate neurons and seems to control the timing of generation of layer V SCPNs by repressing Ctip2 in layer VI and subplate [166, 217]. It was hypothesized that Sox5 acts in combination with different expression levels of Tbr1 and Ctip2 in controlling the specific identities of layer VI and subplate neurons [166]. In Sox5 null mice FOG family member 2 (Fog2) and Ctip2, respectively expressed by CThPNs and SCPNs, are co-expressed by a single population of neurons with mixed SCPN and CThPN features (indicating imprecise differentiation), whereas corticothalamic projections are severely compromised [166, 217]. Another Key TF having a key role in the molecular mechanisms regulating the development of layer VI CThPNs and subplate neurons is Tbr1. T-box TF, highly expressed in subplate and layer VI postmitotic neurons during early corticogenesis [221]. Tbr1 expression is highest in the rostral cortex, but it seems to specify layer VI neurons along the whole cortex [221]. Genetic manipulations have shown that Tbr1 is necessary for the differentiation of layer VI and preplate neurons. In Tbr1 null mice, indeed, the subplate is morphologically indiscernible, subplate specific genes fail to be expressed and CTAs are absent. Moreover, layer VI neurons that would normally develop into CThPNs express aberrantly high levels of Fezf2 and Ctip2, and other SCPN specific genes, and they project to the spinal cord, [143, 221-224]. In addition, Tbr1 misexpression in layer V neurons suppresses Fezf2 expression and prevented layer V neurons from extending axons to subcerebral targets [223, 224]. Thus, Tbr1 gene acts in opposition to Fezf2 and Ctip2 to specify CThPN identity. In support of this, Tbr1 directly binds to Fezf2 locus and represses its activity in layer VI CThPNs to restrict the birth of the corticospinal projections to layer V. Hence, Tbr1 functions, at least in part, by preventing SCPN specification in layer VI [223, 224]. c) Callosal PN fate specification: CFuPNs share a sort of developmental boundary with CPNs, since a subpopulation of CPNs resides in deep layers (V and VI), which are generated during the same temporal window. From the time CFuPN and CPN axons exit the cortical plate, they follow dramatically divergent trajectories, one away from the cortex and the other toward the midline [39, 225]. Thus, some critical controls over CFuPN and CPN development largely function by repressing molecular programs that would instruct the alternative fates. Because the majority of CPN reside in superficial layers, the first known molecular controls over CPN generation and development were laminar specific genes.

  spinal cord and brainstem[150, 229]. Moreover, absence of Satb2 function leads to a loss or a severe reduction of genes characteristic to CPN, including Cdh10, Dkk3, Sip1 and Cux1. In contrast, upper layer neurons in these mice express high levels of Ctip2, and a number of other genes typical ofSCPN, including Clim1, Cdh13 and Grb14 [150, 212]. Furthermore, ectopic Satb2 expression markedly reduces the number of Ctip2-expressing cells and alters the projections of deep layer neurons[150, 212]. These data showed that Satb2 is required for CPN specification by repressing Ctip2 expression.It is known that Satb TFs regulate gene expression by binding AT-rich sequences of matrix attachment regions (MARs), promoting higher-order chromatin organization and facilitating long range interactions between enhancers and promoters[150, 212, 227, 228,[230][231][232]. Chromatin ImmunoPrecipitation (ChIP) experiments demonstrated that Satb2 protein binds to the upstream regulatory region of Ctip2, both in vitro and in vivo. Co-ImmunoPrecipitation (CoIP) experiments revealed that Satb2 interacts with the members of the Nucleosome Remodeling Deacetylase (NURD) complex, such as Histone Deacetylase 1 (HDAC1, belonging to a class of enzymes involved in deacetylation of hyperacetylated histone tails leading to compaction of protein and transcriptional repression[233]) and the Metastasis-Associated 1 family member 2 (MTA2), in the developing cortex. Moreover, Satb2 deletion leads to the hyperacetylation of the Ctip2 locus and reduces HDAC1 and MTA2 levels at this locus (Figure13). Thus, Satb2 is required to recruit the NURD complex to the Ctip2 locus. The NURD complex in turn deacetylates histones in the vicinity, converting the chromatin to an inactivate state. This indicates that Satb2 downregulates Ctip2 expression via the assembly of a NURD chromatin-remodeling complex at the Ctip2 locus[212, 232].Moreover, the transcription co-regulator sarcoma viral oncogene homologue (Ski) has been shown to be a critical component of the repressor complex recruited by Satb2 to initiate HDAC1-dependent chromatin remodeling[234]. Ski protein is expressed in distinct subtypes of neocortical progenitor cells from E10.5 and in projection neurons. In upper layers, Ski and Satb2 are largely co-expressed, while in deep layers the percentage of Ski-positive cells that express Satb2 is lower.

Figure 13 -

 13 Figure 13-Satb2 protein interacts with both the Ctip2 promoter upstream region and histone deacetylase complex and controls chromatin remodeling. Model of Satb2 function in the cortical lamination. Satb2 is required to assemble NURD chromatin remodeling complex on Ctip2 locus. This induces deacetylation of histones and inactivation of Ctip2 expression (modified from [212]).

  stages of CPN development (before E18.5 in mouse), such as Inhba, Btg1, Frmd4b, Epha3 and Ptn, likely act during neuronal subtype specification, differentiation, migration or initial axonal extension. On the other hand, they suggested that genes with expression that rises and falls at mid-stages of CPN development such as Cpne4, Tmtc4, Nnmt, Cav1, Nectin-3 and Chn2, might function at the time when CPN have already crossed the midline, and are extending toward specific targets. Finally, they reported that genes expressed in late CPN development, such as Plexin-D1, Gfra2, TcrB and Dkk3, might function in final stages of CPN maturation and their axonal refinement during adulthood[START_REF] Molyneaux | Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons[END_REF]. In addition to a temporal classification, this work identified CPN genes specific to most CPN in all layers including Lpl, Hspb3, and Cited2, and others discriminating between lower layers CPN, including Plexin D1, Gfra2, Tcrb and Dkk3, and upper layers including Inhba, Limch1, Cpne4, Tmtc4 and Btg1[START_REF] Molyneaux | Novel subtype-specific genes identify distinct subpopulations of callosal projection neurons[END_REF].

Figure 14 -

 14 Figure 14-Ski associates with Satb2 and represses Ctip2 transcription in cortical neurons.Model for Ski function at the Ctip2 locus in callosal projection neurons. Ski is required to assemble a functional NuRD repressor complex containing Satb2, MTA2, and HDAC1 at MAR sites in the Ctip2 locus. In the absence of Ski, Satb2 still binds the regulatory DNA sequences together with MTA2, but recruitment of HDAC1 is impaired. In the absence of Satb2, the NuRD complex is not assembled. Thus, Satb2 and Ski play specific roles in the formation of a functional NuRD complex, and individual loss of these factors prevents transcriptional repression of Ctip2 in callosal projection neurons. Modified from[234].

(

  KO) animals revealed Cux2 function in SVZ formation, as it promotes cell cycle exit of SVZ cells [237]. Moreover, Cux1 and Cux2 also regulate dendrite branching, spine development and synapse formation specifically in layer II/III CPNs [237]. Since most CPNs lie in superficial cortical layers, Brn and Cux genes could be involved in controlling their generation.

Figure 15 -

 15 Figure 15-Satb2-and Ctip2-dependent establishment of cortical connections.

  Unc5C and DCC act as receptors for the secreted ligand Netrin1, mediating either a chemorepulsive (Unc5C-DCC together) or a chemoattractive (DCC alone) response[261][262][263][264]. While Unc5C expression was downregulated in satb2 mutants, DCC expression was upregulated[260]. This study showed that the negative regulation of Unc5C and DCC by Ctip2 and Satb2, respectively, mediates a differential response of neocortical axons to Netrin1. Using Satb2 and Ctip2 compound and double mutants, and by overexpression and down-regulation by in utero electroporation, the authors revealed the roles of Unc5C and DCC in corpus callosum formation. High levels of Unc5C expression, and low levels of DCC expression, instruct neurons to project through the corpus callosum, since in the absence of either Unc5C or Netrin1, callosal axons misproject to subcortical targets mimicking the effect of Satb2 deletion. In contrast, inactivation of Ctip2 or DCC, or the restoration of Unc5C in Satb2 mutants can partially restore the corpus callosum formation. Moreover, DCC-positive cingulate axons act as pioneers for the corpus callosum. The authors suggested a scenario where the source of Netrin1 is the internal capsule, which attracts Unc5C-negative/DCC positive axons while repelling Unc5C positive/DCC negative axons. Hence, callosal projecting deep layer neurons require higher levels of Unc5C and lower levels of DCC, whereas low levels of Unc5C and high levels of DCC instruct neurons to project subcortically (Figure15).Since none of the rescue experiments performed after E14.5 lead to the formation of a corpus callosum the control of Satb2 and Ctip2 over callosal and corticofugal connectivity seems to be dependent on Unc5C and DCC only in deep layer neurons, while upper layer neurons are dependent on other molecular pathways.Hence, it is likely that deep layer neurons act in a cell autonomous manner in deciding between callosal and subcortical fates[260].B. Subcerebral projection neurons; an example of CSMNIn rodents, spinal cord innervation by corticospinal tract (CST) starts relatively late in development, and occurs almost entirely at postnatal stage. Concurrently with axonal outgrowth through the spinal cord, the topographic organization of the motor cortex does not achieve its final configuration before three weeks after birth[149].Similar to the CPN, the forefront of the developing CST is composed of a small number of pioneering axons, while the majority of axons follow as tightly fasciculated bundles. The tract remains fasciculated the entire developmental period, and defasciculate only when the axons have entered the spinal cord where they spread out, at the precise level where they connect with their respective targets in the spinal gray matter[149]. The trajectory of the CST axons results from a combination of various guidance factors acting at different choice points along its journey (Figure16).Early axonal guidance cues are likely to be common between all corticofugal PN subtypes. Initially, the axons growth is directed ventrally from the cortical plate into the intermediate zone at E12.5. The coordinated expression of chemoattractive Sema3C and chemorepellent Sema3A, via the interaction with Npn-1 and Npn-2 receptors on the axons is involved in this step [265]. Moreover, Sema3A signaling might involve the cell adhesion molecule L1 as a part of the receptor complex with Npn-1, during early cortical growth [266, 267]. Diffusible Netrin-1 coming from the underlying ganglionic eminences provides a chemoattractive gradient for early subcortically projecting axons in the IZ [268, 269]. After turning away laterally from the midline, the axons extend through the intermediate zone toward the pallialsubpallial boundary. Once inside the internal capsule, corticospinal axons separate from the corticothalamic axons and enter the cerebral peduncle. At this level, Slit1 and Slit2 contribute to the maintenance of the dorsoventral positioning of the corticofugal axons within the internal capsule, and prevent their growth toward and across the midline [254]. Both Slit receptors, Robo1 and Robo2 are expressed in developing cortical axons, since in their double mutants a massive number of axons abnormally cross the midline [257].The axons pass then through the midbrain to the ventral pons. By birth, they reach the caudal region of the medulla where the majority decussate at the junction with the spinal cord[185, 190]. The Netrin1 signaling through DCC and Unc5 [270],

Figure 16 -

 16 Figure 16-Key events in corticospinal tract guidance.

  CSMNs of sensorimotor cortex and corticotectal PNs of the occipital cortex. In Bhlhb5 null mutants, CSMN failed to send their axons to the spinal cord and together with corticotectal PNs showed a reduced expression of SCPN markers such as S100A10, Mucrystallin and Crim1, indicating a role for Bhlhb5 in their specification[144].

LMO4 structure and protein interactions 1 .

 1 LMO4 belongs to the subclass of LIM domain only (LMO4) of nuclear transcription co-regulators[285]. The LMO subclass of LIM proteins is characterized by a LIM domain, which is a highly conserved cysteine-rich zinc finger-like motif that was found in a variety of nuclear and cytoplasmic proteins, and functions as a docking site for the assembly of multi-protein complexes[286, 287]. The LIM domain does not directly bind the DNA; instead LIM domain proteins function by mediating proteinprotein interactions [288, 289]. The LMO subclass of LIM proteins is characterized exclusively by the presence of two tandem LIM domains [285]. Therefore, they regulate gene expression by acting as "linker" or "scaffolding" proteins thanks to their LIM domains, and thus, they are involved in the formation of multi-protein complexes of DNA-binding factors, and transcriptional regulatory proteins [288-290]. LIM proteins represent a very conserved subclass, found in species ranging from Caenorhabditis elegans, Drosophila, and zebrafish to humans [291-295]. Four LMO4 proteins have been so far identified (LMO1-LMO4) (Figures 17).

  LMO4 is the most recently described member, and was isolated as an interacting protein of Ldb1, also known as NLI or cofactor of LIM proteins (CLIM)[291,[296][297][298]. LMO4 is the most divergent member of the LMO proteins, and shows a wide expression in embryonic and adult tissues throughout development and adulthood, whereas the other 3 members (LMO1-LMO3) are more restricted to specific tissues.LMO proteins function essentially as transcriptional co-regulators, mediating protein-protein interactions of various transcription factors or chromatin remodeling proteins. They were initially proposed as negative regulators of LIM homeodomain (LIM-HD) transcription factors[299][300][301][302][303]. However, mounting evidence indicate that they are also able to activate transcription by nucleating the assembly of complexes with TFs, such as stem cell leukemia (SCL) andGATA [299, 304, 305]. Therefore,

Figure 17 -

 17 Figure 17-Schematic representations of the human LMOs and LDB1. The first LIM domain (LIM1) and the second LIM domain (LIM2) of four LMO proteins are indicated. The homodimerization domain and the LIM interaction domain (LID) of LDB1 are also indicated (Taken from [306]).

Figure 18 -

 18 Figure 18-Schematic representations of LMOs in gene transcriptional regulation.

  as an effector of calcium activity, driving transcription in response to calcium influx in cortical neurons[146]. Calcium influx occurs through voltage-sensitive calcium channels and N-Methyl-D-aspartate (NMDA) receptors, and LMO4 activation involves calcium/calmodulin-dependent protein (CaM) kinase IV and microtubule associated protein (MAP) Kinase, acting downstream of synaptic stimulation by calcium influx[146]. The first LIM domain of LMO4 favors CREBmediated transcription, and disruption of both its LIM domains impairs activation of LMO4 function by calcium influx[146].Furthermore, LMO4 is a mediator of calcium activity not only in cortical neurons, but also in different brain structure including the hippocampus [330In the study identifying LMO4 as a mediator of calcium-related transcriptional activity in cortical neurons[146], it was used an LMO4 conditional KO mice under the control of the Nex promoter[146]. In these conditional KO mice expressing LMO4 only in postmitotic cells, the barrels of the primary somatosensory area were poorly differentiated, smaller than normal ones and blurred[146]. Moreover, these mutants showed an affected distribution of thalamocortical afferents, which do not segregate into distinct columns or give rise to smaller columnar units suggesting that LMO4 loss does not prevent thalamocortical projections from reaching the cortex but does affect their patterning in the cortex[146]. These defects in thalamocortical connections were similar to defects reported after conditional deletion of the NMDA receptor[332]. Since the authors showed that NMDA receptor activation regulates LMO4 dependent transcription, they hypothesized that the effects of NMDA receptor on barrel formation were at least in part mediated by LMO4. Thus, activity dependent regulation of LMO4-mediated transcription plays an important role in the patterning of thalamocortical connections[146].The role of LMO4 in arealization was studied by the mean of other LMO4 conditional Kos: one expressing Cre under the control of the Nestin promoter, which deletes LMO4 in the central nervous system, and another under the control of the Emx1 promoter deleting LMO4 selectively in the cortex. Both CKOs showed a less severe phenotype concerning the barrel field and thalamocortical connections. LMO4 deletion caused a rostral shift of cortical regional markers such as cadherin 8 (Cad8), inhibitor of DNA binding 2 (Id2) and EphrinA5 and its receptor EphA7. However, the relative position of the posteromedial barrel subfield (PMSF) did not shift rostrally or caudally. But the shape of the PMSF was altered, with a clear shrinkage of its structure[325]. However thalamocortical and corticothalamic connections were not affected, even if the innervation of thalamic axons in the mutant cortices was not as clear as controls, implying that LMO4 deletion affects the strength of synaptic connections. Moreover, LMO4 ablation caused behavioral abnormalities related to sensorimotor functions[325]. In summary, LMO4 defines the shape of functional areas in developing cortices and regulates sensory motor control[325].e) LMO4 in neuronal subtype specificationIt was previously stated that the refinement of subtype identity is a progressive mechanism. While mature deep-layer neurons exhibit strikingly divergent patterns of gene expression and axonal projection, newborn postmitotic neurons often extensively co-express transcription factors that later become restricted to different subtypes[START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF].In this regard, it has been shown that LMO4 and the cofactor of LIM proteins (Clim1) progressively delineate cortical PN subtypes during mouse cortex development [333]. Among layer V cortical PNs, callosal and subcerebral neurons progressively adopt distinct and complementary patterns of LMO4 and Clim1 expression. However, during early differentiation steps at E15.5, LMO4 and Clim1 co-localize and are expressed in both presumptive SCPN and CPN in layer V. During mid to late differentiation (from P0 to P6), when layer V PN axons have reached their specific targets, this overlapping expression of LMO4 and Clim1 gradually diminishes. In this phase LMO4 expression becomes specific for CPNs, while Clim1 is confined to layer V SCPNs. This is confirmed by molecular analysis. At P6 LMO4 is excluded from the majority of Ctip2 expressing neurons, while all Satb2 expressing neurons in layer V express LMO4. In contrast, Clim1 is expressed in nearly all Ctip2 expressing neurons. However, a subpopulation of neurons maintains co-expression of LMO4 and Clim1, possibly reflecting ongoing neuronal maturation, or alternatively, highlighting a distinct subpopulation of layer V PNs [333]. Thus, cortical PN identity is progressively refined throughout embryonic and postnatal neuronal differentiation [333]. A more recent report showed that LMO4 and Bhlhb5 have a striking complementary expression patterns in the motor cortex postnatally [145]. The authors divided the motor cortex into rostral motor cortex (MC r ) characterized by a high LMO4 expression and absence of Bhlhb5 expression, and a caudal motor cortex (MC c ) characterized by a high Bhlhb5 expression and absence of LMO4. Thus, LMO4 labels the MC r containing regions controlling facial, neck and forelimb movements, and is excluded from MC c containing regions controlling trunk and hindlimb movement [145]. This study partially contradict previous conclusions from the same group [333]. Indeed, although LMO4 is expressed in CPNs and lacking in CSMNs of the MC r , it is also highly expressed by cortico-brainstem motor neurons (or CBMNs, a subpopulation of SCPNs) residing in layer Va (upper layer V). Moreover, most dual backward PNs (or BPNs, sending a projection backward to the sensory cortex and the other toward either cortical or subcortical targets) are largely restricted to the LMO4 expressing MC r . In the absence of LMO4 function the molecular MC r /MC c boundary was not impaired, however the molecular identity of neurons in MC r was more homogeneous, and rostral layer Va SCPN aberrantly project to the spinal cord instead of the brainstem. Moreover, many BPNs projecting either callosally or subcerebrally fail to send a second backward projection< [145]. Overall, this study demonstrated that LMO4 is a central developmental control over the diversity of motor cortex PN subpopulations, establishing their area specific identity and specialized connectivity [145].

Figure 19 -

 19 Figure 19-Mechanisms underlying cortical thickening, expansion and lamination.

  ,phosphatidylinositol 3'-kinase (PI3K)/ protein kinase B (Akt) and β-catenin mediated Wnt signaling[142]. The Mapk/Erk pathway is involved in the G1-to Sphase transition of cell cycle in different systems[335]. Similarly, β-catenin, a downstream effector of Wnt signaling controls in a dose dependent manner, the duration of the G1 phase in neuronal precursors of the cortical midline[336]. While a short G1 promotes proliferation, a prolonged G1 phase promotes cell responsiveness to intrinsic and extrinsic signals, which trigger the neurogenic program in mammals (reviewed in[337]).COUP-TFI is required for the formation of commissural projections in 2.the forebrain by regulating axonal growth COUP-TFI is expressed in the major developing commissural neurons. In the absence of COUP-TFI function, fibers of the three commissures, the corpus callosum, the hippocampal commissure and the anterior commissure project aberrantly and fail to cross the midline in COUP-TFI null mutants[338].

  shaped cell (MSC) to bipolar shaped cell (BSC) transition, and BSC migration during mid-late stages of corticogenesis[329, 340]. Rnd2 expression is high in multipolar shaped cells and is dowregulated in the upper IZ where BSCs attach to the glia and migrate to the cortical plate. Thus, precise transcriptional control of Rnd2 activity is required for proper radial migration[341].A study from our group, demonstrated a role of COUP-TFI in promoting radial migration of upper layer callosal neurons by repressing Rnd2 expression[341].In the absence of COUP-TFI function, newborn upper layer neurons in the presumptive somatosensory cortex were properly specified but abnormally positioned, suggesting their abnormal migration. The analysis of COUP-TFI mutants showed an increased number of MSCs and a defective cell transition from the IZ to the cortical plate. Normally, the highest percentage of BSCs is found in the upper IZ and the CP, however mutant BSCs were mainly located in the IZ, indicating that although they adopt a bipolar morphology, COUP-TFI -/-neurons fail to reach the cortical plate. This indicated that COUP-TFI is required for both the transition from multipolar to bipolar shape and the intrinsic migratory property of BSCs within the IZ and cortical plate during radial migration of upper layer neuron.This study also revealed that COUP-TFI and Rnd2 have opposite expression gradients in migrating neurons. COUP-TFI KO brains Rnd2 expression expanded caudally and labeled abnormally migrating neurons. Moreover, COUP-TFI was demonstrated to negatively regulate Rnd2 expression in migrating post-mitotic cells favoring newborn neurons to reach the cortical plate. However, the rescue of correct levels of Rnd2 in COUP-TFI mutant brains partially recovered the balance between MSCs and BSCs and strongly promoted BSCs migration to the cortical plate.In addition, acute inactivation of COUP-TFI in single newborn neurons strongly delayed the formation of the corpus callosum. Moreover, Satb2-positive callosal neurons normally having a branched apical dendrite and complex apical tufts in layer I, showed after cell autonomous inactivation of COUP-TFI an altered morphology of their tufts, which barely reached the marginal zone. Moreover their dendrite branching was also strongly altered. Restoring correct levels of Rnd2 rescued the proper contact of these neurons with the pial surface, their apical tuft complexity in layer I and finally their dendritic branching. Overall this work demonstrated a cell autonomous role for COUP-TF1 in migration, axonal pathfinding and dendritic arborization of CPNs, and indicated that fine-tuning of Rnd2 levels by COUP-TFI during radial migration favors CPN maturation[341].

Figure 20 -

 20 Figure 20-Massive expansion of the Frontal motor area and posterior compression of primary sensory areas in COUP-TFI deficient cortex.

  Interestingly, the caudal shifted S1 and V1 in COUP-TF1 CKO express a comparable level of Ctip2 expression with respect to their counterparts in WT animals. Therefore, COUP-TF1 acts in an area restricted manner on the differentiation of the two main classes of corticofugal deep layer neurons, the corticothalamic and the corticospinal motor neurons [134, 140]. In addition, FOXP2/Tbr1 and Ctip2 that are normally expressed in distinct subsets of neurons in layers VI and V respectively, with only rare cells co-expressing both FOXP2 and Ctip2 or Tbr1 and Ctip2, showed increased co-localization in COUP-TF1 CKO. This indicates an abnormal acquisition of mixed corticothalamic and CSMN identity by corticofugal neurons. Moreover, altered balance between Fezf2/Ctip2, that were increased in layer VI and Tbr1 expressing cells was observed since E13.5 and persists at E16.5 when the generation of CFuPN is terminated, indicating that COUP-TF1 is normally involved in determining CSMN versus CThPN identity since early stages of corticogenesis. Thus, in S1 cortex, COUP-TF1 normally represses a CSMN differentiation program during generation of layer VI corticothalamic neurons and in the absence of its function, presumptive CThPN display cardinal molecular features of CSMN differentiation.

Figure 21 -

 21 Figure 21-Increased expression of molecular hallmarks of CSMN in corticofugal neurons of S1 cortex in COUP-TFI CKO mice. (A and B) Coronal sections and (C-V) higher magnification views of frontal (M) and parietal (S1/mS1) cortices of WT and COUP-TFI CKO P8 brains indicate abnormal expression levels of the CSMN markers Fezf2 (A-F), CTIP2 (G-J), Crim1 (K-N), FOXP2 (O-R), and Igfbp4 (S-V) in layer V and radial expansion of these markers toward superficial layer VI (mVI) in mS1 of COUP-TFI CKO cortices. Note that expression of FOXP2 is reduced (Q and R) and expression of Igfbp4 is abolished (U and V) in layer VI in both areas of COUP-TFI CKO cortices (taken from [134]).

A(Figure 1 )

 1 . COUP-TFI fl/fl Emx1-Cre mouse line To assess the intrinsic role of COUP-TFI in cortical area patterning, our group generated a conditional cortex specific COUP-TFI KO mouse line, COUP-TFI fl/fl Emx1 Cre (COUP-TFI CKO) [140]. The generation of this mouse line was described in Armentano et al, 2007 [140] . Homozygous COUP-TF1 flox animals are viable and fertile and mated to the Emx1-IRES-cre mouse line, which has been shown to drive site-specific recombination in dorsal pallium from E10.5. Mice homozygous for COUP-TF1 flox and heterozygous for Emx1-IRES-cre were named COUP-TF1 fl/fl Emx1Cre (COUP-TFI CKO). Mice heterozygous for COUP-TF1 flox (fl/+) and heterozygous for Emx1-IREScre, and Mice heterozygous or homozygous for COUP-TF1 flox (fl/+ or fl/fl) without an Emx1-IRES-cre allele were considered as controls. COUP-TFI CKO mice are viable and fertile and show cortex specific inactivation, as assessed using a COUP-TF1 antibody [140] (Figure 2).

Figure 1 -

 1 Figure 1-Generation of COUP-TFI CKO mice.

Figure 2 -

 2 Figure 2-Cortex-specific inactivation of COUP-TFI in COUP-TFI fl/fl Emx1Cre mice (a-h) Coronal sections at rostral and caudal levels of control (+/+; Emx1Cre) and COUP-TFIfl/flEmx1Cre (fl/fl; Emx1Cre) embryos, at the stage indicated on the left, and immunostained with an antibody to COUPTFI. Note that a few COUP-TFI-positive cells were still present at E10.5 (arrows in b,d) in the pallium (P), whereas no pallial cells expressed COUP-TFI at E11.5 (arrowheads in f and h). Staining in the subpallium (SP) and thalamus (TH) was not affected (f,h), confirming a cortexspecific inactivation of COUP-TFI (Taken from [140]).

  Countings were done manually. Images of coronal sections taken in the somatosensory area (S1BF) or in Motor area (M1) were divided in 6 bins, at P0, going from layer VI to the pia. Bins 1 and 2 represent layer VI, bins 3 and 4 represent layer V, while bins 5 and 6 represent the upper layers. At P7, cortical thickness is increased; the radial surface of analyzed brain regions was thus divided in 10 bins allowing to count cells from layer VI to layer I (1-10). Bins 1, 2 and 3 represent layer VI, bins 4, 5, and 6 represent layer V, and bins 7-10 represent upper layers. The correspondence between bins and layers was determined analyzing the expression of specific laminar markers such as Ctip2, Satb2, Cux1 and others. Counting of single or double labeled cells were normalized to the total number of DAPI cells in each bin.

  designed with an MluI restriction cassette at their 5' end, which allowed digesting the amplified Lmo4 sequence with MluI on both sides(3' and 5').After purifying the PCR product using the QIAGEN PCR Purification Kit (following manufacturer's protocol) LMO4 amplicon was digested as follows: 37°C the digested LMO4 ORF was purified again using QIAGEN Gel Purification Kit (following manufacturer's protocol) and cloned in the expression vector to be used for in utero electroporations. The expression plasmid was previously used in another study to overexpress Fezf2 sequence in post-mitotic cells of the cortex[213], and contains the Cdk5 promoter (upstream of the polylinker), which drives the expression of cloned genes together with an IRES-GFP sequence (Figure3). The IRES sequence allows GFP expression from the polycistronic RNA resulting from CDK5-driven transcription, which contains at the 5' end the sequence of interest and at the 3' end the GFP ORF. To clone MluI digested LMO4 ORF in this plasmid, CDK5-IRES-GFP was digested with the same enzyme and the same protocol described above. However, after digestion and Gel purification, the plasmid ends were dephosphorylated using Calf Intestinal Phosphatase (CIP, BioLabs) to avoid empty plasmid closure during ligation:

Figure 3 -

 3 Figure 3-Schematic of cdk5 LMO4 IRES GFP

Figure 4 -

 4 Figure 4-In utero Electroporation (A) Schematic representation of a micropipette and electrodes. (B) DNA injection into an E15.5 mouse embryo in utero, illuminated with a fibre-optics light source. (C) Electroporation by holding the embryo through the uterus with forceps-type electrodes. Arrows indicate a micropipette for DNA injection (B) and electrodes (C). Scale bar, 5 mm.

5

  seconds at 10μm amplitude. Aliquots were then centrifuged at 14000g and transferred to a new eppendorf leaving debris in old ones.[START_REF] Rubenstein | Regionalization of the prosencephalic neural plate[END_REF]2ml of SDS Dilution Buffer (20mM HEPES pH7.4, 1mM EDTA, 150mM NaCl, 1% Triton X100, PMSF 0.2mg/ml) was added to the supernatants that were then incubated overnight at 4 °C with 3 μg of the following antibodies: HDAC1 (rabbit polyclonal antibody, Millipore), H4K12 (rabbit polyclonal, Abcam), a control antibody (anti-GFP, rabbit polyclonal, Molecular probe) and no antibody. Another aliquot of simple sonication buffer was also added (MOCK) and used as a control (No DNA, No antibody).Then 50 μl of protein A sepharose beads were added to each sample. The samples were subsequently incubated with constant rotation for 3 hours at 4 °C. Samples were centrifuged and supernatant was only collected from the No-antibody aliquot and used as input control. The beads were washed 3 times for 5' at RT with 900μl of Chilled Washing Buffer A (20mM HEPES pH7.4, 1mM EDTA, 500mM NaCl, 0.8% Triton X100, 0.1% SDS, PMSF 0.2mg/ml), 3 times with chilled Washing Buffer B (20mM HEPES pH7.4, 1mM EDTA, 250mM LiCl, 0.5% NP40, 0.5% Deoxycholate, PMSF 0.2mg/ml) and 3 times with chilled TE (20mM Tris pH8.0, 1mM EDTA, PMSF 0.2mg/ml). After the final wash, the precipitated protein-DNA complexes were eluted by incubation with the Elution Buffer (50mM NaHCO3, 1mM EDTA, PMSF 0.2mg/ml) for 1 hour at room temperature. After centrifugation, the supernatants were incubated overnight at 65 °C to reverse the cross-linking. DNA was then extracted with phenol/chloroform, and precipitated with (1/9V NaAC, 1 μl glycogen and 2.5 V Ethanol 100%) ON at -20°C. After 20k g centrifugation, the DNA pellet was washed with Ethanol 70% and resuspended in 20 μl of MQ H2O. 0.5 μl of DNA from each sample was used to perform a PCR for semiquantitative analysis of the ChIP experiment. Primers for Ctip2 locus were used as in[212]. PCR was carried out for 27 cycles, annealing temperature was 60°C. Ctip2 locus primer sequences are as follows: 8 direct 5'-GCTTGGACTCAGTGTACCTC C-3'

  cerebrospinal fluid (ACSF) composed as follows: 126mM NaCl, 3mM KCl, 2mM MgSO4, 1mM NaH2PO4, 25mM NaHCO3, 2mM CaCl2, and 10mM sucrose. Slices were transferred to a thermo-regulated recording chamber and continuously perfused with oxygenated ACSF containing the following synaptic blocke: 50μM 2-amino-5phosphonovaleric acid (APV), 20μM 6,7-dinitroquinoxaline-2,3-dione (DNQX), and 50μM picrotoxin. Visually guided whole cell patch-clamp recordings were made using near-infrared differential interference contrast microscopy. Recording pipettes (with a resistance of 4-7 MΩ) were filled with an intracellular solution composed as follows: 20mM KCl, 100mM Kgluconate, 10mM HEPES, 4mM Mg-ATP, 0.3mM Na-GTP, 10mM Na-phosphocreatine, and 0.1% biocytin. Recordings were performed on layer V cells of primary somatosensory cortex using an AxoPatch 200B amplifier (Axon Instruments, Foster City, CA), filtered at 10 kHz and were not corrected for liquid junction potentials. Data were collected on a DELL PC using custom clampfit software. Analyses of recorded responses were performed using Mini Analysis software.

  calculate action potential characteristics, including firing threshold and afterpotentials, we analyzed responses of cells to threshold currents for generating action potentials. Firing threshold is calculated as the interpolated membrane potential at which dV/dt equals 20 V/s. To measure post spike potentials, we searched for minimum values within 50 ms of the spike and maximum values within 70 ms of this minimum. If the minimum value is more hyperpolarized than both the action potential threshold and the measured maximum value, the fast afterhyperpolarization (fAHP) amplitude is defined as the difference between the firing threshold and the average membrane potential in a small time window about the minimum value. The depolarizing afterpolarization (DAP) amplitude is the difference between the fAHP minimum and the average membrane potential in a small time window about the maximum value.

A

  . Double Satb2/Ctip2-expressing neurons are increased in lower layers of COUP-TFI CKO cortices Previous work from our lab has shown that deep cortical layer neurons of the somatosensory (S1) area are abnormally specified in the absence of COUP-TFI function. Layer VI neurons express high levels of Fezf2 and Ctip2 (two genes involved in the specification of layer V subcerebral projection neuron or SCPN[START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]) and aberrantly project to the spinal cord[134]. Mutant layer V cortico-spinal motor neurons (CSMN), instead, abnormally express both SCPN and corticothalamic projection neuron (PN) markers, and their axons are stalled in the cerebral peduncle failing to reach the spinal cord[134]. This could be due to a switch of fate of COUP-TFI CKO CSMN.To investigate about the genetic mechanisms leading to this abnormal differentiation of layer V neurons and the increase in the number of Fezf2 and Ctip2 positive cells, I started to assess the expression and distribution of several cortical-specific genes in coronal sections of P0 mutant cortices and compared them with control brains of littermates. In particular, I focused my attention on two major transcription factors, Satb2 and Ctip2, normally involved in the specification of two distinct subpopulations of layer V, callosal and subcerebral PNs. It has been reported that, normally, Satb2 binds to MAR sequences on the Ctip2 promoter, and recruits the NURD complex and histone deacetylase 1 (HDAC1) to directly repress Ctip2 expression[150, 212]. Ctip2 immunolabeling confirmed a radial expansion layer V neurons in the presumed somatosensory area of COUP-TFI mutant cortices [134] (Figure1 A, C). The observed increase in Ctip2-positive cells (Figure1 C, D and E) suggested a decreased number of Satb2-positive cells in mutant lower layers. On the contrary, the number of Satb2-positive cells was nearly unaltered and even increased in layer Vb of COUP-TFI CKO (Figure1 C', D' and F). Importantly, the analysis of mutant cortices revealed an unexpected increase in the number of neurons co-expressing high levels of Satb2 and Ctip2, which are mutually exclusive in normal conditions (Figure1 C''', D''' and G). In P0 mutant cortices, indeed, the percentage of double Satb2/Ctip2-expressing neurons constitutes 18 ± 1.8%, and 8 ± 0.5% of the total layer V and VI neurons, respectively, while these percentages stand at around 4 ± 0.3% in layer V and 0.35 ± 0.01% in layer VI of control brains (P= 0.002 in layer V and P=0.0003 in layer VI) (Figure1 G). Together, my data unraveled a non-well characterized neuronal population in P0 somatosensory cortices, expressing two mutually exclusive genes, Satb2 and Ctip2, normally involved in the specification of two divergent PNs subtypes, callosal and subcerebral. This cell population is remarkably increased in lower layers of COUP-TFI CKO P0 cortices. The aim of my work is to characterize this neuronal population at molecular, hodological, morphological and electrophysiological levels.

Figure 1 -

 1 Figure 1-Loss of COUP-TFI function leads to a drastic increase in the number of Satb2/Ctip2 Co-expressing cells in S1 Cortex.

Figure 2 -

 2 Figure 2-High levels of Satb2/Ctip2 Coexpressing cells in the Motor Cortex.

Figure 3 -

 3 Figure 3-Higher distribution of Satb2/Ctip2 co-expressing neurons in the Motor area of the controls and motorized somatosensory area of COUP-TFI CKO brains.

(

  and in establishing the subtype diversity of PNs within the rostral motor cortex [145]. I thus analyzed LMO4 expression by whole mount in situ hybridization (Figure 4 A, A') and immunofluorescence experiments on control and COUP-TFI mutant brains (Figure 4 B, B' and C) and found that its levels drastically increased both tangentially and radially in the mS1 of COUP-TFI mutant cortices at perinatal stages. LMO4 protein is expressed in layer VI and at lower level in layer V of P0 control S1 areas (Figure 4B); this expression predominantly increases in layer V and part of upper layers in the mS1 of COUP-TFI CKOs (in layer V, 60±4.4% in COUP-TFI CKO versus 15±2.4% in controls (P<0.001), in layer VI, 32±2.3% in COUP-TFI CKO versus 26±1.57% in controls (P=0.06) and in lower part of upper layers 51±5.3% in COUP-TFI CKO versus 10±2.8% in controls (P<0.001)) (Figure 4 B' and C), reminding of the increase of double Satb2/Ctip2-positive neurons. Thus, LMO4 increase could account for the increased number of Satb2/Ctip2-positive neurons in the mS1 of COUP-TFI mutant cortices. D. Progressive increase of LMO4 expression levels is correlated with the number of Satb2/Ctip2-positive neurons in the postnatal somatosensory cortex Previous reports have shown that LMO4 has a dynamic and region-specific expression and progressively increases with time during corticogenesis [325]. In order to follow the temporal course of LMO4 expression and, and understand how it correlates with the number and distribution of Satb2/Ctip2-positive neurons in S1, I performed immunofluorescence for LMO4 and for Satb2/Ctip2 at P0, P7 and P21

  Correspondingly, the number of cells expressing Satb2/Ctip2 is poorly represented in layers V and VI in control sections, but highly augmented in COUP-TFI CKO brains (Figure6 A-D'' and M). By P7, layers V and VI neurons have more fully differentiated, and their axons innervate their respective targets[START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF]. At this age, LMO4 expression increases in S1 of control cortices, and seem to reach a more comparable level to the one observed in COUP-TFI CKO brains (Figure5 C and D).However, even if the number of double Satb2/Ctip2-positive neurons gradually increases in control brains, it does not reach the percentages observed in COUP-TFI CKO cortices at P7 (in layer V, 8.1±1.9% in controls versus 21.7±0.18% in COUP-TFI CKO (P=0.004), in layer VI, 8.2±2.5% in the controls versus 23.1±2.4% in COUP-TFI CKO (P=0.02) (Figure6 E-H'' and N). At P21, all cortical neurons have achieved their mature state, their axons reached their final targets, and their collaterals have been fully finished the pruning[219]. At this age almost all cortical neurons

Figure 5

 5 Figure 5-Progressive increase in LMO4 expression in S1 from P0 to P7 to P21.

Figure 6 -

 6 Figure 6-Progressive increase in the proportion of Satb2/Ctip2 positive cells in S1 from P0 to P7 to P21.

Figure 7 -

 7 Figure 7-LMO4 binds to HDAC1 and substracts it from Satb2 in Satb2/Ctip2 Co-expressing cells

Figure 8 -

 8 Figure 8-Increased number of LMO4/Satb2 and LMO4/Ctip2 coexpressing cells in S1 Cortex of COUP-TFI CKO cortices.

Figure 9 -

 9 Figure 9-Satb2/Ctip2 double-labeled cells express LMO4 in WT and COUP-TFI CKO in S1.

  Ctip2 locus in vivo, I performed a chromatin immunoprecipitation (ChIP) assay on genomic DNA extracted from control and COUP-TFI CKO cortices at perinatal stages. I used an antibody against HDAC1 to assess the amount of HDAC1 still bound to Ctip2 locus, and an antibody specific for the acetylated form of histone 4 (H4K12) to verify the state of the chromatin in the same locus. The interactions of HDAC1 with the complexes bound to the Ctip2 locus were detected using a semi-quantitative PCR with primers specific for MAR sequences previously analyzed in the Ctip2 upstream region [212]. The ChIP assay demonstrated a decrease in the levels of HDAC1 bound to the Ctip2 locus, and an increase in the acetylated form of histone H4 (Figure 10B) in COUP-TFI mutants compared to controls. Overall, these data indicate that LMO4 de-repress Ctip2 by binding HDAC1, which is no longer able to repress Ctip2 transcription. (Figure 10 C and D).

Figure 10 -

 10 Figure 10-LMO4 protein binds to HDAC1 and disturbs the NuRD complex on Ctip2 locus derepressing Ctip2 expression.

(

  B) HDAC1 binds less to Ctip2 locus and histones become more acetylated in COUP-TFI CKO. Semiquantitative chromatin immunoprecipitation (ChIP) with Ctip2 locus DNA. ChIP assay was performed using cortices from P0 controls or COUP-TFI CKO brains. A ≈ 500 bp DNA fragment containing upstream part of Ctip2 DNA region (primer pair MAR1) was amplified from samples that were immunoprecipitated with anti-HDAC1, an antibody against acetylated form of the histone H4 (H4K12) and a GFP antibody, no antibody, no DNA (MOCK) and the non bound fraction of No antibody sample (input) as controls. Part (B) shows gel images of semiquantitative PCR for this ChIP. (C) Model of the absence of LMO4, which is dominant in WT situation. Satb2 binds to Ctip2 locus and induces deacetylation of histones and inactivation of Ctip2 expression. (D) Model of the presence of LMO4, which is dominant in COUP-TFI CKO situation. LMO4 sequesters HDAC1 disturbing the formation of the NURD complex and HDAC1 action. Histones on Ctip2 locus are more acetylated and Ctip2 expression is activated in Satb2 positive neurons. WT: wild type, CTR: controls, CKO: COUP-TFI fl/fl Emx1 cre , MW: molecular weight, P0: postnatal day 0, Ab: Antibody, MAR1: Matrix associated region 1, NURD complex: Nucleosome remodeling and deacetylase complex, H4K12: acetylated form of lysine K12 in histone 4.

  expression was altered in upper layers of mutant brains no obvious difference was detected in layer V of COUP-TFI CKO brains (Figure11B, B' and B'').Next, to understand whether the increased number of Satb2/Ctip2 coexpressing neurons observed in the mutant brains could be due to the absence of Ski in double-labeled cells, I performed triple immunofluorescence with Ski, Satb2 and Ctip2 antibodies (Figure11 C-D'''). Surprisingly, I found that Ski is normally expressed in 57.7% of layer V Satb2/Ctip2 co-expressing neurons in S1 of P7 cortices (Figure11 E), indicating that at least in this percentage of neurons, Satb2/Ctip2 colocalization occurs via a Ski-independent mechanism. This process could be orchestrated, then, by LMO4, as suggested by my previous experiments.It has been demonstrated that even in the absence of interactions with Ski and HDAC1, Satb2 remains bound to the Ctip2 promoter region[234]. In order to test whether LMO4 interferes with the assembly of the NURD complex directly binding Satb2, or if it rather interacts independently with HDAC1 and Ski, I performed reciprocal co-imunoprecipitation between LMO4 and Satb2. Both reciprocal coimmunoprecipitation showed no protein interactions between LMO4 and Satb2 either in control or in COUP-TFI mutant brains (Figure11 A), indicating that LMO4 probably binds and subtracts the components of the NuRD complex before their binding to Satb2. Further ChIP experiments on Ctip2 locus are required to validate this hypothesis.

Figure 11 -

 11 Figure 11-LMO4 disturbs the NURD complex and derepresses Ctip2 in a Ski independent mechanism. (A) Satb2/Ski interaction decreases while LMO4 Ski1 remains unaltered in COUP-TFI CKO. No interaction between LMO4 and Satb2 was found in controls and mutants. Nuclear extracts from P1 controls and COUP-TFI CKO cortices were immunoprecipitated using either anti-LMO4 or anti-Satb2

Figure 12 -

 12 Figure 12-A strong decrease in Satb2/Ctip2 positive neurons in the absence of LMO4 function.

  '', C-C'' and D-D''), compared to the contralateral non-electroporated hemisphere (Figure14 B) and to control electroporation with the scrambled construct (Figure14 E-E'', and F-F'').Importantly, LMO4 downregulation remarkably reduces Ctip2 expression in COUP-TFI CKO cortices (Figure15 A-A'', C-C'' and E) compared to the contralateral non-electroporated hemisphere (Figure15 B) and to the control electroporation with the scrambled shRNA (Figure15 D and F). No obvious changes were observed in the number of Satb2 expressing cells (Figure14 D'' and F''), indicating that the downregulation of LMO4 has no effect on Satb2 expression. These data further support a role for LMO4 on Ctip2 transcriptional control and strongly suggests that ectopic Ctip2 expression in COUP-TFI CKO brains is mainly due to a transcriptional de-repression of Ctip2 by LMO4.

Figure 14 -

 14 Figure 14-Downregulation of LMO4 expression in COUP-TFI mutants using LMO4 shRNA (A-A'' and C-F'') COUP-TFI CKO E13.5 brains were electroporated in utero with a control (scrambled) and LMO4-specific shRNA-IRES GFP expressing vectors. (A-A'', C-C'', and E-E'') Coronal sections of electroporated brains at P0 immunostained using an antibody against LMO4 and higher magnification views (D-D'' and F-F'') indicate that LMO4 shRNA strongly downregulates LMO4 expression compared to contralateral non-electroporated hemisphere (B) and brains electroporated with scrambled (E-E''). Arrows indicate the downregulation of LMO4 expression in brains electroporated compared to their correspondent control regions. Error bars: 200 μm (A-A'' and B), 100 μm (C-C'' and E-E'') and 50 μm (D-D'' and F-F''). E13.5: embryonic day 13.5, P0: postnatal day 0, COUP-TFI CKO: COUP-TFI fl/fl Emx1 Cre , NCX: neocortex, Str: striatum, mS1: motorized S1, UL: upper layers, V: layer V and VI: layer VI.

Figure 15 -

 15 Figure 15-Downregulation of LMO4 expression reduces Ctip2 expression in COUP-TFI mutants (A-A'' and C-F''') COUP-TFI CKO E13.5 brains were electroporated in utero with a control (scrambled) (E-E'') and LMO4-specific shRNA-IRES GFP expressing vectors. (A-A'', C-C'') Coronal sections of electroporated brains at P0 immunostained using an antibody against Ctip2 and higher magnification views for Ctip2 and Satb2 (D-D''' and F-F''') indicate that LMO4 downregulation strongly decreases Ctip2 expression compared to contralateral non-electroporated hemisphere (B) and brains electroporated with scrambled, while Satb2 expression remains unaltered. Arrows indicate the downregulation of Ctip2 expression in brains electroporated compared to their correspondent control regions. Error bars: 200 μm (A-A'' and B), 100 μm (C-C'' and E-E'') and 50 μm (D-D''' and F-F'''). E13.5: embryonic day 13.5, P0: postnatal day 0, COUP-TFI CKO: COUP-TFI fl/fl Emx1 Cre , Str: striatum, mS1: motorized S1, UL: upper layers, V: layer V, Va: upper layer V, Vb: lower layer V and VI: layer VI.

Figure 17 -

 17 Figure 17-Satb2 Ctip2 positive cells project to brainstem or callosaly to contralateral hemisphere.

  order to assess whether Satb2/Ctip2 positive cells expressing Bhlhb5 are also CBMN, I performed double immunofluorescence for LMO4 and Bhlhb5 on retrogradely labeled coronal sections of wt S1 cortices. These immunofluorescence demonstrate that all CSMN are positive for Bhlhb5 but not for LMO4 (Figure18 C-D'''). In contrast, SCPN are either positive for LMO4, or Bhlhb5 or double positive for LMO4 and Bhlhb5. Since LMO4 is excluded by CSMN, SCPN expressing both LMO4 and Bhlhb5 must be CBMN (Figure18 A-B'''). Thus, Satb2/Ctip2 positive corticobrainstem PNs can be either LMO4 positive or LMO4/Bhlhb5 positive further confirming the previous molecular characterization of this cell population.

Figure 18 -

 18 Figure 18-Corticobrainstem neurons express LMO4 while corticospinal express Bhlhb5 instead. (A-A''') Retrogradely labeled coronal sections taken in the somatosensory area of P7 WT with CTB 555 (red) injected in the pons labeling broad class of subcerebral PN (SCPN): Corticobrainstem and cortiscospinal projection neurons. Sections containing retrogradely labeled SCPN (including corticobrainstem and corticospinal neurons) are immunostained for LMO4 and Bhlhb5 and higher magnification views (B-B''') indicating that a remarkable percentage of labeled SCPN are LMO4 positive or Bhlhb5 positive or both. White arrows indicate SCPN expressing LMO4; pink arrows indicate SCPN expressing Bhlhb5. (C-C''') Retrogradely labeled coronal sections taken in the somatosensory area of P7 WT brains, with CTB 555 (red) injected in the spinal cord labeling corticospinal motor neurons (CSMN). Sections containing retrogradely labeled CSMN are immunostained for LMO4 and Bhlhb5 and higher magnification views (D-D''') indicating that CSMN do never express LMO4 but rather they are all Bhlhb5 positive. Pink arrows indicate CSMN expressing only Bhlhb5. Scale bars: 200 μm (A-A''', C-C'''), 100 μm (B-B''', D-D'''). P7: Postnatal day 7, WT: wild type, SA: somatosensory area, SCPN: subcerebral projection neurons, CSMN: corticospinal motor neurons, II-III: layers II-III, IV: layer IV, V: layer V, and VI: layer VI.

Figure 20 -

 20 Figure 20-Sox5 and Bhlhb5: layer V SCPN specific genes. (A-A''', D-D''') Retrogradely double-labeled coronal sections taken in the somatosensory area of P7 WT with red and green beads injected in the pons and the somatosensory area respectively labeling broad class of subcerebral PN (SCPN): Corticobrainstem and cortiscospinal projection neurons and callosal PN (CPN) in the contralateral hemisphere. (A-A''') Sections containing retrogradely labeled SCPN (including corticobrainstem and corticospinal neurons) and CPN are immunostained for Sox5 and higher magnification views (B-B''', C-C''') indicating that all SCPN are Sox5 positive, while Sox5 is excluded from all CPN. White arrows indicate SCPN expressing Sox5, and pink arrows indicate CPN Sox5 negative. (D-D''') Sections containing retrogradely labeled SCPN (including corticobrainstem and corticospinal neurons) and CPN are immunostained for Bhlhb5 and higher magnification views (E-E''', F-F''') indicating that a remarkable percentage of labeled SCPN are Bhlhb5 positive, however layer CPN are Bhlhb5 negative. White arrows indicate SCPN expressing Bhlhb5, and pink arrows indicate CPN Bhlhb5 negative. Scale bar: 100 μm. P7: Postnatal day 7, WT: wild type, S1: Primary somatosensory area, CPN: callosal projection neurons, SCPN: subcerebral projection neurons, II-III: layers II-III, IV: layer IV, V: layer V, and VI: layer VI.

  B). The algorithm then generates a curve representing the logarithm of the number of intersections divided by the surface (Log N/S) of each created circle in function of the logarithm of the radius (Log R) of each circle (Figure 22 C). Such a curve typically represents neuronal morphology. By applying the Sholl analysis, three distinct morphologies came out among the YFP-positive neurons, corresponding to Satb2/Ctip2, single Ctip2 and single Satb2 positive cells.Moreover, analysis of YFP-labeled apical dendrites aimed to identify further morphological differences, such as apical dendrite thickness, bifurcation point of the apical dendrite (Figure23 B), apical tuft complexity and other parameters (Figure23A, B) is ongoing. In collaboration with mathematicians of the Morpheme team at the iBV, we are currently generating a software that would automatically detect morphological differences among layer V neurons and classify them according to different parameters, such as sphericity and volume of the soma, number of secondary dendrites, their diameters and angles between them, distance to the bifurcation point of the apical dendrite, distance from the soma center to the pia, and complexity of the apical tuft.

Figure 21 -

 21 Figure 21-Morphological differences in soma shape among Satb2/Ctip2 co-expressing neurons and single Satb2 or Ctip2 positive neurons. (A) Coronal hemisection of P21 WT Thy1-YFP-H brains, labeling with YFP layer V cortical neurons, immunostained for Ctip2 (Red) and satb2 (shown only in higher magnification) indicating the position of YFP labeled neurons, in layer Vb. (B-D''') Higher magnifications views in layer V in the somatosensory area revealing 3 neuronal population: Double Satb2/Ctip2 co-expressing neurons (B-B'''), single Ctip2 positive neurons (C-C''') and Single Satb2 positive neurons (D-D'''). Arrows indicate YFP neurons with their molecular code (Double Satb2/Ctip2, single Ctip2 or single Satb2 positive neurons. (E-G) Higher magnifications for the three detected different shapes showing differences in the morphology of the soma between Double Satb2/Ctip2 co-expressing neurons (Shape I) (E), single Ctip2 positive neurons (Shape II) (F) and Single Satb2 positive neurons (Shape III) (G).

Figure 23 -

 23 Figure 23-Apical dendrite morphology; another parameter distinguishing different shapes among layer V cortical neurons. (A) Image of the somatosensory area of a P21 WT Thy1-YFP-H brains, labeling with YFP layer V cortical neurons, their axons, apical dendrite reaching the layer I, and secondary dendrites. (B) Different layer V YFP labeled neurons with their apical dendrites. Arrows and dashed lines indicate bifurcation point of apical dendrites revealing positional laminar differences of its primary branching in different cortical layers. Scale bar: 100 μm (A, B).

  ')that exhibited different spike frequency adaptation.Interestingly, quantification of the spike frequency adaptation and of the FI (Firing current) curve show that Satb2/Ctip2 type 2 neurons are very different from the other three subpopulations. The firing frequency of single Ctip2 type 1 and 2 and double Satb2/Ctip2 type 1 remain nearly constant, as demonstrated by their high adaptation ratio; however, the extent of adaptation of double Satb2/Ctip2 type 2 is much larger (very small adaptation ratio) and clearly different from the other three subpopulations (Figure24 J). In response to a series of depolarizing currents, the three subpopulations show relatively uniform mean firing rates. The slopes of their FI curves are larger than those of the Satb2/Ctip2 (type 2) subpopulation (Figure24 I, J). These results indicate that the two sub-populations of Satb2/Ctip2 positive neurons have distinct firing properties, the first subpopulation of Satb2/Ctip2 positive neurons (type 1) shows intrinsic electrical activity similar to single Ctip2 positive neurons, whereas Satb2/Ctip2 type 2 positive cells showed remarkably intrinsic electrophysiological distinct features.

Figure 24 -

 24 Figure 24-Electrophysiological properties of Satb2/Ctip2 co-expressing neurons.

  Downregulation of LMO4 is sufficient to reduce the expansion of Ctip2 2. expression in layer V of COUP-TFI mutant S1 cortices. Absence of COUP-TFI induces a clear radial expansion of Ctip2-positive neurons in layer V of S1, which reproduces the distribution of subcerebral projection neurons observed in motor/frontal areas [134]. My study strikingly showed that downregulating LMO4 expression by in utero electroporating an shRNA construct against LMO4, strongly decreases Ctip2 expression in the mS1 area of COUP-TFI CKO cortices (Figure 15 A, C and D), whereas Satb2 expression remains unchanged (Figure 15 D'' and F''). Interestingly, Ctip2 expression is completely downregulated in layer Va. Since not all Ctip2 positive cells are also Satb2 positive, Ctip2 is ablated from Satb2 postitive and negative cells. These data indicate that LMO4 not only de-represses Ctip2, but also promotes its expression by other mechanisms. Moreover, Ctip2 expression disappears from layer VI of COUP-TFI mutants after, even if layer VI neurons do not express the LMO4 shRNA electroporated at E13.5. Two hypotheses could explain this observation. First, LMO4 could regulate Ctip2 expression by "extrinsic" mechanisms, through regulation of diffusible signaling molecules (as it was shown for Sip1 [358]), negatively regulating Ctip2 expression in layer VI. Another plausible explanation involves impairment in neuronal migration. A defect in migration was previously reported in COUP-TFI mutant cortices [341]. Thus Ctip2 positive cells in layer VIa could represent a delayed migration of a layer V neuronal population (Figure 15 E). Accordingly, electroporation of scrambled construct shows a defective migration of Ctip2 positive cells, which are spread along the radial extent of the cortex (Figure 15 E'). LMO4 shRNA unexpectedly rescues the migration of delayed cells, and simultaneously down-regulates Ctip2 expression in these cells (Figure 15 C-C''). This hypothesis seems plausible since LMO4 has been reported to facilitate Ngn2 mediated radial migration [326]. Interestingly, it was similarly to what observed in COUP-TFI mutant P0 cortices. Thus, LMO4 overexpression at E13.5 in already born Satb2-positive cells (as shown in electroporated control cells) most probably de-represses Ctip2 in these cells and gives rise to Satb2/Ctip2 co-expressing neurons at an earlier developmental stage.

  CPN sub-population of Satb2/Ctip2 co-expressing neurons in the S1, while Sox5 and Bhlhb5 expression are confined to the cortico-brainstem sub-population of Satb2/Ctip2-positive cells. Other downstream targets of Satb2 and Ctip2 could be also involved, particularly axon guidance molecules. Unc5C and DCC are downstream effectors of Satb2 and Ctip2 in the choice of callosal versus subcerebral fate in lower layer neurons [260]. Unc5C and DCC are negatively regulated by Satb2 and Ctip2, respectively. High levels of Unc5C and low levels of DCC are required to project axons to the corpus callosum. In contrast, high levels of DCC and low levels of Unc5C are required to project axons sub-cerebrally [260]. Thus, in Satb2/Ctip2 coexpressing neurons the expression of a third transcription regulator, Er81 or Bhlhb5/ Sox5, would probably be a key event to ultimately decide which axon molecule is predominantly expressed. I plan in the future to assess the expression of DCC, Unc5C and other guidance molecules in double Satb2/Ctip2-expressing neurons.

  .....

	CThPNs: Corticothalamic PNs LID: LIM interaction domain Sema: Semaphorin
	Ctip2: COUP-TF1 interacting protein 2 LIM-HD: LIM homeodomain Sey: small eye
	CTR: control LMO4 CKO: LMO4 flox fox Emx1 Cre Sfrp2: secreted frizeled related protein
	Cux1: cut-like homeobox1 LMO4: Lim domain only 4 Shh: sonic hedgehog
	Cux2: cut-like homeobox2 M1: primary motor Sip1: SMN Interacting Protein 1-Delta
	DAP: Depolarizing afterhyperpolarization MAP: microtubule associated protein Ski: ski sarcoma viral oncogene homologue
	DCC: deleted in colorectal carcinoma MAPK: Mitogen-activated protein kinases Slit 2: Slit homolog 2
	DEAF1: deformed epidermal regulatory factor 1 MARs: matrix attachment regions SNPs: short neural precursors
	dLGN : dorsolateral geniculate nucleus MC c : caudal motor cortex Sox5: SRY-box-containing gene 5
	DNQX: 6,7-Dinitroquinoxaline-2,3-dione MC r : rostral motor cortex SP: Subplate
	DV: dorsoventral MDGA1: MAM domain containing glycosylphosphatidylinositol anchor 1 Sp8: trans acting transcription factor 8
	E: embryonic day MGv: ventral medial geniculate nucleus Stat3: Signal transducer and activator of transcription 3,
	Ebf2: early B-cell factor 2 mS1: motorized S1 SVZ: subventricular zone
	EC: entorhinal cortex MSCs: multipolar shaped cells Tbr1: T-box brain protein 1
	EGFP: enhanced GFP, MSN: medium spiny neurons TCA: thalamocortical afferents
	Egfs: epidermal growth factors MTA2: metastasis-associated 1 family, member 2 TFs: transcription factors
	Egr1: early growth response protein 1 mVI: motorized sixth layer Tgfα: Transforming Growth Factor α
	Emx CKOs: COUP-TFIfl/fl EmxCre mice NCAM: neural cell adhesion molecule Tle4: transducing like enhancer of split 4
	Emx2: empty spiracles homeobox Ne: Nestin promoter Unc5C: Unc-5 Homolog C (C. Elegans)
	Eph: ephrin Nex CKOs: COUP-TFIfl/fl NexCRE mice V1: primary visual
	Er81: Ets-Related Protein 81 NFT: neurofibrillary tangles VL: ventrolateral nucleus
	ERK: Extracellular signal-regulated kinases NGN2: neurogenin 2 VPN: ventral posterior nucleus
	ERα : Estrogen receptor α NMDA: N-Methyl-D-aspartate VZ: ventricular zone
	ES: embryonic stem Npn1: Neuropilin 1 Wnts: vertebrate orthologs of drosophila wingless
	ESC: embryonic stem cells NS: pimary neurospheres WT: wild type
	ETS: E26 transformation-specific NuRD Nucleosome Remodeling Deacetylase YAC: yeast artificial chromosome
	Etv1: ets variant 1 OSVZ: outer subventricular zone progenitor cell YFP: yellow fluorescent protein
	Fezf2: fez family zinc finger 2 Otx1: Orthodenticle homeobox 1
	Fgfs: fibroblast growth factors P: Postnatal day
	FI: Firing current Pax6: Paired box gene 6
	Fog2: FOG family member 2 PCR: Polymerase chain reaction
	Foxg1: Forkhead Box G1 PI3K: phosphatidylinositol 3'-kinase
	Foxp2: Forkhead box P2 PLA: proximity ligation assay
	GABA: γ-aminobutyric PMBSF: posteromedial barrel subfield
	GCSF: granulocyte colony-stimulating factor PNs: projection neurons
	GCSF: granulocyte colony-stimulating factor POU: Pit-Oct-Unc domain TFs (Brn1 and Brn2)
	GFAP: Glial fibrillary acidic protein PPARγ: peroxisome proliferation activated receptor-γ
	h-COUP-TFI: human ortholog of COUP-TFI Ppp1r1b: protein phosphatase 1, regulatory subunit 1B
	HDAC1: histone deacetylase 1 PT: pyramidal tract
	HDACs: histone deacetylases RA: retinoic acid
	Id2: inhibitor of DNA binding 2 RGCs: radial glial cells
	IGF1: Insulin like growth factor 1 Rnd2: Rho family GTPase 2
	Igfbp4: insulin-like growth factor binding protein 4 Robo: Roundabout, Axon Guidance Receptor
	IL-6: interleukin 6 RORβ: RAR-related orphan receptor beta
	IPC: intermediate progenitor cell Ryk: receptor-like tyrosine kinase
	IT: intratelencephalic S1: primary somatosensory
	IZ: intermediate zone SAGE: serial analysis of gene expression
	KO: knock out Satb2: Special AT-Rich Sequence-Binding Protein 2
	LDB1: LIM domain binding protein 1 SCL: stem cell leukemia
	Lhx: LIM/homeobox protein SCPN: subcerebral PN

single contralateral projection reside in layer II, III, layer V and to a lesser extent in VI, whereas CPN with dual projections reside preferentially in deep layers of the neocortex [160]. For instance, CStrPNi reside almost exclusively in layer Va [161]. While deep layer CPN have long distance dual axonal projections, superficial

  layer CPN participate in local circuitry within cortical columns.

	caudal neocortical regions, subpopulations of CPN send dual projections to
	contralateral or ipsilateral S1 (Backward PN; BPN), and rostrally, to contralateral or
	ipsilateral frontal areas (Frontal PN; FPN) [160-162].
	CPN with a

a) Subplate neurons (SP)

	persist throughout life in layer VI and establish permanent connections between the
	cortex and the sensory and motor thalamic nuclei. As previously mentioned, all
	cortical areas receive thalamic input and send projections to the thalamus [171],
		. Subcortical and subcerebral projection neurons are confined to deep cortical
	layers (layers V, VI, and subplate). CFuPNs are exclusively generated during the first
	few days of murine neocortical development.
	Subplate neurons are born around E11.5 in the mouse. They form the deepest cortical
	layer, the subplate, and send pioneering subcortical projections toward the thalamus,
	forming the first corticofugal tract [168, 169]. Although subplate and corticothalamic
	PNs share the same targets, these two neuronal subtypes have distinct functions and
	fates. In most species, SP neurons die postnatally, but they are thought to play a
	critical developmental role in instructing corticothalamic and thalamocortical
	connectivity [168, 170].
	b)	Corticothalamic neurons (CThPNs)
	Corticothalamic PNs are born around E12.5. Contrary to subplate neurons, CThPNs

  axons. When they are still migrating to the cortical plate, CPNs send axons away from the cortex. They are in part guided by guidance factors such as Semaphorin (Sema) most probably by providing a structural framework to follow by direct axon-axon contact[240][241][242][243][244]. This process is mediated by Neuropilin 1 (Npn1)[245, 246], which is also expressed by cingulate pioneering axons and has been shown to be involved in corpus callosum formation[246][247][248].

	It has been also shown that Netrin-1 initially attracts callosal pioneering
	axons, but it is not attractive for neocortical callosal axons [249]. Instead, Netrin-1
	attenuates slit-roundabout axon guidance receptor (Robo) repulsion in pre-crossing
	callosal axons to allow them to cross the midline of the developing brain [247].
	Once arrived at the midline, callosal axons are guided by midline glial
	structures such as the glial wedge and the indusium griseum, by neurons in the
	3A, which repels axons away from the cortical marginal zone [238]. Once they reach Even if adhesion molecules and axon guidance receptors are well studied, the
	the intermediate zone, callosal axons turn toward the midline rather than projecting relationships between transcription factors and these effectors that determine the
	laterally as corticofugal axons do. This key decision point is regulated by the action of terminal differentiated state of a neuron are mostly unknown. A recent study showed
	Sema3A [239]. Then, CPN axons approach the midline in a ventral trajectory through the contribution of Unc-5 Homolog C (C. Elegans) (Unc5C) and deleted in colorectal
	carcinoma (DCC) in the corpus callosum formation. But most importantly, the authors

the cingulate cortex and abruptly turn to cross the midline at the corticoseptal boundary. This process is initiated by pioneering axons from the cingulate cortex that begin the process of midline crossing and act as pioneers for neocortical CPN axons and guide them subcallosal ring [250], and by short-range guidance molecules of the ephrin (Eph) family [251]. The glial wedge is a bilaterally symmetrical glial structure that releases a cocktail of repulsive molecules, such as Slit homolog 2 (Slit2), Draxin, and Wnt 5A preventing the ventral growth of CPN axons into the septum [252-258]. While, the indusium griseum, which is a region of the hippocampus constituted by neurons and glia positioned dorsally to the corpus callosum and expressing slit2, acts as a dorsal repulsive barrier for CPN axons [253]. Upon encountering the contralateral glial wedge, CPN axons turn dorsally to enter the contralateral cingulate cortex and extend into the contralateral cortex toward their homotopic regions. This mechanism is still largely unknown; although it has been proposed that callosal axons follow the trajectory of the radial glia as they extend their axons to appropriate targets [245]. Finally, the pattern and maintenance of CPN projections is likely to be sculpted by activity-dependent mechanisms [259]. When callosal axons fail to cross the midline, they remain ipsilateral and form probst bundles, which are longitudinal axon fascicles, product of callosal axon misguidance [149]. demonstrated that these two Netrin-1 receptors are directly repressed by Satb2 and Ctip2, respectively [260].

  ethanol 70% and re-centrifuged at the same speed. RNA was air-dried to remove ethanol, and then re-suspended in 40 µl of sterile H 2 O. This antibody recognizes the digoxigenin of the riboprobes and it is conjugated to an Alkaline

	Phosphatase.
	Revelation of the signal: The excess of antibody was removed from brains by
	washing them in B1 buffer at RT first for 5', then twice for 1h, and finally for 2 days
	The reaction was carried with 15µg of plasmids, 5µl of the correspondent 5x buffer and 3µl of the enzyme (Ozyme) in H 2 O for 2 hours in a total volume of 50 and verified in gel electrophoresis. The linearized plasmid was then purified on MicroSpin (Roche) columns using the protocol provided by the manufacturer. The transcription of RNA probes was carried using 1µg of linearized DNA in a 20µl mix containing 2µl of transcription buffer 10x (Roche), 2µl of Dig labeling Mix (Roche), 1µl of RNAase inhibitor (Roche) and 2µl of the RNA polymerase Sp6 for 2h at +37°C. Then, 2µl of DNAase (Roche) were added and the mix was left at +37°C for other 30' to destroy the linearized DNA in the solution. DNAse action was stopped adding 2 µl of EDTA 25mM. Probes were then precipitated at -80°C for 30' by Sample preparation: After PFA fixation, brains were progressively dehydrated in ethanol 25%, 50%, 75% and 100% in PBS-tween 0.1% by washing 2 times in each solution for 10' at +4°C. Brains were stored at -20°C in pure ethanol and then progressively rehydrated to PBS-tween 0.1% before usage. EDTA 5mM, 2%, 50µg/ml, CHAPS 0.5%, Tween 2%, Yeast RNA 50µg/ml, in H 2 O) first 10' at RT, then pre-hybridized 1h at +70°C. Probes were diluted in the same hybridization buffer up to a concentration of 200ng/ml, hybridization was carried out at +70°C over night. Antibody incubation: The excess of probes was washed with hybridization buffer twice for 5' and then 30' at +70°C, then the brains were equilibrated in a mix of 50% TST (NaCl 0.5M, Tris HCl pH7.5 0.01M, Tween 0.1% in H 2 O) and 50% hybridization solution at +70°C, then in 100% TST at RT for 10'. To eliminate unbound RNA, brains were incubated 30' in RNAase A 10µg/ml solution in TST at RT, then samples were washed in TST for 10' at RT, in hybridization buffer for 10' at RT, and then twice in hybridization buffer for 30' at +65°C. Brains were equilibrated in B1 buffer (Maleic acid 100 mM, Nacl 150 mM in H 2 O) (four washes: 5', 5', 10' and 1h) at RT. The blocking was done with the B2 buffer (20% sheep serum in B1 changing the buffer every day. Before the revelation, brains were incubated twice for 20' in B3 buffer (Tris HCl 100mM, MgCl 2 50mM, NaCl 100mM, Tween 0.1% in H 2 O) at RT. Revelation was performed using NBIT-BCIP (SIGMA) 0.2% tween as a substrate of alkaline phosphatase giving a blue precipitate where the Digoxigenin antibody bound hybridized riboprobes. The reaction was left going either at RT or at +4°C until the signal reached the proper intensity. After a time varying from 1 to 3 days the reaction was stopped washing 3 times the hybridized brains with PBS-tween adding 100µl H E. Whole mount In situ hybridization 0.1% for 10'.

2 O, 10µl LiCl 4M and 300µl of Ethanol 100% to the mix. To separate precipitated RNA, the mix was centrifuged 15' at 20000 g at +4°C. The pellet was then washed in 100µl Hybridization: Brains were treated with H 2 O 2 0.5%in PBS-Tween 0.1% for 1h at RT. Then, brains were washed twice with PBS-Tween 0.1% and incubated with Proteinase K 10µg/ml in PBS-Tween 0.1%, for 30'. After brief rinse in PBS-Tween 0.1% the brains were post-fixed with P.F.A. 4%, glutharaldheyde 0.1% in PBS-Tween 0.1% for 20' in ice and in gentle rocking. Then, the brains were washed 5' in PBS-Tween 0.1% and incubated in hybridization buffer (Formamide 50%, SSC 1.3X, buffer) for 1h at RT and followed by incubation with the α-Dig-U-AP antibody (ROCHE) (diluted to 1/2000 in B1 2% sheep serum) ON at +4%C. De-staining process: during the revelation, aspecific precipitate may impinge on the quality of the signal. The de-staining process removes some precipitate increasing the quality of the signal. All the following passages were performed at RT for 1h in gentle rocking. They consist in equilibrating brains in a mix of 50% methanol and 50% PBS-Tween 0.1%, followed by 3 washes in methanol 100%, and an equilibration in a mix of B.A.B.B. (Benzyl alcohol 33%, Benzoil benzoate 67%) and 50% methanol, finally followed by washes in B.A.B.B. for several days until brains reach a good quality of the signal. De-staining process was stopped by inverting all previously described steps. Samples were stored at +4°C in PBS (0.1% tween, 0.1% PFA).

Acknowledgments

First of all I would like to thank my Ph.D supervisors Michèle Studer and Christian Alfano, for there guidance, support and encouragement during the four years of my Ph.D. Michèle, thank you for offering me the opportunity to work on this project, for all our fruitful discussions and advice during the four years. Christian, I have enjoyed our many valuable discussions during these four years and I have learned a lot from you. I appreciate all your contributions of time and ideas to make my Ph.D experience productive.

controls versus 29±1.6% in the CKO) (Figure 6I-L'', O andR). Thus, the progressive increase in LMO4 expression levels correlates with a progressive rise of Satb2/Ctip2 positive cells in lower layers.

Together these data suggest that in S1 COUP-TFI might control the timing of onset of LMO4 expression, which in turn would allow Satb2/Ctip2 co-expression in lower layer neurons. Absence of COUP-TFI might anticipate the specification of Satb2/Ctip2 positive cells due to the precocious expression of LMO4.

Chapter 4: Discussion

shown that migratory defects observed in COUP-TFI mutants depend on disregulation of Rnd2, which is a downstream target of Ngn2 during radial neuronal migration.

LMO4 overexpression anticipates the birth of Satb2/Ctip2 co-expressing 3.

neurons.

Complementary to the previous approach, in utero electroporation of a post-mitotic expressing LMO4 construct in E13.5 wt brains, strongly up-regulated LMO4 expression in the S1 of P0 brains, where normally LMO4 is weakly expressed. As previously mentioned electroporation of a control GPF-expressing plasmid failed to target Ctip2-expressing neurons, indicating a probable earlier birthdate of Ctip2-and Satb2/Ctip2-positive cells. However, a remarkable percentage of the GFP-positive cells were Satb2 positive (Figure 13 G-I'''). Thus, absence of GFP positive neurons expressing Ctip2 (alone or with Satb2) at P7 in control electroporated cells implies that these cells might be born earlier than E13.5. This apparent discrepancy compared to previous birthdating analyses [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF] might be explained by technical limits inherent to the electroporation method. Indeed, ventricle-injected DNA can be electroporated only in progenitors surrounding the ventricular surface (apical progenitors), while BrdU molecules can label both apical and intermediate progenitors. These data suggest, interestingly, that at E13.5 apical progenitors do not produce anymore Ctip2 positive cells, while intermediate progenitors continue to amplify this neuronal population. Moreover, this unexpected result confirmed that Lmo4 acts by derepression of Ctip2 expression in Satb2 expressing cells.

Lmo4 overexpression, indeed, induced Satb2/Ctip2 co-expression in a population of cells that in control electroporations expressed solely Satb2 (Figure 13 D-F'''). This seems very plausible, since Satb2/Ctip2 co-expressing neurons are a population that increases over time, together with the increase in LMO4 expression.

My data show that the percentage of Satb2/Ctip2 co-expressing neurons increases from 4± 0.3% at P0 to 8.1±1.9% at P7 in layer V neurons, and from 0.35± 0.01% at P0 to 8.2±2.5% at P7 in layer VI neurons (Figure 6). The number of double Satb2/Ctip2-expressing neurons increases even more after P7, a stage where normally cortical neurons are fully committed, suggesting late specification of this cell population. Thus, LMO4 overexpression at E13.5 leads to an anticipated specification of Satb2/Ctip2 co-expressing neurons among the Satb2-positive cell population, own progressive downregulation [166,217]. Finally the transcription factor Er81 is expressed throughout layer V in both CPNs and SCPNs in the rat cortex [348].

Thus, since Satb2/Ctip2 neurons expressed all these subtype-specific genes with different percentages, I divided this cell population into two major subpopluations: one with subcerebral characteristics expressing Sox5 and Bhlhb5, which are known to be involved in corticofugal and CSMN respectively, and the other with callosal properties and expressing LMO4 and Er81, but not Bhlhb5 and Sox5 (Figure 16), and which seems to be less represented than the subcerebral one.

My molecular analysis on retrogradely-labeled callosal and subcerebral PNs revealed that LMO4, which is expressed in almost all Satb2/Ctip2 co-expressing neurons, is expressed by both CPNs and SCPNs, whereas Er81 is expressed only by layer V callosal neurons. Differently, Sox5 and Bhlhb5 are expressed only by SCPN, and excluded from layer V callosal neurons. In summary, combining retrograde labeling with molecular analysis, my data revealed that Satb2/Ctip2 co-expressing neurons can be classified into two major cell sub-populations, a callosal subpopulation expressing LMO4 and Er81 and another subcerebral sub-population expressing Sox5, Bhlhb5 and LMO4 (Figures 19 and20).

B. Connectivity

My data demonstrate that Satb2 and Ctip2 co-expression does not give rise to one hybrid cell population projecting both intracortically and subcerebrally, but rather to two distinct sub-populations projecting either to the contralateral cortex (CPN) or to the brainstem (cortico-brainstem PNs, CBPN) (Figure 17). My analyses on retrogradely labeled brains showed that cortico-spinal motor neurons (CSMN) express Bhlhb5 but not LMO4 as previously described [145]. In contrast, cells labeled by CTB injection in the rostral pontine region can be either LMO4 or double LMO4/Bhlhb5 positive. This is not surprising since injection of the tracer in this brainstem region will label not only CBPNs but also CSMNs whose axons axons are en route to the spinal cord. However, two points should be discussed in this issue.

First, it has been previously shown that LMO4 and Bhlhb5 have a complementary expression pattern [145], however it is important to note that my data revealed that LMO4 and Bhlhb5 also co-localize in a remarkable percentage of layer V neurons,

Chapter V-Conclusion

Future Perspectives