Keywords: Reprogramming, Lmo4, Fezf2, Neocortex, Corticofugal projection neurons a. Upper layer CPN marker CUX1 ..

The mammalian neocortex is tangentially organized into functional areas and radially subdivided into six layers of neuronal populations with distinct projections, morphology and expression patterns. While neurons in the upper layers project within the neocortex or towards the contralateral hemisphere, such as callosal projection neurons, most lower layer neurons, named corticofugal projection neurons, innervate subcerebral targets via the corticospinal tract. Injuries or loss of these neurons can lead to severe neurological disorders, such as Amyotrophic Lateral Sclerosis or other diseases and/or lesions implicating the corticospinal tract. Since the adult mammalian brain lacks a significant regenerative capacity and thus, damage or loss of neurons is permanent, methods for restoring neurons are urgently needed.

Recent studies revealed an unexpected plasticity of post-mitotic neurons, which can be converted into cell-types of other neuronal lineages. This approach, called direct neuronal reprogramming, bypasses an intermediate pluripotent state. Direct reprogramming is fast, likely to keep epigenetic hallmarks and it can be conducted in vivo. Forced expression of transcription factors, known to act as master regulators of cell fate during development, is one of the best strategies for directly converting one cell-type into another one. However, it is still not clear to which extent neurons, generated by direct reprogramming, acquire authentic molecular signatures of the desired cell type, down to the point of precise subtype specification. The transcription factor FEZF2 is known for its role in cell fate specification of layer 5 subcerebral projection neurons and high ectopic Fezf2 expression can convert upper layer or striatal neurons into a corticofugal fate, even if at low efficiency.

During my PhD thesis, I used Fezf2 and the nuclear co-adaptor Lmo4 to efficiently reprogram upper layer projection neurons into corticofugal lower layer projection neurons, at both, embryonic and postnatal stages. The successful reprogrammed cells downregulated upper layer markers, while lower layer markers were drastically increased. Additionally, by using advanced tracing methods, we showed that reprogrammed neurons projected towards subcerebral targets, including the thalamus, cerebral peduncle and spinal cord. These data demonstrate an unexpected synergistic role of Lmo4 with Fezf2 in neuronal reprogramming and reveals an effective cocktail for the conversion of neuronal cells into corticofugal neuronal subtypes.

First of all, I want to thank Michèle Studer for her supervision of my thesis. Thank you for offering me the opportunity to work on a great project in your lab. Thank you also for your great support, your kindness and your guidance during the last 3 and a half years. Furthermore, I would like to thank Christian Alfano and Kawssar Harb for supporting me in the very beginning of my thesis and for handing me over their knowledge and preliminary data of my project.

I then want to thank Thomas Lamonerie for accepting to chair the jury of my PhD defense. Also, I want to thank Fanny Mann and Benedikt Berninger for reviewing my thesis and my defense. Thank you all for your time.

I want to thank my wonderful lab colleagues for a great time during the last years. It was a pleasure to work with you -every day, I was happy to come to the lab. Therefore, thank you Eya Setti, Michele Bertacchi, Alessandro Simi, Chiara Tocco and Maria Anna Di Bonito.

The environment in the institute is to mention, as all colleagues are exceptionally helpful and show a great sense of kindness. Thank you, members of the Lamonerie lab, the Collombat lab, the Schedl lab, the Fürthauer lab, the Sandoz lab, the Delaunay lab, the Acceuil of Science Naturelle and Biochemie, Agnès Loubat from the Cytometrie platform, the Prism platform and all members of the animal house.

Then I want to thank my wonderful friends here in Nice. I had such a good time, thanks to you guys: Aidan Falvey, Cristina Paraschivescu, Sanya Kuzet, Racha Fayad, Anthony Ruberto, Hereroa Johnston and Tom McMahon.

A very special thanks goes to Salsabiel. Your friendship is very precious to me and I cannot thank you enough for your support during the last years. I wish you all the best in the world for your future.

Ich habe das große Glück einer wunderbaren Familie anzugehören, dafür bin ich sehr dankbar. Vielen Dank an meinen Bruder Mario, meine Schwägerin Anna und die Kleinen Louis, Lotte und Jule. Vielen Dank an meine Schwester Ina und meinen Schwager Marc mit dem kleinen Bruno. Ebenfalls vielen Dank an meine tollen Großeltern Ursula und Gerhard.

Mein größter Dank geht an meine Eltern, ich kann mich unendlich glücklich schätzen Euch zu haben.

Reprogrammation efficace en neurones de projection corticofuges aux stades embryonnaires et postnatals chez la souris Jury:

Président du jury

Thomas LAMONERIE, Professeur, Université Côte d'Azur

Rapporteurs

Fanny MANN, DR1 CNRS, Institut de Biologie du Développement de Marseille Benedikt BERNINGER, Professeur, King's College London Titre : Reprogrammation efficace en neurones de projection corticofuges aux stades embryonnaires et postnatals chez la souris

Résumé

Le néocortex des mammifères est tangentiellement organisé en aires fonctionnelles et radialement divisé en six couches de neurones qui présentent chacune des projections, des morphologies et des patterns d'expression distinctes. Alors que les neurones de projection des couches supérieures projettent leurs axones dans d'autres couches du cortex ou vers l'hémisphère controlatéral, tel que les neurones calloseux, la plupart des neurones des couches inférieurs, nommés neurones corticofuges, innervent des structures sous-corticales à travers la voie pyramidale. La perte de neurones corticofuges peut conduire à des désordres neurologiques sévères, tel que la sclérose latérale amyotrophique ou d'autres maladies et/ou lésions impliquées dans le tractus cortico-spinal. Étant donné que le cerveau adulte des mammifères ne dispose pas d'une capacité de régénération efficace et que, par conséquent, la perte de neurones devient permanente, des nouvelles méthodes de remplacement de neurones sont indispensables.

Des études récentes ont révélé une plasticité surprenante des neurones post-mitotiques, qui peuvent être convertis en types cellulaires d'une lignée neuronale différente. Cette approche, appelée reprogrammation directe, contourne un état de pluripotence intermédiaire, est assez rapide et susceptible de conserver une signature épigénétique. L'expression forcée de facteurs de transcriptions, connu pour agir comme principaux régulateurs du destin cellulaire pendant le développement, est la meilleure stratégie pour convertir un type cellulaire en un autre. Cependant, il est encore difficile de comprendre dans quelle mesure des neurones, généré par reprogrammation directe, acquièrent les caractéristiques moléculaires du type cellulaire souhaité, jusqu'au stade de la spécification précise du sous-type. Le facteur de transcription FEZF2 est connu pour son rôle dans la spécification des neurones de projections sous-corticales de la couche 5, et une surexpression ectopique de Fezf2 peut convertir les couches supérieures ou les neurones striataux en neurones corticofuges de projection, même si cela est à faible efficacité.

Durant ma thèse, j'ai utilisé Fezf2 et le co-adaptateur nucléaire Lmo4 pour reprogrammer efficacement les neurones calloseux de la couche supérieure en neurones corticofuges de la couche inférieure, à la fois, au stade embryonnaire et postnatale. Les cellules reprogrammées avec succès régulent négativement les marqueurs de la couche supérieure, alors que les marqueurs de la couche inférieure sont surexprimés considérablement. De plus, en utilisant des méthodes de traçage avancées, les cellules reprogrammées ont la capacité de projeter vers des cibles sous-corticales, tels que le thalamus, le peduncule cérébrale et la moelle épinière. Ces données démontrent un rôle synergique inattendu de Lmo4 avec Fezf2 dans la reprogrammation neuronale et révèlent un cocktail efficace pour la conversion de cellules neuronales en sous-types de neurones corticofuges de projection.

Mots clés : Reprogrammation, Lmo4, Fezf2, Néocortex, Neurones corticofuges de projection

Table of Contents

List of Figures

INTRODUCTION

During evolution, one part of the brain experienced a drastic development in complexity and size and is one of the major reasons for the evolutionary success of mammals: the cerebral cortex. It covers the outer portion of the cerebrum and is anatomically comprised of the archicortex, the paleocortex and the neocortexits largest component (O'Leary, Chou and Sahara, 2007). The neocortex is the evolutionary newest part of the cerebral cortex and its cytoarchitecture is a distinguishing feature of mammals; in humans, it covers up to 80% of the total volume (Rakic, 2009;Kaas, 2011). The neocortex represents the major and critical organ for sensory input processing, movement, memory and language and is additionally responsible for higher cognitive functions, such as abstract thinking and self-awareness (Kaas, 2011). Due to these intrinsic features it is a major organ of interest for fundamental and medical research, but still represents a major challenge because of its high complexity.

Therefore, gaining a better understanding on the molecular, cellular and systemic level is crucial to unravel its mystery and thus, this work on the mouse neocortex is intended to contribute to it.

I. The Mammalian Neocortex: Cytoarchitecture and Development

A. Cytoarchitecture of the neocortex 1. Cellular components

The neocortex is composed of neuronal cells and glia. Glutamatergic projection neurons (PNs) make up almost 80% of the total neuronal population in the neocortex, they are generating the neocortical output and are responsible for the connection of the neocortex to subcortical, subcerebral or intracortical targets. The other major population is composed of interneurons (INs), which make up around 20% of the neuronal population (Lodato and Arlotta, 2015). They are largely inhibitory and modulate the activity of neuronal circuits by release of caminobutyric acid (GABA). INs share a common feature of connecting within the neocortex via short-range projections even though they appear to be a very heterogenous class with diverse morphology, connectivity, biochemistry and physiological properties (Lim et al., 2018).

Non-neuronal cells are the third category of cells that are present in the neocortex and are named glia. Their role besides supporting neuronal circuits is very various. Three main subcategories of glial cells exist: Microglia, Oligodendrocytes and Astrocytes. Microglia are known as the 'immunocompetent' cells with phagocytic abilities. They cover a huge part of the brain parenchyma and not only survey the brain tissue for any damage, but are also implicated in the maintenance of the diverse cell populations that comprise the CNS (Schafer and Stevens, 2015). Oligodendrocytes are the myelin-producing cells insulating axons to allow fast axon potential propagation. Some oligodendrocytes are not producing myelin and may have other functions, as they are present in the grey matter of the cerebral cortex (Kang et al., 2013). Astrocytes are the most abundant glial cell population in the neocortex, their function in the neuronal circuit is various and ranges from water and ion homeostasis, participation in synapse signalling or maintenance of the blood brain barrier (Jäkel and Dimou, 2017).

The investigated cell type in this thesis is the excitatory projection neuron and therefore the following part of the introduction will be focused on this cell type.

Tangential organisation of the neocortex

The neocortex is tangentially organised into functional areas, each having a unique cytoarchitecture, distinct connections and specific functions. For the neuroscientist Brodmann, "each area represents an organ" of the adult brain (Brodmann, 1909), implying that each area is well distinguished from another one by rather distinct cytoarchitecture borders and has a unique function within the brain. Four major areas can be distinguished in all eutherian mammals (Kaas, 2011) and are defined as primary areas due to their function as first "receiver" of sensory inputsauditory area A1, visual area V1 and somatosensory area S1 -or as first "executor": the motor area M . The sensory areas are processing information from the cochlea (A1), the retina (V1) and the body (S1), while M1 controls voluntary movement of different body parts (O'Leary, Chou and Sahara, ).

Radial organisation of the neocortex

Tangentially, the neocortex is subdivided into functional areas, whereas in its radial dimension the neocortex is organised into six layers (see Fig. 1). Each layer is composed of a different pool of neurons and, each layer varies in its cellular composition, its projections and its thickness depending on their areal identity (Lodato and Arlotta, 2015).

Layer (L)1 represents the first sheet just underneath the pial surface (the inner most membrane that envelopes the cortex) and mainly consists of axons, dendrites and sparse cells named Cajal-Retzius cells (Kaas, 2011). The following layers are grossly classified by their projections within the central nervous system (CNS, see Fig. 1): L2-3 are mainly composed of neurons which extend their axons to the contralateral cortical hemisphere, named callosal projection neurons (CPNs) and associative projection neurons, projecting within the neocortex. Most of the CPN axons cross the midline through the corpus callosum (CC), which represents the major "bridge" between the two cortical hemispheres. Some CPNs send their axons through the anterior commissure (AC), which represents a second "bridge" with fewer cortical axonal projections, connecting both hemispheres (Greig et al., 2013). In fewer numbers, CPNs are also residing in L5 and L6. Neurons in L4 are the main recipient of thalamocortical afferents and the majority of L4 neurons project within one cortical hemisphere to L2/3 (Lodato and Arlotta, 2015).

PNs in L5 and L6 project away from the cortex to distinct subcortical or subcerebral structures.

These projection neurons are named corticofugal projection neurons (CFuPNs) and can be further subdivided into subcerebral projection neurons (SCPNs) -residing in L5and, corticothalamic projection neurons (CThPNs), in L6. SCPNs are projecting towards distinct subcerebral targets in function of their areal location: from V1 to the superior colliculus (corticotectal PNs), from S1 to the brainstem (mostly pons, cortico-pontine PNs) and from M1

to the spinal cord (Corticospinal motor neurons, CSMNs). CThPNs project to specific thalamic nuclei, in a topographic manner, meaning that CThPNs from distinct areas project to specific nuclei in the thalamus (Molyneaux et al., 2007;Greig et al., 2013;Lodato and Arlotta, 2015). This is a rough classification and gives only a general idea of neuronal population differences within layers. For example, associative projection neurons are present in each layer and extend their axons within a single cortical hemisphere (Molyneaux et al., 2007). Additionally, some neurons send projections to multiple targets and therefore can be classified in several classes. Among them are CPNs, projecting to both the contralateral hemisphere and ipsilaterally within the cortex, whereas intrathelencephalic corticostriatal PNs, extend callosal axons to the ipsilateral but also to the contralateral striatum (Shepherd, 2013). SCPN often project to multiple targets, as they send collaterals to diverse corticofugal targets (Shepherd, 2014). Also, some SCPN extend backward projections, sending axons to subcerebral targets and within the cortex (Molyneaux et al., 2007;Greig et al., 2013;Lodato and Arlotta, 2015).

C. Corticofugal projection neurons (CFuPNs)

project outside the cortex to subcortical targets including corticothalamic projection neurons (CThPNs), which reside in layer 6, and subcerebral projection neurons (SCPNs), which reside in layer 5.

D.

Neurons that send projection to multiple targets can sometimes be classified into more than one category. Examples include CPN with frontal projections, which extend axons to the contralateral hemisphere and to ipsilateral frontal cortex. SCPN with backward projections, which extent axons to subcerebral targets and to ipsilateral caudal cortex. Intratelenecpahilic corticostriatal projection neurons, which extend projections to contralateral hemisphere and to ipsilateral striatum.

CC Corpus Callosum, Crb cerebellum,

LGN lateral geniculate nucleus of thalamus, OB olfactory bulb, OT optic tectum, PO pons, SC spinal cord, Th thalamus, VL ventral lateral nucleus of thalamus, VP ventral posterior nucleus of thalamus. Taken from Greig et al. (2013).

Connections of the neocortex

Axonal trajectories are important messenger propagation routes to ensure communication between higher cognitive upper layer neurons and executive lower layer neurons. PNs can be characterized by their axonal projections, revealing their functional role in the cortex. For simplification, associative PNs and callosal PNs are grouped together as intratelencephalic (IT) PNs, as they project to the contralateral hemisphere or within the telencephalon (cortex and striatum) or both (bilateral IT neurons). Names of SCPNs and CThPNs are not changed.

a. Cortico-cortical projections

Inputs on cortical neurons originate mainly from other cortical neurons of the same hemisphere (associative projection neurons) or from neurons projecting from the contralateral hemisphere. The axonal pathway from one hemisphere to its contralateral region through the CC is a relatively recent feature in cortical evolution and is unique to placental mammals (Aboitiz, Morales and Montiel, 2003). Other interhemispheric commissures exist and are more ancient, the hippocampal commissure and the AC, but the CC is by far the largest and the only one which serves solely the communication between the two neocortical hemispheres (Aboitiz, Morales and Montiel, 2003). Axons in the CC arise mainly from CPNs which are abundantly located in L2 -3 (around 80% in rodents), in L5 (around 20%) and some in L6 (Fame, MacDonald and Macklis, 2011). All CPNs extend an axon to the contralateral hemisphere, but they can also innervate local cortical neurons within the hemisphere and/or the striatum. In fact, mainly lower layer CPNs possess dual projections to the contralateral side and ipsilaterally within the forebrain, while upper layer CPNs project to cortical neurons in L2 -3, to stellate cells in L4 and they also send collaterals to L5 and L6 cortical neurons (See Fig. 2, Petreanu et al., 2007). Thus, CPN function is mainly related to integration and connection of neuronal circuits within the neocortex.

b. Corticofugal projections

The sub-classification of CFuPNs is complex regarding their connectivity and is not yet completely understood. The complexity arrives from the fact that many CFuPNs target several structures via branches along their trajectory. This is especially the case for L5 SCPNs, which can project to the midbrain, striatum, thalamus, and subthalamic nuclei, en route to their dorsal-most destinations in the brainstem and the spinal cord (Shepherd, 2013). Additionally, SCPNs also form ipsilateral connections within the cortex, but do not cross through commissures to the contralateral hemisphere (Shepherd, 2013). Less complex projections are found of L6 CThPNs, which are projecting to the thalamus only. Here, the major trajectories of CFuPNs will be presented.

c. The corticospinal tract (CST)

The control of voluntary movements in mammals is based on sophisticated motor and sensory communication in the CNS. Axonal trajectory of the CST enables fast and controlled motor execution that has been carried out by corticospinal motor neurons (CSMNs) in the neocortex.

The functions of the CST comprise control of afferent inputs, spinal reflexes and motor neuron activity (Lemon and Griffiths, 2005). Axons in the CST originate from L5 SCPNs in the primary motor and somatosensory cortex and in less extent from parietal, cingulate, visual and prefrontal regions (Miller, 1987;Akintunde and Buxton, 1992;Tennant et al., 2011;Kamiyama et al., 2015). The canonical path of the CST in mammals starts from axonal projections of L5 SCPNs in the neocortex, which then travel into the internal capsule (IC), further on to the cerebral peduncle (CP), into the brainstem and some reach the spinal cord (see Fig. 2). The axonal bundles of the CST remain at the ventral position until reaching the medulla in the brainstem. In rodents, 80 -95% CST axons cross the midline at the junction between the brainstem and spinal cord, and pass from a ventral to a dorsal position, forming the pyramidal decussation (Welniarz, Dusart and Roze, 2017;Armand, 1982;Schreyer and Jones, 1982;Rouiller et al., 1991;Joosten et al., 1992). Therefore, the majority of the axon trajectory continues in the contralateral part of the SC and terminates in the ventral part of the dorsal funiculus, located between the dorsal horn and the midline (Kuypers, 1964). The remaining 5 -20% uncrossed CST project in the ventral funiculus. However, both crossed CST and uncrossed CST originate from the same cortical regions (Galea and Darian-Smith, 1994;Brösamle and Schwab, 1997;Lacroix et al., 2004), but control different muscles: the crossed CST is involved in fine movements of distal extremities, whereas the uncrossed CST targets proximal or axial musculature (Welniarz, Dusart and Roze, 2017). The terminations of crossed CST are located in the dorsal and intermediate horns (grey matter) of the contralateral spinal cord, uncrossed CST terminations on the ipsilateral side show the same pattern (Welniarz, Dusart and Roze, 2017). Interestingly, CSMNs of adult rodents do not form direct connections with motor neurons in the spinal cord (Alstermark and Ogawa, 2004), whereas higher primates have monosynaptic connections between CSMNs and motor neurons, allowing fine hand dexterity control (Lemon, 2008). However, this monosynaptic connection is present in juvenile mice and is eliminated by signalling molecules and is therefore lost in the adult mouse (Gu et al., 2017). Thus, the CST connection to motor neurons in the spinal cord might be formed by polysynaptic transmission via interneurons and propriospinal neurons (Lemon, 2008). Overall, L5 SCPNs extend their axons within the CST and project to several corticofugal targets via branches or collaterals of their axons, en route to their dorsal-most locations, the brainstem and the spinal cord.

d. Cortico-striatal connections

The striatum of the mammalian cerebrum is a critical component of motor and reward systems, including motor and action planning, motivation and reward perception (Yager et al., 2015). It receives glutamatergic input from the cortex and dopaminergic input from basal ganglia structures. Connectivity between the cortex and the striatum is directional, meaning that cortical projections form mono-synaptic input, whereas neurons in the striatum communicate indirectly with the cortex via polysynaptic downstream circuits (Shepherd, 2013). The cortical neurons innervating the striatum originate from L5 IT or SCPNs and L6 IT neurons. Moreover, cortico-striatal projections can either originate from L5 IT neurons or SCPNs, but not from both (Shepherd, 2013). L5/6 IT neurons projecting to the striatum have the unique attribute of being both corticofugal and callosal, because they project to the striatum and they additionally project to the contralateral hemisphere (Macklis et al., 2012).

Interestingly, the striatum is special in receiving both L5/6 IT (bilateral) and SCPN (ipsilateral) inputs, differently from other cerebral areas. In the striatum, L5/6 IT neurons and SCPNs make synaptic contacts primarily with spines of striatal projection neurons, also called medium spiny neurons (MSNs, Reiner, 2010), which is by far the most abundant neuron type in the striatum (Yager et al., 2015). MSNs themselves project to basal ganglia structures such as globus pallidus and substantia negra pars reticulate. Taken together, the cortical input the striatum receives originates from several types of PNs, which includes L5 and L6 IT neurons and SCPNs.

e. Connections between cortex and thalamus

The thalamus is a prominent structure in the diencephalon and is the major receiver and relay of sensory inputs, which propagates in a topographic manner to the cortex. The thalamus and the cortex are strongly interconnected and both are important for proper development of one another and represent an integrated processing unit, which regulates thalamic transmission of peripherally derived data for cortical processing (Leyva-Díaz and López-Bendito, 2013). The cortex innervates the thalamus, by corticothalamic axonal (CTA) projection and the thalamus projects to targets in the cortex, by thalamocortical axonal (TCA) projection.

The cortical innervation of the thalamus originates from CThPNs of L6 and to a smaller extent of SCPNs in L5. But, the projection patterns of L6 CThPNs differ from L5 SCPNs. For instance, L6 CThPNs project to all thalamic nuclei (including both first-order and higher-order relays), whereas SCNPs only project to higher-order thalamic nuclei (Hoerder-Suabedissen et al., 2018). First-order relays get their driving input from subcortical sources and form a reciprocal feedback pattern with CThPNs. Moreover, CTA of CThPNs innervate thalamic nuclei, which in turn send TCA to the same cortical region in L6 from where the input originated, depending on their area identity (Briggs and Usrey, 2008). These projections from CThPNs modulate how sensory information is relayed to the cortex [START_REF] Olsen | Gain control by layer six in cortical circuits of vision[END_REF]Lam and Sherman, 2013;Crandall, Cruikshank and Connors, 2015). Most of TCA terminate in L4, thereby innervating local circuit neurons, named stellate cells. In addition, also L1 and L2 -3 receive TCA input (Caviness and Frost, 1980). Thus, the neocortical primary areas are connected via CThPNs with the thalamic nuclei in the following way: M1 is connected with the ventrolateral (VL) nucleus, S1 with the ventroposterior nucleus (VP), V1 with the dorsal lateral geniculate nucleus (dLGN) and A1 with the ventral part of the medial geniculate nucleus (MGv, Guillery, 1967;[START_REF] Hoogland | Organization of the projections from barrel cortex to thalamus in mice studied with Phaseolus vulgaris-leucoagglutinin and HRP[END_REF] Welker and Van der Loos, ; O'Leary, Chou and Sahara, ).

In contrast, higher-order relays get their driving input from SCPNs (Usrey and Sherman, 2018).

SCPNs from S1 innervate posterior medial nucleus, V1 SCPNs innervate pulvinar and A1 SCPNs project to dorsal division of medial geniculate nucleus (Usrey and Sherman, 2018). These projection patterns imply that L6 CThPNs provide a more modulatory input to the thalamus and SCPNs provide driving inputs (Usrey and Sherman, 2018). Thus, thalamic nuclei receive input from L6 CThPNs and L5 SCPNs. CThPNs project to the thalamus and receive reciprocal thalamic afferents. SCNPs project solely to higher-order thalamic nuclei, providing cortical input. Overall, PNs of the neocortex can be classified by their axonal projections, which in turn indicates their functional role. Upper layer IT neurons project within the cortex while some lower layer IT neurons also project to the striatum. Moreover, lower layer SCPNs project to multiple subcerebral targets and CThPNs solely innervate thalamic nuclei (see Fig. 2).

Figure 2: Long-range axonal projections define two classes of corticostriatal projection neuron

A. SCPNs (blue) project to ipsilateral striatum, thalamus, subthalamic nucleus (STN), and many brainstem and spinal cord regions. Intratelencephalic (IT) neurons project ipsi-or bilaterally (via corpus callosum, Cal), within the cerebral hemispheres to cortex (IT-CCol, orange), and many of these also to striatum (IT-CStr, red) Shepherd (2013).

SCPN

CThPN

B. Development of the neocortex 1. Corticogenesis

Early in development, the telencephalic wall is composed of neuroepithelial cells. They divide symmetrically, thereby generating two more neuroepithelial cells, intended for expansion purposes. These epithelial cells are multipotent and represent the first neuronal population in the neocortex (Haubensak et al., 2004;Greig et al., 2013;Tan and Shi, 2013) ). IPs act as transit-amplifying cells, undergoing asymmetric division to self-renew, limited proliferative divisions, and more often dividing symmetrically to produce two neurons (Haubensak et al., 2004;Noctor et al., 2004;Wu et al., 2005;Sessa et al., 2008). A fourth class of progenitors, the short neural precursors, reside in the VZ, producing post-mitotic neurons directly within the VZ (Gal, 2006;Stancik et al., 2010). Neocortical progenitors begin to produce excitatory PNs around E10.5 (Angevine and Sidman, 1961;Rakic, 1974). Earliest-born cortical neurons migrate past the VZ and form the preplate (PP), which is then split into two structures: the marginal zone (MZ) and the subplate (SP). The marginal zone will give rise to L1 neurons. Between these two structures, the cortical plate (CP) will develop and give rise to the other layers of the neocortex. The first newborn neurons migrate into the CP via somaltranslocation, whereas later newborn neurons move along a glial scaffold via glia-guided neuronal migration. Throughout the rest of corticogenesis, newly born neurons migrate into the CP, organizing themselves in an "inside-out" fashion (see Fig. 3). This implies that earlyborn neurons form lower layers 6 and 5, and late-born neurons populate upper layers 4 -2 (Greig et al., 2013). After neurogenesis is complete, neural progenitors transit to a gliogenic mode, generating astrocytes and oligodendrocytes.

Development of the callosal projection neurons (CPNs)

The birthdate of CPNs is in line with their future location in the radial column of the neocortex, such as CPNs of L6 are born at E12.5 together with CThPNs, L5 CPNs at E13.5 and L2-3 CPNs from E15.5 to E17.5 (Molyneaux et al., 2007). As newborn CPNs are migrating to the CP, they already start to send axons towards premature zones of the developing CC. In ex vivo experiments, Semaphorin 3 has been shown to repel axons away from the cortical marginal zone (Polleux et al., 1998) and guide premature CPN axons towards the midline (Zhao et al., 2011). Parallel to the birth of CPNs, the two telencephalic hemispheres start to fuse with help of glial cells (Lindwall, Fothergill and Richards, 2007) (Norris and Kalil, 1991).

Due to the different birthdates, axons of lower layer CPNs cross the midline before axons of upper layer CPNs. When reaching the contralateral side, CPN axons turn dorsally and extend into the neocortex towards homotopic targets (Fame, MacDonald and Macklis, 2011).

Important signalling molecules responsible for proper guidance of CPN axons are members of the Slit/Robo, Wnt, Netrin and Ephrin families. The specific mechanisms of the callosal axon guidance to their homotopic regions of the contralateral side are largely unknown. However, callosal fibers that fail to cross the midline due to wrong guidance cues or absence of molecular factors remain ipsilateral and form Probst bundles (Fame, MacDonald and Macklis, 2011). Fame, MacDonald and Macklis (2011).

Figure 4: CPN development and diversity

Development of the CST

Axons of the CST originate from SCPNs, residing in lower layer 5B of the cortex. Axons of SCPNs, once generated, have to travel a long distance towards the spinal cord and do so by early outgrowth of the cortex. At E14, CST axons are already reaching the IC and at E17, CST axons are present in the brainstem. At P0, CST cross the midline after the pyramidal decussation and reach the spinal cord at postnatal day (P) 2 (see Fig. 5).

Initially, corticofugal projections, including axons from SCPNs and CThPNs, grow out of the cortex and make a lateral turn away from the midline, thus diverging from callosal projections (Welniarz, Dusart and Roze, 2017). This long trajectory implies proper guidance by extrinsic signals. Guidance cues accompany pioneer axonal projections and ensure they reach their targets. After CST axons have left the cortex, Semaphorin family members SEMA3A and SEMA3C together with NETRIN-1, attract CST axons away from the midline (Srivatsa et al., 2014). After CST axons have passed the pallial/subpallial boundary (PSPB) -a molecular and cellular border between pallial and subpallial structures -they make a medial turn to enter the IC, attracted by Slit guidance ligand 1 and 2 (SLIT1/2) and ROBO1/2 receptors (Bagri et al., 2002;Lopez-Bendito et al., 2007). Passing the IC, CST axons, with the help of NKX-2, enter the brainstem in the caudal medulla reaching the pyramidal decussation (Welniarz, Dusart and Roze, 2017). At the level of the pyramidal decussation, CST axons are starting to cross the midline. Several molecules are involved in the corticospinal midline crossing of CST. NETRIN-1, DCC and UNC5H3 are crucial factors, as loss of these molecules in animal models leads to a failure of most CST axons to cross the midline. The exact mechanisms are still not known and it is uncertain how the small proportion of CST axons are instructed not to cross the midline (Welniarz, Dusart and Roze, 2017). What is known is that once the CST cross the midline, they are prevented from re-crossing by repellent protein EPHRIN-B3, secreted from the midline (Kullander et al., 2001). However, a small fraction of CST axons does not cross the midline at the pyramidal decussation and instead project to the ipsilateral spinal cord (Welniarz, Dusart and Roze, 2017 see Fig. 6).

Figure 5: Mechanisms and time course of mouse corticospinal tract (CST) development

The

Development of CTA

Newborn post-mitotic cortical neurons directly start to extend their axons before even leaving the IZ. Characteristic for corticofugal axons, they are attracted by Sema3C expressed in the IZ (Bagnard et al., 1998;Skaliora, 1998). Furthermore, the SEMA3 gradient within the IZ guides CTA towards the lateral side of the cortex (Bagnard et al., 1998). Then, CTA pass through the IZ and reach the lateral part of the IC at E13 -E15.5 (Jacobs et al., 2007). At E15.5, CTA cross the PSPB (Carney et al., 2009). At this point, ventrolateral oriented CTA tend to turn medially in order to reach the IC in the subpallium (Agmon et al., 1995). As it is the case in the development of the CC, also pioneer axons of the very first-born CThPNs in the subplate act as pioneer axons to guide later-arriving CTAs. Guidance cues of the Slit and Robo family ensure CTAs remain in the IC until they reach the diencephalon-telencephalon boundary (DTB, Leyva-Díaz and López-Bendito, 2013). At the DTB, CTA enter the premature thalamus and meet cells of the perireticular thalamic nucleus and the thalamic reticular nucleus (RTN), at E16 (Jacobs et al., 2007). Then, CTA finally reach the thalamus (E18.5 -P2.5) and start to innervate their specific nuclei, a process which can take several days (Jacobs et al., 2007). Therefore, the CTA form proper connections at early postnatal stages, for instance, the first CTA to reach the dLGN appear at P4.5 (Jacobs et al., 2007).

Development of TCA

The reciprocal connection between the cortex and the thalamus starts between E12 -E18 (Leyva-Díaz and López-Bendito, 2013). TCA follow a canonical pattern in order to reach the cortex. They run rostrally to the telencephalon, make a sharp turn at the DTB and follow through the striatum to reach the developing cortex (Leyva-Díaz and López-Bendito, 2013).

TCA are already present in the IC at E13. Interestingly, they even reach the cortex before their appropriate target neurons are even born. Just before birth of the embryo, TCA grow into the CP and form branches and synapses in their appropriate layer (Leyva-Díaz and López-Bendito, 2013).

In summary, the development of the neocortex is organized in an "inside-outside" fashion,

where PNs first populate lower layers and upper layers are populated at later embryonic stages. While PNs migrate to their destined locations, they already start to send axons to their appropriate targets and thus, establish first major connections at early postnatal stages.

C. Genetic regulation of PNs position and identity 1. Positional information in progenitors

The establishment of areas is driven by an interplay between intrinsic and extrinsic mechanisms throughout the development of the neocortex (see Fig. 7). First, broad patterning of areas is initiated by a complex interaction between morphogens and molecular signals secreted from opposing patterning centres of the neocortical primordium (Grove and Fukuchi-Shimogori, 2003;Alfano and Studer, 2013). Beginning at E9.5 in mice, fibroblast growth factors (FGF) 8 and 17 are secreted from the commissural plate, Wnt and Bmp signalling molecules are diffused from the cortical hem and SFRP2 and some Egf signalling molecules derive from the cortical antihem (Greig et al., 2013). Their spatial-and time-dependent secretion establishes a gradient expression of TFs in the VZ, which act in a complementary manner, thereby shaping boundaries in the developing telencephalon. Namely, transcription factors PAX6 (paired box 6), EMX2 (empty spiracles homolog 2), SP8 (trans-acting transcription factor 8) and COUP-TFI (chicken ovalbumin upstream promoter transcription factors 1) are expressed in progenitors of the VZ. [START_REF] Bishop | Regulation of area identity in the mammalian neocortex by Emx and Pax[END_REF]Zhou, Tsai and Tsai, 2001;Sahara et al., 2007), the starting point of corticogenesis. While Pax6 is expressed in a high rostrolateral to low caudomedial manner, Emx2 is expressed in a low rostrolateral to high caudomedial manner. Moreover, Sp8 is expressed in a high rostromedial to low caudolateral manner, whereas Coup-TFI is expressed in a high caudolateral to low rostromedial manner.

Progenitors located at different medio-lateral and rostro-caudal coordinates express distinct levels of these TFs, which establishes a fate map of cortical areas in the VZ. Loss-of-function and gain-of-function studies revealed the areal identity establishment driven by these TFs.

Here, Fgf8 has been shown to function as key organizer of area identity. Upon overexpression of Fgf8, rostromedial areas of the cortex are expanded caudally (Fukuchi-Shimogori and Grove, 2001;Suzuki-Hirano et al., 2010). In contrast, reduced Fgf8 expression in hypomorphic mutants causes caudal areas of the cortex to expand rostrally (Garel, 2003).

Strong caudal expression of Emx2 and Coup-Tf1 promotes specification of sensory areas. In

Nestin-Emx2 transgenic mice, Emx2 is expressed more uniformly throughout the VZ, leading to an increase in the size of visual cortex, and a concomitant size decrease and rostrolateral shift of somatosensory and motor areas. In contrast, upon absence of one allele of Emx2, motor areas expand, and sensory areas shift caudomedially (Hamasaki et al., 2004). Similarly, in Coup-tf1-conditional null mice, motor areas expand dramatically to occupy a large portion of cortex, while sensory areas are displaced to a narrow occipital band that contains compressed, but properly-configured, sensory representations (Armentano et al., 2007;Greig et al., 2013). Rostrally, expression of Pax6 and Sp8 drives specification of motor identity. Both Sp8 and Pax6 conditional null mice, as well as Pax6sey/sey ("small eye") hypomorphic mutants, exhibit a drastic loss of motor areas [START_REF] Bishop | Regulation of area identity in the mammalian neocortex by Emx and Pax[END_REF]Muzio et al., 2002;Zembrzycki et al., 2007). Gain-and loss-of-function in utero electroporation experiments, however, independently support a role for Sp8 in cortical area identity, both by cell-autonomous repression of Coup-tf1 in neocortical progenitors and indirectly by induction of Fgf8 (Sahara et al., 2007;Borello et al., 2014).

These complementary expression patterns allow coordination of areas in any tangential

dimension and form a first rough area pattern, known as the 'protomap' (Rakic, 1988) have a miniaturized body map representation which can also be observed in its corresponding thalamic nuclei (Zembrzycki et al., 2013). On the contrary, ablation of TCA inputs to the developing V1 is leading to a loss of V1 identity, which is not anymore distinguishable from higher order visual areas (Chou et al., 2013). At postnatal stages, TCA innervations are responsible for refining the initial 'protomap', influencing the size and identity of specific cortical areas (Antón-Bolaños, Espinosa and López-Bendito, 2018). Greig et al. (2013).

Genetic regulation of PN diversity

In the beginning of corticogenesis, undifferentiated neurons share molecular sub-type identities. With proceeding developmental stages, neurons start to differentiate and sub-type identities become more distinct from each other. The sub-type identities of post-mitotic PNs are established by cross-repressive molecular controls. Molecular boundaries for example exist between CFuPN and CPN subpopulations. Furthermore, among CFuPNs, molecular controls diverge SCPN and CThPN subpopulations (see Fig. 8).

a. Molecular fate specification of CThPNs

Corticofugal projection neurons are among the first class of neurons to be generated during cortical neurogenesis (E12.5-E13.5) and are populating L6 and L5.

Tbr1

Important for the post-mitotic specification of CThPNs is the transcription factor T-Box Brain Protein 1 (TBR1). Its expression starts in the first post-mitotic neurons, the preplate neurons, followed by subplate neurons. Studies using genetic manipulations revealed that Tbr1 is necessary for the differentiation of L6 neurons. In Tbr1 -/-mice, early-born neurons (E11.5) fail to express a subset of L6 marker such as Transducin Like Enhancer Of Split 4 (TLE4) and Forkhead box protein P2 (FOXP2). Instead these cells express markers associated with L5 neurons, such as B-cell lymphoma/leukemia 11B (CTIP2) and FEZ Family Zinc Finger 2 (FEZF2, McKenna et al., 2011). On the other hand, ectopic expression of Ctip2 and Fezf2 in L6 neurons represses the connectivity of CThPNs to the thalamus and instead cells send their axons to subcerebral targets, characteristic of L5 neurons (McKenna et al., 2011). Chromatin immunoprecipitation assays revealed that TBR1 directly binds Fezf2 and inhibits its expression in vivo, whereas Ctip2 does not seem to be directly regulated by TBR1, as overexpression of Tbr1 in dissociated cells (E12.5 and E13.5) does not change the expression of Ctip2. In a complementary manner, Fezf2-deficient mice show an aberrant expansion of TBR1 expression in L5 at the expense of SCPNs. (McKenna et al., 2011).

Sox5

Another TF involved in the specification of CThPNs is SRY-Box 5 (SOX5). Its genetic inactivation in mice shows a similar phenotype as in Tbr1 -/-mice (Kwan et al., 2008;Lai et al., 2008). SOX5 is expressed at high levels in the CP and SP during the specification of CFuPNs. Its expression is restricted to post-mitotic cells and is present in L6 TBR1-positive post-mitotic neurons but not present in progenitors of the SVZ (Lai et al., 2008). Genetic inaction of Sox5 in mice alters the molecular identity of L6 neurons leading to a failure of these neurons to express TBR1 or the CThPN marker Zinc Finger Protein ZFPM2 (also named FOG2), but instead the SCPNs markers CTIP2 and FEZF2 are expressed (Kwan et al., 2008). In contrast to Tbr1 -/-cortices, the corticothalamic projections of L6 neurons are not affected in Sox5 -/-mutant mice. Instead, an additional subcerebral projection tract appears, which runs within the external capsule and reaches the cerebral peduncle and the pons (Lai et al., 2008).

These studies suggest a reciprocal TF mechanism, where SOX5 acts upstream of TBR1, specifying post-mitotic CThPNs. Both TFs can inhibit the expression Fezf2, thereby indirectly inhibiting the expression of Ctip2 and thus, specification of SCPNs.

b. Molecular fate specification of SCPNs

Fezf2 and Ctip2

FEZF2 is expressed in a subset of committed progenitors during CFuPN generation and remains expressed in post-mitotic SCPN and CThPNs (Eckler and Chen, 2014). In the absence of FEZF2, Ctip2 expression in L5 is downregulated, whereas Tbr1 is upregulated in putative L5 neurons (see Fig. 8). The low-level CTIP2-expression in L6 is not affected though, suggesting a precise layer-specific or time-specific regulation of the repressing machinery of TFs. Interestingly, not only SCPN lineage specification is perturbed in Fezf2 -/-mice, also expression of CThPNs markers such as FOXP2 and Protein Phosphatase 1 Regulatory Inhibitor Subunit 1B (DARPP32), which are decreased in L6 neurons, revealing a function of FEZF2 in CThPN specification (Chen et al., 2008;McKenna et al., 2011). Together with changes in molecular identity, axonal projections are also affected, as typical SCPN projections in Fezf2 -/-cortices are disturbed.

More specifically, L5 projections to the tectum, pons and spinal cord are reduced and fail to reach their final targets, while more axons tend to project to the thalamus (Molyneaux et al., 2005;Chen et al., 2008). On the other hand, overexpression of Fezf2 in progenitors at E13.5

induces Ctip2 expression at similar levels as in L5 neurons, indicating that FEZF2 is acting upstream of Ctip2 (Guo et al., 2013). As overexpression of Fezf2 leads to an induction of Ctip2, Fezf2 absence results in downregulation of Ctip2 in L5 (Hirata et al., 2004). While Fezf2overexpressed cells show a clear migration deficit -they remain in a heterotopic layer beneath the corpus callosum -they anyhow differentiate and project to subcerebral targets (Molyneaux et al., 2005). Interestingly, in Ctip2 -/-mice, L5 neurons fail to reach the spinal cord, indicating that Ctip2 is critical for SCPNs to extend projections to the SC [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF].

Taken together, Sox5 seems to act upstream of Tbr1 as determinant of CThPNs via inhibition of SCPN determinant Fezf2 and indirectly Ctip2. On the contrary, Fezf2 is inhibiting Tbr1 and promotes Ctip2 expression to regulate SCPN specification. Additionally, Fezf2 seems to be involved in CThPN specification.

c. COUP-TFI

Another transcription factor implicated in the post-mitotic lineage specification during cortical neurogenesis is COUP-TFI. Interestingly, Coup-TfI -/-mice show superficial CThPNs, which express abnormal high levels of Ctip2 and Fezf2 (see Fig. 8). Also, Lmo4 expression (highly expressed in motor cortex) is abnormally upregulated in L5 of the somatosensory cortex (Alfano et al., 2014). Surprisingly, the expression pattern of TBR1 is not altered. This observation adds up to the function of COUP-TFI as sensory area identity determinant not only in progenitors, but also in post-mitotic neurons [START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]. Thus, COUP-TFI acts as an area-and laminar-specific regulator, suggesting that the two processes are tightly linked.

d. Fate specification of CPN

Satb2

The peak of CPN generation during cortical neurogenesis appears around E14.5 and the majority of newborn CPN neurons migrate to upper L2-4. Some CPNs are also generated at earlier stages, together with CFuPNs, thus, sharing a developmental boundary. However, their axonal projections are diverging as soon as they exit the CP. The transcription factor special AT-rich sequence binding protein 2 (SATB2) has been found to be critical for CPN specification.

SATB2 is expressed in a subset of neurons in L2-5 during differentiation and can be found in CPNs as well as in associative projection neurons (Alcamo et al., 2008;Chen et al., 2008;Leone et al., 2015). Genetic inactivation studies, using Satb2-deficient mice, revealed that CPN identity markers such as Cadherin 10 (CDH10) or Cut Like Homeobox 1 (CUX1) are drastically reduced. Instead, the SCPN identity marker CTIP2 and others are upregulated in layer 2-4.

Additionally, callosal projections through the CC are severely reduced, and axons project alternatively to subcerebral targets in pathways, normally innervated by CTIP2-positive SCPNs (Britanova et al., 2008). High expression of Satb2 promotes expression of upper layer genes, including Brn2, Cux2 and Svet1 (Tarabykin et al., 2001;McEvilly et al., 2002;Sugitani et al., 2002;Nieto et al., 2004;Zimmer et al., 2004) which are essential for proper CPN development and represses lower layer genes such as Ctip2 (Britanova et al., 2008). In contrast, a study of the B. Chen group showed that low expression of Satb2 activates Fezf2 expression in SCPNs, while strong Fezf2 expression inhibits Satb2 (McKenna et al., 2015). Also, CFuPNs showed lowor medium Satb2 expression, in accordance with the studies of Arlotta et al. (2015) and Leone et al. (2014). This possible combinatory effect of determination genes is further supported by research from our lab. A subpopulation of lower layer neurons co-expresses Ctip2 and Satb2

during cortical neurogenesis and this subpopulation is present until late postnatal stages in somatosensory and motor area (Harb et al., 2016).

To conclude, CPN and CFuPN fate determination is highly regulated by fate determination genes. These TFs mainly repress each other and thereby delineate CThPN, SCPN or CPN identity. In this process, SCPN-determinant Fezf2 is likely to have a key role, as it interacts with both, CPN and CThPN fate specification TFs.

Figure 8: Competing molecular programs direct differentiation of newly-postmitotic projection neurons into one of three broad subtype identities A. The subtype identities of postmitotic PNs 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, preventing neurons of one sub-type from taking on characteristics of another sub-type, are established by the action of cross-repressive molecular controls. One boundary exists between neurons specified as SCPN and those specified as CThPN, and another exists between CFuPN and CPN. Early in corticogenesis, undifferentiated neurons have largely overlapping sub-type identities (top). As development proceeds, neurons differentiate, and sub-types become more distinct from each other (bottom).

B. Known molecular controls represent key nodes of an elaborate transcriptional network, 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 sub-types to shift, with neurons partially or completely acquiring features characteristic of other sub-types. In some mutants, neurons acquire CFuPN identity generally, rather than a well-defined CThPN or SCPN identity. The boundaries between CFuPN and lower layer or upper layer CPN may shift independently of one another., represented by the dashed line between lower layer and upper layer CPN.

CFuPN, corticofugal projection neuron; CPN, callosal projection neuron; CThPN, corticothalamic projection neuron; SCPN, subcerebral projection neuron. Taken from Greig et al. (2013).

Layer-specific molecular markers in the neocortex

This section will give an overview of prominent molecular markers in the neocortex, which are expressed in distinct layers throughout development. The presentation of these markers is limited and intended to help the reader for the result section.

a. Upper layer CPN marker CUX1

CUX1 belongs to the family of homeodomain TFs and is characterized by four DNA-binding domains and a homeodomain that contains a nuclear localization sequence (Aufiero, Neufeld and Orkin, 1994). CUX1 can act as repressor or activator, depending on the target gene (Weiss and Nieto, 2018). In the cortex, CUX1 is highly coupled to Cux-ortholog CUX2, with whom it shares 48% amino acid identity (Quaggin et al., 1996). Both have similar expression profiles, they can act in a complementary manner but differ in some functional roles.

Cux1 mRNA in the cerebral cortex can first be observed in VZ and SVZ where it is expressed in cortical precursors (Nieto et al., 2004;Zimmer et al., 2004). Then, CUX1 is exclusively expressed in upper-layer CPNs (Weiss and Nieto, 2018). This expression profile is maintained during the subsequent postnatal cortical development and remains until adulthood. The level of expression differs in an area-and layer-specific manner. CUX1 is drastically decreased in upper layers of motor area compared to upper layers of S1. In the postnatal and adult S1 cortex, CUX1 levels are higher in layer 4 than in layer 2-3 (Nieto et al., 2004;Ferrere et al., 2006). The functional role of Cux1 seems to be merely restricted to late stages of differentiation of CPNs. Cux1-mutant mice show no migration defects or aberrant specification of upper layer neurons (Cubelos, Sebastián-Serrano, Kim, Moreno-Ortiz, et al., 2008;Cubelos, Sebastián-Serrano, Kim, Redondo, et al., 2008). This suggests, that Cux1 action is not crucial for fate choices or determination of cortical precursor cells (Weiss and Nieto, 2018). Instead, Cux1 mutant mice show reduction in dendritic length and branching, as well as a reduction in spine density and aberrant spine morphology (Cubelos et al., 2010). On the other hand, only CUX1 seems to be important for axonal projection of CPNs, but not CUX2.

Upon Cux1 deletion, the characteristic topographic projection of CPNs through the CC is drastically reduced (Rodríguez-Tornos et al., 2016). Taken together, CUX1 is a very suitable marker for upper layer CPNs at early postnatal stages throughout adulthood. Moreover, CUX1 labels upper layers, thereby delineating the cortex from the lower layers.

b. SCPN marker CTIP2

CTIP2 is a zinc finger TF which acts as transcriptional repressor (Avram et al., 2000;Senawong et al., 2003). CTIP2 starts to be present in the cortical plate during the peak of SCPN production between E12 -E14 (Fig. 3). However, Ctip2 does not seem to be critical for precursor specification of SCPNs, as it is not expressed in the VZ and SVZ (Chen et al., 2008). Ctip2 is a major downstream effector of Fezf2 and is highly expressed in all post-mitotic SCPNs, regulating the extension of axons towards subcortical targets [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]Chen et al., 2008). The expression pattern of CTIP2 in L5 SCPNs is maintained from early postnatal to adulthood [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. The transcription of Ctip2 is repressed by TFs SATB2 or SOX5, which promote CPN or CThPN fate determination in post-mitotic neurons, suggesting that Ctip2 is a critical target of transcriptional regulation in developing PNs (Fig. 8)

Genetic inactivation of Ctip2 revealed its function as regulator of axonal outgrowth and collateral refinement. In Ctip2 -/-mice, SCPNs are still born and migrate to L5, but CSMNs have defects in axonal fasciculation, outgrowth and pathfinding, resulting in failed spinal cord innervation. Axonal projections aberrantly stop at the pons [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF] and instead some axons project to ectopic targets on the dorsal side. Other axonal projections, such as contralateral connections by CPNs are not affected, revealing a specific SCPNs function of Ctip2 [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. Consistently, ectopic CTIP2 expression in E15.5 upper layer neurons is sufficient to redirect axonal projections to subcerebral targets: internal capsule, thalamus and spinal cord (Chen et al., 2008). Moreover, Ctip2 is also highly expressed in medium spiny neurons (MSNs) in the striatum. Here, Ctip2 regulates proper differentiation and their organization as patch matrix, which is traversed by axonal projection of CSMNs. Furthermore, Ctip2 is implicated in regulative expression of guidance molecules in MSNs, indicating a noncell autonomous function (Arlotta et al., 2008). Overall, Ctip2 is involved in the development of immature post-mitotic SCPNs and its expression pattern makes it a reliable marker for L5 SCPN identity.

c. CThPN marker FOG2 (Friends of GATA2)

FOG2 is a non-DNA binding co-repressor that regulates transcription by interaction with other proteins, for example with family members of COUP-TF (Huggins 2001). Its function has been investigated in few studies, such as heart development (Tevosian et al., 2000) or gonadal differentiation (Tevosian et al., 2002), where its function is implicated in cell differentiation.

In the cortex, FOG2 expression starts in post-mitotic cells at E12.5 and expression continues until E15.5 in the CP. At early postnatal stages (P6), FOG2 is specifically maintained in L6 and the only other expression can be found in the cingulate cortex, while after P7 its specific L6 expression levels drastically decreases (Galazo, Emsley and Macklis, 2016). Even though it has been used as a L6 marker in some studies (McKenna et al., 2011;Rouaux and Arlotta, 2013), a functional role of FOG2 has only recently been described in the cerebral cortex (Galazo, Emsley and Macklis, 2016). Interestingly, the specificity of FOG2 is remarkably limited to CThPNs only and is not expressed in any other cortical subclasses, such as in SATB2-(for CPN identity) or CTIP2-(for SCPN identity) expressing neurons in lower layers. Furthermore, FOG2

is strongly expressed in all thalamocortical retrograde labelled CThPNs in L6, at late embryonic and early postnatal stages. Loss-of-function experiments revealed no difference in arealization or lamination in the developing cortex or any mis-expression of molecular identities (Galazo, Emsley and Macklis, 2016). These data indicate that FOG2 is crucial neither for precursor fate determination nor for proper molecular identification of post-mitotic CThPNs during cortical neurogenesis. However, Fog2-cKO mice show reduction of CThPNs projections to their natural targets in the motor cortex but not S1 cortex, indicating that FOG2 might have a guidance role in axonal projections of CThPNs. Additionally, a subpopulation of L6 neurons in the motor cortex aberrantly expresses high levels of CTIP2. Consistently, gain-of-function experiments by mis-expressing FOG2 in SCPNs decreases the CTIP2-expression levels (Galazo, Emsley and Macklis, 2016). Thus, FOG2 and CTIP2 seem to inhibit each other in a reciprocal manner, most likely at early postnatal stages when refinement of post-mitotic neurons takes place. Taken together, even though used in earlier studies as L6 marker, the study by Galazo et al. (2016) broadens the knowledge of FOG2 as a very specific CThPNs marker.

d. CFuPN marker PCP4

The purkinje-cell protein 4 (PCP4) is a 7.6-kDa IQ motif-containing protein acting as a calmodulin regulator (Johanson et al., 2000). Calmodulin is an important protein kinase involved in synaptic plasticity (Xia and Storm, 2005), mainly expressed in the granular cells of the cerebellum and in the cortex (Cabin, Gardiner and Reeves, 1996). It is known that PCP4 positively regulates neurite outgrowth and neurotransmitter release (Harashima et al., 2011).

In addition, deregulation of PCP4 has been observed in various human disorders, including

Alzheimer (Slemmon et al., 1994) and Huntington's disease (Utal et al., 1998). This suggests, that precise regulation of PCP4 level expression is associated with normal brain function.

In the cortex, PCP4 is expressed in neurons of nearly all cortical areas, including frontal, temporal, parietal and occipital regions of the brain. Additionally, PCP4 expression is found in subcortical structures such as hippocampus, caudate putamen and thalamus (Renelt, von Bohlen und Halbach and von Bohlen und Halbach, 2014). Radially, PCP4 RNA has been found in lower layers 5 and 6 of the whole cerebral cortex, with high expression levels in primary sensory areas (Watakabe et al., 2012). Furthermore, tracing studies on thalamocortical afferents of the lateral geniculate nucleus showed PCP4 RNA expression in corticothalamic neurons residing in layer 6 of V1 in the rat. Besides the indication of PCP4 being expressed by CThPNs, another study indicated PCP4 expression in SCPNs [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. Upon purification of CSMNs, CPNs and CThPNs at different developmental stages, [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF], were able to identify gene expression of PCP4 in CSMNs, with high level expression at late developmental stages (P6 -P14). In addition, it has been shown that FEZF2 binds to the proximal promoter of PCP4 in cultured cortical neural progenitors, suggesting a direct transcriptional regulation of PCP4 by Fezf2 (Lodato et al., 2014). Taken together, PCP4

expression is indicated for late developmental CSMNs and CThPNs, projecting to the lateral geniculate nucleus, therefore representing a specific molecular marker of CFuPNs.

e. CThPN marker DARPP32

Dopamine and CAMP-Regulated neuronal Phosphoprotein 32 (DARPP32) is a protein that plays a crucial role in the physiological activity of dopaminoceptive neurons. Dopamine and other neurotransmitters act on dopaminoceptive neurons by changing the phosphorylated state of DARPP32 (W. W. Wang et al., 2004). Major cell recipients of dopaminergic transmission from various regions of the brain are MSNs in the striatum. Here, DARPP32 is important for cortical excitatory projection of MSNs, which are GABAergic projection neurons and represent the vast majority of cells in the striatum (Gerfen, 1992). Around 95% of MSNs express DARPP32 whereas it is absent in all other cells in the striatum (Ouimet et al., 1984;Anderson and Reiner, 1991;Ouimet, Langley-Gullion and Greengard, 1998). Interestingly, also CTIP2 is strongly expressed in MSNs during development and loss-of-function of Ctip2 results in failed MSN differentiation and decreased expression of distinct striatal molecular identifiers (Arlotta et al., 2008). Darpp32 is one among those molecular identifiers that exhibits low expression when Ctip2 is lost, indicating a possible role of Ctip2 in regulating Darpp32 expression in MSN. Beside other brain structures, also the neocortex exhibits Darpp32 expression. In the cortices of WT mice at postnatal stages, only lower layers show DARPP32

protein expression (W. W. Wang et al., 2004). This is in accordance with ISH data from the Allen brain atlas, where strong RNA-expression can be detected in lower layers during postnatal development of the neocortex. More precisely, Darpp32 expression has been linked

to layer 6 CThPNs in the rat cingulate cortex, thus, indicating a specific expression pattern for CThPNs (Ouimet, 1991). Taken together, Darpp32 is highly expressed in striatal MSNs and in lower layer 6 of the cortex, where it has been suggested to be expressed by CThPN.

Areal specialization of post-mitotic projection neurons

Although TFs expressed in the VZ are involved in impinging a pre-identity to cortical progenitors during development, recent studies have shown that expression in early postmitotic neurons seem to be key in establishing and maintaining areal specification. Among some of these TFs is Basic Helix-Loop-Helix Family Member E22 (BHLHB5), the LIM Domain Only 4 (LMO4), Pre-B-cell leukemia homeodomain 1 (PBX1), the nuclear receptor COUP-TFI, LIM Homeobox 2 (LHX2) and B Cell CLL/Lymphoma 11A (CTIP1, Kashani et al., 2006;Joshi et al., 2008;Huang et al., 2009;Alfano et al., 2014;Perez-Garcia et al., 2015;Greig et al., 2016).

a. BHLHB5

Bhlhb5 is expressed in a high-caudomedial to low-rostrolateral gradient in the CP in layer 2-5 neurons of primary sensory areas (Joshi et al., 2008). Bhlhb5-deficient mice show normal cortical lamination but the molecular identity in the caudal motor and somatosensory regions is affected. Regionalized markers, such as Cdh8 that are normally expressed in layers of rostral motor area, are ectopically expressed in S1 of Bhlhb5 mutant mice. In addition, the sensory area promoting Coup-TFI is abnormally expressed in S1 cortex. The barrels of S1 appear to be disorganized in Bhlhb5 -/-cortices. In L4, barrel fields are organized of walls and of a cell-free hollow, which is innervated by axons from the thalamic VPM nucleus. In Bhlhb5-mutant mice, these clear separated barrel walls appear blurry and reveal indistinct borders. Another effect in Bhlhb5-deficient mice can be observed in the number of CSMNs. Fewer CSMNs are located in rostral motor cortex and more CSMNs are located in the caudal motor cortex. Lastly, Lmo4

expression is lost in the somatosensory area, which is expressed in L4 of S1 cortex (Joshi et al., 2008). Thus, BHLHB5 acts as post-mitotic regulator of sensory identity by controlling barrel formation in L4 and CSMNs in L5.

b. LMO4

Developmentally regulated genes that constitute a program of combinatorial molecular genetic controls, mediate the specification and differentiation of subtypes of cortical PNs.

Lmo4 has been shown to establish rostral motor cortex PN subtype diversity (Cederquist et al., 2013). In this study, the motor cortex has been subdivided into rostral The total knockout of Lmo4 in mice leads to embryonic lethality (Lee et al., 2005) and the few pups that survive, die within two hours after birth (Huang et al., 2009). To overcome this severe phenotype, studies on loss-of-function of Lmo4 used conditional knock out mice (Kashani, Qiu, et al., 2006;Huang et al., 2009). Upon restricted deletion of Lmo4 in the CNS, expression of cortical regional markers CDH8 and ID2 (Bishop, Goudreau and O'Leary, 2000) were altered. The expression of Cdh8 in upper layers of the anterior cortex was reduced in Lmo4 cKO cortices and L5 Id2 expression was shifted rostrally. Deletion of Lmo4 in post-mitotic neurons leads to perturbations in the development of the barrel fields, as Lmo4 cKO cortices showed poorly differentiated and blurred barrels (Kashani, Qiu, et al., 2006). Furthermore, TCAs in Lmo4 cKO mice do not segregate into distinct terminal patches. These results show that conditional deletion of Lmo4 does not prevent thalamocortical projections from reaching the cortex, but it does affect their patterning in the target tissue (Kashani, Qiu, et al., 2006).

Interestingly, upon deletion of Lmo4 in the cortex, mice showed impaired sensory perception and motor control, revealing a role in sensory motor control. Taken together, Lmo4 is important for the regulation of the shape of cortical functional areas, development of the barrel subfield and is implicated in sensory and motor functions. Moreover, Lmo4 shows to be important for post-mitotic SCPNs identity and projections as well as barrel field maintenance in S1 cortex.

c. PBX1

Another TF that has been shown to control areal specification in both, progenitors and postmitotic cells, is Pre-B-cell leukemia homeodomain 1 (PBX1, Golonzhka et al., 2015). In adult mouse brains, Pbx1 is implicated in the homeostasis of adult neurogenesis in the SVZ (Grebbin et al., 2016). In the developing cortex, Pbx1 has been shown to be expressed throughout the telencephalic VZ and SVZ at E12.5 with a caudorostral gradient (Toresson, Parmar and Campbell, 2000;Golonzhka et al., 2015). At E18.5, its expression follows a rostral high to caudal low expression in upper layers (Golonzhka et al., 2015). At later stages, Pbx1 seems not to be expressed in the cortex anymore (P14 -P56), revealed by ISH data (Allen Brain Atlas), suggesting a primary developmental role. However, Pbx1 cKO in progenitors and post-mitotic cells show an aberrant areal specification, as revealed by downregulation of Lmo4 expression in the rostral region of the brain with more severe affection when deleted in progenitors than in post-mitotic cells (Golonzhka et al., 2015). Interestingly, Reelin is mis-expressed in the deep cortical plate (E15.5) and an inversion of cortical layers in the rostral cortex can be observed at P8, shown by ectopic expression of lower layer markers in upper layers (Golonzhka et al., 2015).

d. Other post-mitotic area regulators

Among being an area pattering gene in progenitors, Coup-TFI is also expressed in post-mitotic neurons, where it promotes sensory area identity (Alfano et al., 2014). Also, Lhx2 or Ctip1 have been shown to regulate sensory identity in post-mitotic neurons (Perez-Garcia et al., 2015;Greig et al., 2016).

The motor area as "output" region

The frontal motor area (M1) is the major center of output signals in the neocortex and is supposed to be the most recent area in placental mammals (Kaas, 2011). The motor area is different from other primary areas, as it does not receive direct input from peripheral signals.

Interestingly, it also has some cytoarchitectural unique features compared to primary sensory areas. The adult primary motor cortex contains a large number of CSMNs and has a thick layer 5, whereas S1 is characterized by a thick layer 4 [START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]. The prominent staining of L4 stellate PNs in primary sensory areas is lacking in the motor area (Brodmann, 1909). Stellate cells are the main recipients of thalamocortical inputs, have extensive and largely unidirectional projection to the superficial cortical layer (Yamawaki et al., 2014).

Stellate cells can easily be detected as they exhibit strong Rorβ-expression in S1, but only faint expression in M1, in accordance with a very thin L4 in this area. Interestingly, the Rorβ expression is complementary to Ctip2 expression: Ctip2 is more broadly expressed in L5 in the motor cortex, while its expression is narrower in the somatosensory area (see Fig. 9, Dehay and Kennedy, 2007;[START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]Harb et al., 2016). These observations suggest that the motor area has a considerably less amount of associative projection neurons but instead an increased number of SCPNs, showing broad expression of Ctip2. Given its functional role in the neocortex as center of planning voluntary movements, its cytoarchitecture resembles its function and the motor area can be seen as the center of neocortical "output".

known as Fezl and Zfp312, Shimizu et al., 2010;Weng, Golden and Lee, 2010). They have been first discovered in the anterior neuroepithelium of Xenopus and zebrafish embryos (Hashimoto, Itoh, et al., 2000;Hashimoto, Yabe, et al., 2000;Matsuo-Takasaki et al., 2000).

Both members share an N-terminal Engrailed homology 1 (Eh1) repressor domain, allowing protein-protein interaction, which has also been shown to interact with the Groucho/TLE family of transcriptional co-repressors (Hashimoto, Yabe, et al., 2000;Eckler and Chen, 2014).

Furthermore, both Fez members contain six C2H2 zinc-finger DNA binding motifs at the Cterminal. Fezf1 and Fezf2 exhibit high levels of homology, particularly within their DNA binding and protein-protein interaction domains (see Fig. 10).

Figure 10: The Fez family of transcription factors.

A Schematic of FEZF1 and FEZF2 proteins highlighting the strong evolutionary conservation of the Engrailed homology 1 (Eh1) and zinc-finger DNA binding domains. Conservations is reported as percent homology to Homo sapiens FEZF1. Taken from Eckler and Chen (2014).

a. Fez expression during embryogenesis and early postnatal stages

At E8.5, Fezf2 and Fezf1 exhibit similar and overlapping expression patterns in the developing forebrain, notably, also in the olfactory system. At E10.5, both Fez members are still expressed in an overlapping pattern in the forebrain and the olfactory system. Fezf1 is expressed in neural progenitors throughout the forebrain and olfactory pit, Fezf2 is also expressed in progenitors of the forebrain, but within the olfactory pit it is restricted to progenitors that will give rise to the vomeronasal organ (VNO) (Hirata, 2006a;Eckler et al., 2011;Eckler and Chen, 2014) , . As development proceeds, both factors start to demarcate distinct neuronal structures. At E15.5, Fezf1 is broadly expressed in the main olfactory epithelium (MOE) and its expression vanishes from the cerebral cortex, but remains in the developing amygdala, ventral thalamus and hypothalamus (Hirata, 2006b(Hirata, , 2006a;;Kurrasch et al., 2007;Watanabe et al., 2009;Eckler et al., 2011). In contrast, Fezf2 is expressed within the VNO, neocortical progenitor cells and newly born deep-layer projection neurons, with high expression levels in L5 and L6 neurons. In addition, Fezf2 is expressed in subcortical structures, such as thalamic eminence, prethalamus, developing amygdala and hypothalamus (Inoue et al., 2004;Hirata, 2006bHirata, , 2006a;;Watanabe et al., 2009;Eckler et al., 2011). At birth, expression of these factors mostly mimics their adult patterns (see Fig. 11). Expression of Fezf1 is confined to progenitor cells and sensory neurons of the MOE as well as neurons of the hypothalamus and amygdala (Eckler and Chen, 2014). Fezf2 on the other hand is expressed at high levels within L5 neurons of the neocortex and at lower levels within L6 neurons. In subcortical structures, Fezf2 is expressed in the VNO and neurons in the hypothalamus and amygdala (Kurrasch et al., 2007;Eckler et al., 2011). The dynamic and overlapping expression patterns of both genes underscore their essential function during development of multiple structures within the nervous system.

b. Fez genes during development of the olfactory system and diencephalon

As indicated by their expression patterns during embryogenesis, Fezf1 and Fezf2 play important roles in the development of the olfactory system. Genetic loss-of-function studies show that Fezf1 is required for maturation of neurons in the olfactory system and their axonal projection to the olfactory bulb (Eckler et al., 2011). Absence of Fezf2 during embryogenesis leads to decreased proliferation and apoptosis of sensory neurons in the olfactory system (Eckler et al., 2011). Single knockout of either Fezf1 or Fezf2 in the developing diencephalon (composed of prethalamus, thalamus, hypothalamus and pretectum), has no effect on diencephalic patterning. However, double mutant mice reveal redundant roles in patterning of these structures: the prethalamus is completely lost, accompanied by a rostral expansion of the pretectum and marked reduction in thalamic size (Hirata, 2006b). In addition, regional markers such as GBX2 and LHX1 revealed that the rostral diencephalon fail to be specified and thus allowing rostral expansion of the caudal diencephalon. These observations indicate a redundant function of Fezf1 and Fezf2 during early diencephalon patterning.

c. Fez genes play unique and redundant roles in forebrain neurogenesis

Expression patterns of Fezf1 and Fezf2 transcription factors in the developing cerebral cortex suggest a functional role at early developmental stages. Indeed, both Fez genes are implicated in controlling the neurogenic program of neocortical progenitors (Shimizu et al., 2010). In double mutant mice, Hes5 expression was increased across the forebrain. Hes5 belongs to the Hes genes, vertebrate homologs of Drosophila hairy and enhancer of split, which encode bHLH transcriptional repressors. HES proteins repress the expression of bHLH proneural genes, which promote neurogenesis (Ross, Rao and Kadesch, 2004;Kageyama, Ohtsuka andKobayashi, 2007, 2008). Hence, depletion of Fezf1 and Fezf2 leads to decreased neurogenesis and subsequent decreasing numbers of PNs born during early corticogenesis (see Fig. 12).

Interestingly, single knock-out of Fezf1 or Fezf2 does not lead to any defects in cortical neurogenesis, suggesting a redundant regulation of Hes5 transcription. Fezf1 is only expressed during early stages of cortical development. In contrast, Fezf2 is expressed in neocortical progenitors throughout cortical neurogenesis. Fezf2 is expressed in RGCs, which give rise to all major PN subtypes as well as glial cells (Guo et al., 2013). This indicates that Fezf2 expression in early neocortical progenitors does not direct these cells towards a specific cell fate. Nevertheless, as addressed before, Fezf2 is essential for the specification of SCPNs during cortical neurogenesis via inhibition of alternative fate genes.

E. Lmo4

Lmo4 is a member of the subclass LIM domain only (LMO), a family of nuclear transcription co-regulators (Sang et al., 2014) and is characterized by the presence of two tandem LIM domains and no other functional domain (Deane et al., 2004, see Fig. 13). The LIM domain is a highly conserved cysteine-rich zinc finger-like motif, found in a variety of nuclear and cytoplasmic proteins (Sang et al., 2014). The LIM domain does not directly bind to DNA but instead mediates multiple protein-protein interactions (Kenny et al., 1998;Matthews et al., 2008) and can serve as docking site for the assembly of multiprotein complexes (Sánchez-García and Rabbits, 1994;Dawid, Breen and Toyama, 1998). Therefore, LMO proteins regulate gene transcription by functioning as 'linker' or 'scaffolding' proteins due to their LIM domains and are involved in the formation of multiprotein complexes of DNA-binding factors and transcriptional regulatory proteins (Dawid, Breen and Toyama, 1998;Kenny et al., 1998;Bach, 2000;Sang et al., 2014, see Fig. 13).

LMO proteins are a conserved subclass, found in several species such as C. elegans, Drosophila, Zebrafish and humans (Rétaux and Bachy, 2002;McCollum et al., 2007;Patterson et al., 2007;Matthews et al., 2008). Four members of the LMO subclass have been identified so far: Lmo1 -Lmo4. The latter one, Lmo4, was the most recent described family member and was isolated as an interacting protein of LDB1 (also known as NLI or CLIM, Grutz, Forster and Rabbitts, 1998;Kenny et al., 1998;Sugihara, 1998;Racevskis et al., 1999). It is the most divergent member of the LMO subclass and is widely expressed throughout embryonic development and adult tissue. In contrast, the expression patterns of Lmo1, Lmo2 and Lmo3 are more tissue restricted (Sang et al., 2014). The functional role of LMO proteins has been extensively studied in cancerous tissue (Boehm et al., 1990) and observations revealed the main role of LMO proteins as nuclear transcriptional co-regulators, mediating protein-protein interactions of various transcription factors or chromatin modelling proteins which may have either positive or negative effects on gene transcription (Sang et al., 2014).

LMO4 is composed of 165 amino acid residues and has a broad expression spectrum in human tissue and in mice, where its expression can be found in T lymphocyte lineage, cranial neural crest cells, somite, dorsal limb bud mesenchyme, motor neurons and Schwann cell progenitors (Kenny et al., 1998). Furthermore, Lmo4 plays important roles in several developmental systems, such as epithelial, mammary, ear and neural development (Sugihara, 1998;Hahm et al., 2004;Lee et al., 2005). Also, Lmo4 is implicated as an oncogene, as it is overexpressed in more than 50% of primary breast tumours and its overexpression markedly reduces differentiation of mammary epithelial cells (Visvader et al., 2001;W. W. Wang et al., 2004).

So far, Lmo4 has been shown to interact with various proteins or protein complexes. For instance, it interacts with the ovarian tumour suppressor protein BRCA1, thereby inhibiting the transcriptional activity of Brca1 in yeast and mammalian cells (Sum et al., 2002). In the developing cortex, LMO4 and its binding partner BLI/LDB1/CLIM2 interact with NEUROGENIN2, thereby forming a multi-protein transcription complex. Subsequently, this complex is recruited to enhancers of neurogenin2 sequence and activates its transcription (Asprer et al., 2011). Therefore, Lmo4 can act as activator or repressor depending on the proteins it interacts with and depending on the cellular context (see Fig. 14.)

Interestingly, Lmo4 has also been shown to bind to estrogen recptor α (ERα) and MTA , thereby forming a multi-protein complex consistent of LMO4, ERα, MTA and histone deacetylases (HDACs) in vivo (Singh et al., 2005). MTA1 is a component of the NURD complex, a group of associated proteins with ATP-dependent chromatin remodelling and histone deacetylase activities (Xue et al., 1998). LMO4 here functioned as repressor of ERα in an HDACdependent manner, suggesting that LMO4 may play a role in chromatin remodelling complexes. Indeed, in the postnatal mouse cortex LMO4 is sequestering HDAC1, a critical component of the NURD complex, thereby preventing its binding to the Ctip2 locus. Therefore, LMO4 acts as de-repressor, allowing Ctip2 expression (Harb et al., 2016, see Fig. 15).

Figure 15: Schematic model of the putative mechanism by which Lmo4 de-represses Ctip2 expression

The locus of Ctip2 is repressed by Satb2 which is linked to the NuRD complex including HDAC1. Lmo4 sequesters HDAC1 thereby de-repressing the Ctip2 locus and allowing Ctip2 expression. Taken from Harb et al. (2016).

a. Expression pattern of Lmo4

As mentioned above, Lmo4 is an important factor for neocortical development and is implicated in arealization and neuronal subtype specification. In the very early stages of cortical development, Lmo4 is expressed in the VZ and the preplate (E11.5, Asprer et al., 2011).

Later on, at E15.5, expression of Lmo4 is found in the SVZ and the hippocampus. At birth (P0), expression of Lmo4 is merely expressed in post-mitotic neurons of the cortical plate.

Tangentially, Lmo4 expression is found in the rostral (motor area) and caudal part (visual area) of the cortex, while its expression is almost absent in between, where the presumptive somatosensory area is located. At P5, Lmo4 is increasingly expressed in upper layers 3 and 4 of the somatosensory area. At P10, Lmo4 is expressed in all layers of the entire cortex (Huang et al., 2009) and this expression pattern is retained in the adult brain (Allen Brain Atlas). The dynamic expression pattern during cortical development indicates a role for Lmo4 in defining boundaries between areas and post-mitotic neuron layer-specification.

b. Lmo4 in cortical subtype specification

The expression of Lmo4 in the developing neocortex is highly dynamic and region-specific.

During early stages of cortical neurogenesis (E11.5 -E18.5), Lmo4 starts to be expressed in post-mitotic neurons of the PP and the CP (Azim et al., 2009;Harb et al., 2016). Tangentially,

Lmo4 is highly expressed in the rostral/frontal and caudal/occipital part of P0 cortices but at lower levels in the presumptive S1 area (Huang et al., 2009). By the time S1 is fully specified (P10), Lmo4 is expressed in all layers across the entire cortex (Huang et al., 2009). At the cellular level, Lmo4 is co-expressed with markers of various cell identity in the CP. At E15.5, Lmo4 is expressed in SATB2-positive cells, but also in FEZF2/CTIP2-positive cells as well as in SOX5-positive cells, and this co-localization is maintained until P0. At late postnatal stage P6, Lmo4 is excluded from most Ctip2-expressing neurons and nearly all Lmo4-expressing neurons have acquired CPN identity (Azim et al., 2009).

Lmo4 together with Clim1 has been shown to progressively delineate cortical PN subtypes during development (Azim et al., 2009). At E15.5 Lmo4 and Clim1 are both expressed postmitotically in the CP, in an overlapping subset of PNs. Interestingly, a subset of CP neurons expresses both Fezf2 and Lmo4, therefore indicating that a subpopulation of neurons destined to project subcerebrally co-express Lmo4 in early development stages (Azim et al., 2009).

II. Cell reprogramming A. General Introduction

The ability of a differentiated cell to keep its identity is of eminent importance since each cell plays a unique function within a network of cells that in turn contributes to proper functioning of distinct biological systems. During embryogenesis, differentiating cells become committed to a highly specialized one and due to epigenetic barriers, its ability to convert into a cell type of another lineage is prevented. Epigenetic modulations are modifications of gene expressions, which can lead to their silencing (barrier) or activation. Plasticity of a cell therefore can be described as the ability of a cell to switch to new identities (Filip et al., 2005).

This ability of switching to new identities is decreasing as a cell differentiates; therefore, the plasticity is reduced over time.

A problem can arise when one cell or several cells are defective, which can affect the local network and ultimately, the whole system. During evolution various species have found different solutions to either regenerate or repair defective cells. While in Hydra, the regeneration potential is so large that it can basically regenerate itself after being fully dissociated (Technau et al., 2000), the regeneration capacity of mammals is very limited. If multipotent or pluripotent stem cells are lacking, terminally differentiated cells cannot simply be replaced. Therefore, biomedical research has great interest in generating specific cell types that can replace defective ones. For example, converting cells into cell-types of defective ones could restore the function of a network. The pioneer study of Gurdon (Gurdon, 1962a) revealed that it is possible to de-differentiate mature cells into an immature state with pluripotent stemness. The feasibility of reprogramming cells led to subsequent observations in different ways and approaches. The study of Davis (1987) elegans. In the following section, I would like to decipher differences in reprogramming approaches and their according nomenclature.

B. Nuclear reprogramming

The first attempt to reprogram cells has been conducted by Gurdon et al. (1962) even though the intention was different from the reprogramming approaches nowadays. At this time, the aim of Gurdon and colleagues was to understand whether somatic (non-reproductive) cells maintained their genome integrity after differentiation. The experimental approach worked as follows: the nucleus of a somatic cell was isolated, while the chromosomes of an unfertilized egg (Xenopus laevis) were destroyed by UV irradiation. Then, the donor nucleus was transferred to a recipient egg via micropipette injection and as a result, a frog containing the genetic origin of the donor could properly develop. This technique paved the way for the first reproductive cloning experiments in mammals, a prominent example is the sheep called Dolly, which represents the first successful cloning of a mammal (Wilmut et al., 1997). These studies indicated that cells retain their genetic makeup and that the plasticity of a cell is strongly dependent on its environment. They also suggested that expression of genes is a dynamic process during development and that silencing of pluripotent genes can be experimentally reversed. Besides ethical issues, reprogramming via somatic nuclear transfer has limitations and disadvantages. The reprogramming efficiency using adult somatic cells is very low (1 -2% normal adult animals), some genes are not properly transcribed after reprogramming (approx.

5%, Humphreys et al., 2002;Vassena et al., 2007) and the normality of nuclear transplant embryonic development decreases with nuclei harbouring increased differentiation states (Gurdon, 1962b). Additionally, as not all genes are properly transcribed, clones usually show abnormalities (Yang et al., 2007) and cross-species nuclear reprogramming in mammals leads to death after few rounds of meiosis (Gurdon and Wilmut, 2011). However, nuclear transfer reprogramming has enlightened our understanding of genetic integrity of differentiated cells and paved the way of cell reprogramming.

C. Cell fusion reprogramming

Another approach of nuclear reprogramming is represented by the fusion between somatic and pluripotent cells (Yamanaka and Blau, 2010). Upon fusion of the membranes, the nuclei of both cells can stay separated in the "newly-hybrid" cell and without cell division form a heterokaryon. Another outcome is a synkaryotic cell with fused nuclei (hybrid genome) of both cell types, which undergoes cell division (Yamanaka and Blau, 2010). Interestingly, a pluripotent cell (e.g. embryonic stem cell) functions in a dominant way, thereby activating silent genes including pluripotent factors such as Oct4 (Tada et al., 2001). This approach is useful for elucidating molecular programs that are responsible for the reset of pluripotent genes, for example DNA methylation (Yamanaka and Blau, 2010). Furthermore, heterokaryons of mixed species allow deep comprehensive examination of nuclear reprogramming, as gene expression changes can be monitored on the bases of species-specific differences (Yamanaka and Blau, 2010). One major limitation of this approach lies in the fusion of nuclei, as tetraploid cells are unsuitable for therapeutic approaches (Ficz and Reik, 2013). However, cell fusion reprogramming is useful for examining early reprogramming mechanisms, such as rearrangement of methylation sites or chromatin alterations.

D. Cell reprogramming by cell extract treatment

Cells can also be reprogrammed by exposition to protein cocktails from pluripotent stem cells (Taranger et al., 2005). To this purpose, the membrane of differentiated cells (of interest) are permeabilized and incubated with protein extracts of pluripotent stem cells. The membranes can be re-sealed and plated on cell dishes. These cells then start to express previously silent stem cell genes (Taranger et al., 2005). This supports the notion, that changing the

E. Natural occurring de-differentiation and trans-differentiation

De-differentiation of cells is a common regenerative mechanism reported for many species including humans (Jopling, Boue and Belmonte, 2011). For example, liver or blood cells are continuously generated from somatic cells. More specifically, some differentiated liver cells are reverted to a less-differentiated state, proliferate and then re-differentiate again into the original cell-type. Blood cells are generated by somatic stem cells which, after activation, proliferate and give rise to differentiated blood cells (Jopling, Boue and Belmonte, 2011). This natural de-differentiation allows local regeneration and is crucial for maintenance of tissues.

Another phenomenon occurring naturally is trans-differentiation: conversion of a somatic cell into a cell from another lineage has been reported to occur naturally in certain species (Jopling, Boue and Belmonte, 2011;Beyret and Belmonte, 2014). The term transdifferentiation will be used in this work to name the natural phenomenon, whereas reprogramming will be used as term for experimental conversion of cells. Transdifferentiation in newts has been reported long time ago, at the end of the 19 th century (Call, Grogg and Tsonis, 2005). Upon removal of the lens, pigmented epithelial cells of the iris de- PDA (Beyret and Belmonte, 2014). This behaviour is occurring with invariant precision and therefore allowed the researches to examine the mechanisms responsible for the efficiency of this event. Briefly, H3K4 methyltransferase Set1 associates with subunits of a complex, which normally ensures embryonic stem cell fate and thereby initiates de-differentiation of the Y cell. Subsequently, this complex cooperates with H3K27me2/3 demethylase and a transcriptional regulator of terminal motor neuron identity Unc-3, to re-differentiate the Ycell into a motor neuron. These sequential histone-modifications demonstrate how important epigenetic mechanisms are for trans-lineage conversion of cells and reveal useful information for experimental reprogramming approaches.

F. Reprogramming to induced Pluripotent Stem Cells (iPSCs)

The field of reprogramming became more popular thanks to the milestone-publication of [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]Yamanaka in 2006 (Takahashi andYamanaka, 2006). Virally induced overexpression of four transcription factors -Oct4, Sox2, Klf4 and Myc (OSKM)converted mouse fibroblasts into pluripotent stem cells, which re-acquired ES cell properties. The pluripotency of these iPSCs were examined by teratoma formation, a benign tumour characterized by rapid growth in vivo carrying remnants of all three germ layers. These tumours developed in mice after injection of reprogrammed cells and differentiated into cells of all three germ layers, including neural tissue, cartilage and columnar epithelium (Takahashi and Yamanaka, 2006). This study demonstrated for the first time that somatic cells can be reprogrammed to a pluripotent state by overexpression of ES-type TFs able to silence somatic maintenance factors. Upon reprogramming to iPSCs, these cells can be re-differentiated into any somatic cell type (Halley-Stott, Pasque and Gurdon, 2013). The discovery of iPSCs boosted the field of reprogramming and a tremendous amount of discoveries has been made ever since, too numerous to be covered in this thesis work (see Heinrich, Spagnoli and Berninger, 2015;Masserdotti, Gascón and Götz, 2016;Takahashi and Yamanaka, 2016;Torper and Gotz, 2017). Nevertheless, some major findings, advances and limitations of iPSCs are described below.

After the publication of murine iPSCs, the subsequent study of reprogramming human fibroblasts into iPSCs followed just one year after (Takahashi et al., 2007;Yu et al., 2007). From that moment on, experiments on human pluripotent cells exploded, since ethical issues had been widely extinguished: a problem that existed before by working on human ES cells, as they had to be derived from human embryos. Somatic cells from patients can be derived and reprogrammed into iPSCs, which then are differentiated into the disease-relevant cell-type.

These cells are then examined for disease aetiology and pathological mechanisms can eventually be revealed (Shi et al., 2017). Thanks to new genetic modification tools, such as CRISPR-Cas9, iPSCs of a patient can be modified and used as internal control. Patient-derived iPSCs can further be used as a disease model to identify potential drugs for therapy. Another application has been demonstrated by the repair of genetic mutations in murine iPSC and their subsequent transplantation in the diseased mouse model from which the iPSCs originated.

These "newly regenerated" progenitors were able to counteract the disease (Hanna et al., 2007). This approach is now starting to become reality for treating diseases in humans. In Japan for example, macular degeneration has been treated with iPSC-derived retinal pigment epithelium, even though with moderate success (Mandai et al., 2017). In addition, thanks to the iPSC technology, 3D-organoids have been generated from patients to study genetic diseases, thus adding another way of modelling genetic diseases via iPSCs (Lancaster et al., 2013;Di Lullo and Kriegstein, 2017).

Nevertheless, a major concern for iPSC technology has been the delivery of OSKM factors into the somatic cells, as the viruses used by Takahashi integrates into the host genome (Yamanaka and Blau, 2010). Advances have been made to overcome this hurdle by using integration-free delivery methods such as synthetic mRNA transfection (Warren et al., 2010), episomal plasmid vectors (Okita et al., 2008) or Sendai-virus infection, a virus which does not integrate into the host genome (Fusaki et al., 2009). To date, iPSCs can be easily derived from different cell sources and thus, somatic cells can be more efficiently reprogrammed than initially reported (Kogut et al., 2018), and large numbers of iPSCs can be produced in fully controlled bioreactors in defined culture media (Martin, 2017). In addition, the process of differentiating iPSCs has been improved by using small molecules and biochemical reagents (Martin, 2017).

The undoubtedly success of iPSCs increased our knowledge of cell plasticity, their underlying mechanisms -such as epigenetic landmarks or chromatin accessibility, the discovery of pluripotent factors and the subsequent application opportunities. Despite the advances in cell reprograming by iPSCs, some hurdles still remain. A major problem in iPSC technology is that several targeted cells do not fully reprogram and therefore can undergo defective differentiation (Takahashi and Yamanaka, 2016). Partially reprogrammed cells exhibit epigenetic landmarks of somatic cells but depict ES cell characteristics, such as proliferation, onset of pluripotency-associated genes or teratoma-forming activity (Takahashi and Yamanaka, 2016). This partial cell reprogramming could explain the widely reported low full conversion rate of iPSC reprogramming. In fact, the reprogramming mechanism seems to be split in two steps. Initially, OSKM factors bind to genes, which are responsible for somatic identity and thus, silence them. The second step is the induction of pluripotent genes, which seems to be largely inefficient due to the presence of repressive methyl-histone marks (Takahashi and Yamanaka, 2016). As a result, even if many somatic cells will silence their differentiated genetic repertoire, pluripotency fails to be activated by OSKM factors, leading to a partial reprogramming. The homogeneity of fully reprogrammed cells is important for further clinical usage to ensure aberrant development of iPSCs, which could lead to cancerous formations in the host organism (Takahashi and Yamanaka, 2016). Another problem arises from the variability of individual iPSC lines regarding their potential to differentiate into fully functional cells of a defined lineage (Hockemeyer and Jaenisch, 2016). This cause is derived from epigenetic memories, which could be somatic methylation patterns that remained in reprogrammed iPSCs. In turn, using iPSCs as disease model can be tricky if small phenotypic differences between control and patient iPSCs are not the cause of the disease, but rather come from variations between individual iPSC lines (Soldner and Jaenisch, 2012;Hockemeyer and Jaenisch, 2016). Another question is, to which extent differentiated iPSC-derived cells mimic in vivo cell maturity. So far, the vast majority of iPSC-directed differentiations produce populations that are more similar to foetal or neonatal cells than to mature cells (Berry et al., 2018). This implies that the right time for iPSC-differentiated cell transplantation has to be carefully evaluated. For instance, a mature cell would be equipped with full functionality, but might not be plastic enough to integrate into the system and vice versa. In conclusion, iPSC technology is an exciting field, which not only increased our understanding of cellular plasticity but also gives us a powerful tool to design and generate almost any kind of cell-type in vitro.

Nevertheless, it shows limitations that need further investigations to overcome.

G. Direct reprogramming

Generation of desired functional cell types is one major goal of regenerative medicine. For this purpose, differentiation of iPSCs has been extensively studied and applied in certain disease situations (FUSAKI et al., 2009;Warren et al., 2010;Zhou et al., 2010;Shi et al., 2017).

However, because of the limitations described above, there has been great interest in developing alternative strategies. One of these alternatives is represented by direct reprogramming, which can be defined as inductive conversion of a cell into another one without passing through an intermediate pluripotent state. This definition delineates direct reprogramming from conversion of iPSCs. The first direct reprogramming study has been reported by Davis (Davis, Weintraub and Lassar, 1987), describing conversion of embryonic mouse fibroblasts into myoblasts by overexpression of MyoD. This study demonstrated for the first time, that a single TF is able to change somatic cell fate of a committed but not fully differentiated cell. Subsequent studies revealed TF-driven cell fate conversion between related lineages, such as glia to neuron, pancreatic to liver cell or B cell to macrophage (Shen, Slack and Tosh, 2000;Heins et al., 2002;Xie et al., 2004;Berninger et al., 2007). It has been assumed that it may be easier to reprogram cells from the same germ layer, but further studies were able to demonstrate that cells can be reprogrammed across germ layers by overexpressing a distinct combinations of TFs (Vierbuchen et al., 2010). Therefore, direct reprogramming is not limited to cell conversion within lineages; it is indeed able to transform cells across unrelated lineages or germ layers.

The direct reprogramming toolbox has been improved by alternative molecules besides TFs and increased our understanding in regard to cell plasticity. For instance, epigenetic regulators, such as chromatin modifiers or DNA methylases, have been shown to have an impact on the reprogramming process (Takeuchi and Bruneau, 2009;Dhawan et al., 2011).

Epigenetically repressed genes of the target cell-type have to be accessible for the fate determinant factor to be able to convert the target cell. Thus, chromatin modifiers can play an important factor in the cell conversion process. However, it is still not clear on the outcome an epigenetic regulator can have during reprogramming. While, chromatin remodelling complexes can facilitate the conversion of a cell (Takeuchi and Bruneau, 2009), in some case it is hindering reprogramming. For instance, inhibition of chromatin modifiers, like histone deacetylases, facilitated the conversion of germ cells to neurons, which was driven by neuronspecific factors [START_REF] Tursun | Direct conversion of C. elegans germ cells into specific neuron types[END_REF]Patel et al., 2012;Xu, Yi and Chien, 2012). Additionally, the deficiency of DNA methyltransferase Dnmt1 led to a conversion of pancreatic β cells into α cells (Dhawan et al., 2011). Therefore, it is likely that alteration in the epigenetic state of a cell is become crucial for subsequent reprogramming process.

Another important input in direct reprogramming comes from miRNA. Overexpression of miRNAs have been shown to successfully convert human fibroblasts into cells expressing neuronal markers (Yoo et al., 2011). Additionally, also cardiac lineage conversion has been demonstrated by using miRNAs (Jayawardena et al., 2012). Despite the powerful role of miRNAs in direct reprogramming, miRNA-mediated conversion is less efficient than TFmediated direct reprogramming (Xu, Yi and Chien, 2012). As miRNAs function mainly as translational repressors, the overexpression of a certain miRNA may misbalance the expressions of cell fate genes leading to cell fate change. Also, miRNAs could be implicated in epigenome alterations, thereby altering the epigenetic state of the cell, which as for chromatin modulators, can lead to cell fate changes. Indeed, a recent study demonstrated that upon overexpression, pro-neuronal miRNA -9/9*124 induced extensive remodelling of the epigenome, including simultaneous activation of a pan-neuronal program and the reconfiguration of chromatin accessibilities (Abernathy et al., 2017). These observations suggest an effective role for miRNA in remodelling the epigenome of a cell, which can then be useful for TFs to convert cells more efficiently.

Recently, small molecules gained more and more interest for inducing somatic cell fate changes. Small molecules are low molecular weight organic compounds. The function of small molecules can range from targeting signalling pathways, epigenetic remodelling or influencing metabolic processes during direct reprogramming (Xu et al., 2008;Li and Ding, 2010;Federation, Bradner and Meissner, 2014). They have been used merely to improve transcription factor-induced reprogramming but are also able to change cell fate on their own, even across germ layers (Li et al., 2015). For instance, generation of induced neurons (iNs) from mouse fibroblasts has been reported by using a cocktail of small molecules, such as Forskolin, ISX, CHIR99021 and I-BET 151. Apparently, these small molecules suppressed a fibroblast-specific program, while endogenous neurogenic TF were activated (Li et al., 2015).

The advantages of small molecules over genetic manipulations are numerous. They are costeffective, easily synthesized and standardized and, their biological effects are mostly reversible (Qin, Zhao and Fu, 2017). In clinical studies, small molecules could be used as drugs promoting endogenous regeneration in patients. Altogether, small molecules hold great promises as a replacement for genetic modifications in direct reprogramming. Nevertheless, their efficiency has to be improved and further studies are important to reveal more powerful types of small molecules.

Another notable factor in direct lineage reprogramming is the metabolic state of a cell. It is known that iPSCs and embryonic stem cells (ESCs) differ from differentiated cells regarding their metabolic state (Zhang et al., 2012). ESCs and iPSCs have an elevated dependence on glycolysis under aerobic conditions compared to highly or lowly respiring (metabolic reactions and processes that take in cells to convert biochemical energy) differentiated cell types (Zhang et al., 2012). During reprogramming to iPSCs, the cell undergoes an active process of metabolic resetting which can influence the reprogramming efficiency. For example, experimental inhibition of glycolysis lowers the reprogramming efficiency whereas increasing glycolysis enhances iPSC reprogramming efficiency (Zhou et al., 2010;Folmes et al., 2012;Panopoulos et al., 2012;Zhang et al., 2012). These observations reveal an important role for the metabolic state of a cell during reprogramming. This might become even more important, when a cell with a certain metabolic state is converted into a cell with a different metabolic state. This was elegantly described during reprogramming of astrocytes and fibroblasts (anaerobic glycolysis and β-oxidation) into neurons (oxidative, McKay et al., 1983;Tsacopoulos and Magistretti, 1996). Gascón et al. (2016) discovered that inhibiting ferroptosis, an irondependent form of non-apoptotic cell death (Dixon et al., 2012), promoted survival and neuronal conversion of astrocytes. Thus, lipid peroxidation, a hallmark of ferroptosis, was investigated and revealed increasing presence during neuronal reprogramming. In addition, factors inhibiting oxidative stress led to improvements in cell survival, maturation and efficiency (Gascón et al., 2016). This milestone publication demonstrated nicely, that metabolic changes during reprogramming occur and that upon inhibiting oxidative stress, lineage reprogramming (in this case astrocyte-to-neuron) will become more efficient and stable over time. Taken together, the experimental intervention with factors influencing the metabolic state of a cell in favour of the target cell can improve the reprogramming process.

Notably, whether direct reprogramming (here in fully differentiated cells) actually reverts the target cells to transient higher potent state during conversion, or whether it leads to a direct switch between differentiated cell states was not deeply investigated so far. Thanks to the study of Karow et al. (2018), a better insight regarding this topic was provided. By deciphering transcriptomes at the population of single cell level, the trajectory underlying conversion of adult human pericytes into iNs was reconstructed (Karow et al., 2018). The transcription factors ASCL1 and SOX2 (AS) upregulated various cell signalling genes during the very early process of reprogramming. These cell signalling genes were further investigated and showed to be markedly enriched in germinal zones of the developing CNS containing neural stem cells in mouse embryos. Following the transcriptomics at later stages of reprogramming, Karow et al. (2018) could show that the trajectory can then bifurcate to give rise to differentiated neuronal lineages. Thus, AS-mediated reprogramming unfolded developmental programs, followed by genes instructing neuronal fates during later stages of the conversion process.

These results indicate that cells are undergoing a transient stem cell-like state before converting into a differentiated cell.

The advantages of direct reprogramming over the aforementioned alternative approaches are numerous. For example, direct reprogramming bypasses the procedure of resetting the cell to a de-differentiated state before changing cell fate. This is advantageous as it may be less likely to introduce genetic mutations, which can be a general concern in iPSC reprogramming (Liang and Zhang, 2013). Furthermore, direct reprogramming does not need the introduction of oncogenes like Oct-3/4 used in iPSCs, therefore reducing the risk of possible tumorigenesis for in vivo applications. Another advantage for direct reprogramming is that it does not reset the age of the starter cell, which is especially important for disease modelling, e.g. when using patient derived cell biopsies (Mertens et al., 2015;Tang et al., 2017). Notable, direct reprogramming appears to be faster and more cost-effective than iPSC generation, as it also does not necessarily need an in vitro culture system using rather expensive reagents.

A big advantage of direct reprogramming is that it can be conducted in vivo. Some studies showed OSKM-mediated reprogramming in vivo too, which resulted in pluripotent gene expression (Senís et al., 2018). The issue here is the control over the induced pluripotent cells and the cancerous appearance of teratomas. Direct reprogramming allows to overcome these hurdles as it does not generate pluripotent cells as well as it does not necessarily need oncogenic factors. It therefore may be used in the future for cell replacement therapy, bypassing difficulties of transplantation (e.g. rejection of the transplanted tissue) (Xu, Du and Deng, 2015). The advantage of in vivo reprogramming is the provision of a niche that supplies factors to support the functional maintenance of target cell types, which might also facilitate functional conversion (Xu, Du and Deng, 2015).

Major limitations and hurdles of direct reprogramming are mostly common with other reprogramming approaches and are described below:

First, the selection of the starter cell-type may ease or impede the conversion process. It is likely to assume that lineage-related cells might be easier to convert into each other as they share a common origin. For example, glia are efficiently converted into functional neurons by single overexpression of one transcription factor (Heins et al., 2002;Berninger et al., 2007;Heinrich et al., 2010). In contrast, conversion of one neuronal subtype into another one appeared to be rather difficult and has been achieved only in immature cells (Rouaux and Arlotta, 2013;Gascón et al., 2017). Furthermore, the same reprogramming factor can trigger different outcomes when it is induced in different cell-types. For instance, Neurogenin-2 has been shown to convert astrocytes into glutamatergic neurons (Heinrich et al., 2010;Masserdotti et al., 2015) , while in foetal fibroblasts it triggers cell conversion into a cholinergic motor-neuron like cell fate (Liu et al., 2013). These observations highlight the importance of the crosstalk between the reprogramming factors and the cellular context in which they operate.

Another important hurdle in reprogramming is to improve the efficiency of converting cells, as it was the case for the initial protocol of iPSC generation (Takahashi and Yamanaka, 2006).

Subsequent studies revealed factors which can facilitate conversion, such as Glis1 or Mbd3, and barriers whose removal drastically increased reprogramming efficiency (Maekawa et al., 2011;Rais et al., 2013;Di Stefano et al., 2014). A prominent factor hindering reprogramming efficiency is p53. For example, expression of a dominant-negative form of p53 or its knockdown improves direct conversion of mouse embryonic fibroblasts into dopaminergic neurons (Liu and Qian, 2014;Jiang et al., 2015). Thus, efficiency of direct reprogramming can be improved by adding factors facilitating conversion or by blocking factors hindering conversion.

Furthermore, generating different types of fully functional mature cells remains a major challenge (Xu, Du and Deng, 2015). A common concern is that the direct converted cells fail to silence the expression programs of the initial population, indicating that it still possess an immature phenotype (Cahan et al., 2014). For instance, mouse hepatocyte-like cells induced by hepatic fate determination TFs remained functionally immature, reflected by incomplete hepatocyte differentiation and expression of hepatoblast markers (Huang et al., 2011;Sekiya and Suzuki, 2011). Thus, fate determination factors alone may not be sufficient to achieve functional maturation of reprogrammed cells. Combination of lineage-specific TFs and factors promoting cell maturation could represent an effective combination to achieve functional maturation by direct reprogramming. Lastly, it is crucial to evaluate how far the reprogrammed cells resemble the endogenous population. Several methods have been used

to evaluate converted cells: analysis of marker gene expression, global gene expression and epigenetic patterns, silencing of exogenous factors and the epigenetic state of master genes (Cohen and Melton, 2011). As these assays are primarily based on whole cell populations and therefore cannot reveal the heterogeneity of converted cells, further deep analysis such as singe-cell RNA sequencing, could be applied for gaining additional information.

A straightforward test to evaluate converted cells is to assay their function in vivo. Several studies demonstrated successful integration and survival of reprogrammed cells after transplantation in mice (Ring et al., 2012;Bredenkamp et al., 2014). The function of converted cells can be further tested in mouse disease models. For example, mouse fibroblasts reprogrammed into thymic epithelial cells formed a complete and functional thymus in vivo, which supported T cell development in an athymic mouse model (Bredenkamp et al., 2014).

Overall, from the field of reprogramming, cells can be seen as maintaining a stable state (represented as islands in Fig. 16) which can be reverted upon changes of the epigenetic environment and induction of factors able to induce a certain cell fate ("boats"). Converting cells into cell-types from close related lineages can be instructed by using few TFs ("orange and yellow sails"), whereas reprogramming of cells from different germ-lines ("difficult routes, storms and reefs") might need a more complex combination of several TFs ("blue and red sails"), or combination with miRNA or small molecules.

Figure 16: The Cook Islands model applied to neuronal reprogramming

In the Cook Islands model, each island represents a different stable state. In essence, a boat, namely a cell, can move from island to island freely and in reprogramming such transitions are instructed by the combinatorial expression of specific transcription factors (depicted in the model as different colours in the sails of the boats). More complicated routes need bigger sails with several transcription factors, for example the reprogramming of fibroblasts to striatal or dopaminergic neurons. By contrast, small sails, representing fewer transcription factors, are sufficient to push boats to more closely related neuronal sub-types, for example from astrocytes to glutamatergic or GABAergic neurons. Certain routes are easier than others due to various factors such as distance and/or the relative water level, representing the various barriers to reprogramming as they occur both in vitro and in vivo. Other routes are particularly difficult due to the presence of strong opposing sea currents (see fibroblasts to GABAergic neurons), sudden storms (see glutamatergic to Layer V) or reefs (fibroblasts to dopaminergic). The presence of these hurdles may require novel routes to be taken, or more potent and equipped boats; that is, new strategies for reprogramming may be required to overcome cell type-specific barriers to reprogramming. Taken from Masserdotti, Gascón and Götz (2016).

Direct neuronal reprogramming in vivo

The great advantage of direct reprogramming over other reprogramming approaches is the straightforward conversion in vivo. This strategy may represent an interesting alternative for regenerative medicine as it aims to replace damaged or lost cell to restore organ function.

Especially organs with low intrinsic capacity of self-renewal in the adult organism, such as the pancreas, the heart or the CNS, are in need of strategies to replace cells that haven been damaged by disease or injury. Here, the focus will be on the brain, as it is the main interest of my thesis work.

The adult mammalian brain lacks a significant regenerative capacity and is therefore especially vulnerable to damaging events resulting in the loss of neural cells. Notably, there are few regions in the brain, which retain a neural stem cell niche even during adulthood. Neural stem cells (NSCs) give rise to newborn neurons throughout life, a process called adult neurogenesis.

Two main locations in the adult brain possess NSCs: the SVZ of the lateral ventricles and the dentate gyrus of the hippocampus. NSCs in the SVZ give merely rise to interneurons which migrate to the olfactory bulb, whereas NSCs in the SGZ (subgranular zone) of the dentate gyrus generate granule neurons (Braun and Jessberger, 2014). Both processes are contributing to olfactory-and hippocampus-dependent learning and memory processes (Deng, Aimone and Gage, 2010). Although these stem cell niches do not seem to be designed for neuronal renewal after injury, they could be eventually exploited for brain repair. The idea is to boost NSCs proliferation and guided chemokine-directed migration towards lesioned tissue (Li et al., 2012).

An alternative strategy for self-repair aims at fate converting resident cells into the desired cell type (Heinrich, Spagnoli and Berninger, 2015), named direct reprogramming in vivo. The field of developmental biology here serves as a fundamental source of knowledge for the identification of potential reprogramming candidates. Again, TFs are mostly used for reprogramming, as their fate specification in development is known and thus, can possibly predict the outcome of converting a cell. For an experimental design of in vivo reprogramming, defining the careful sequential steps that are essential for setting up a direct reprogramming experiment is crucial (Heinrich, Spagnoli and Berninger, 2015): first, the cell type source for reprogramming has to be characterized; second, the factors required to obtain a certain celltype after conversion must be identified, as well as a safe and efficient delivery method; third, reprogrammed cells of the same molecular identity and phenotype as the desired cell type, while erasing its original molecular identity, must be obtained. So far, these considerations are shared by in vitro approaches for direct reprogramming, as mentioned above. However, two other considerations are more specific to in vivo reprogramming approaches. Host environments can differ in their permissiveness to accommodate new cells and may sometimes create barriers against their functional integration. Cell networks with limited selfregenerative potential may have constraints for new cells and identifying these barriers can help overcoming them. Lastly, the full functional integration of reprogrammed cells is the final step to be accomplished (Heinrich, Spagnoli and Berninger, 2015). Therefore, factors

promoting the functional integration have to be identified and then, integration into the system has to be carefully examined. So far, several studies reported successful direct reprogramming in vivo. Below, the presented studies are divided into reprogramming in an injured environment and reprogramming under physiological conditions, as both conditions differ in the plasticity of targeted cells.

a. Direct neuronal reprogramming in vivo in the injured brain

Despite their fatal impact, brain injuries also provide an opportunity for reprogramming, as they produce glial cells, known to be cells with a certain plasticity (Gascón et al., 2017). For instance, during the acute phase of injury, astrocytes become hypertrophic, resume proliferation and upregulate expression of the intermediate filament proteins glial fribrillary acidic protein (GFAP) and vimentin in a process called reactive gliosis (Torper et al., 2013;Z. Guo et al., 2013;Aravantinou-Fatorou et al., 2015;Brulet et al., 2017). After injury, astrocytes become proliferative, and in some cases begin to express markers of neural stem/progenitor cells and neurogenic differentiation (Anderson, Ao and Sofroniew, 2014;Bayraktar et al., 2015;Duan et al., 2015;Nato et al., 2015;Brulet et al., 2017). In addition, other glial cells, such as NG2 cells and microglia undergo reactive responses to injury and form a defence system against the invasion of micro-organisms and cytotoxins from the surrounding tissues (Pekny and Nilsson, 2005;Sofroniew and Vinters, 2010;Robel et al., 2011). Once activated, many reactive glial cells stay in the injury site and secrete neuroinhibitory factors and eventually form a glial scar inside the brain (Sofroniew and Vinters, 2010). Nevertheless, many studies took advantage of the environmental change that occurs upon brain injury. Guo et al. (2014) demonstrated, upon brain injury, reprogramming of reactive glial cells, including astrocytes and NG2 cells, into functional neurons in the adult mouse cortex (Guo et al., 2014). After stab injury, reactive proliferative glial cells were infected by a retrovirus encoding for NeuroD1 (a proneural TF) and then analysed for neuron-like properties. Infected astrocytes were converted into glutamatergic neurons, while NG2 cells were converted into both glutamatergic and GABAergic neurons, demonstrating again the importance of the starting cell-type for the reprogramming outcome. Interestingly, NeuroD1-converted neurons expressed Ctip2 and Tbr1, lower layer markers of the cortex. Additionally, NeuroD1-driven reprogramming of glial cells was successfully employed in an Alzheimer's disease mouse model (Guo et al., 2014). These results show that reprogramming in the injured brain, mechanically or diseased, can be potentially achieved and reactive glial cells can be eventually converted into functional neurons.

Another study from Heinrich et al. (2014) demonstrated successful conversion of NG2-glia into induced neurons (Heinrich et al., 2014). By performing stab wound injury and subsequent injection of retrovirus -coding for Sox2 alone or in combination with Ascl1 -forced reactive NG2 cells could be converted into DCX or NeuN-positive neuronal cells (immature neuronal marker and mature neuronal marker, respectively). Interestingly, Sox2 was able to convert NG2 cells into iNs alone but did not succeed to do so without stab wound injury (Heinrich et al., 2014). Thus, Sox2-mediated reprogramming of NG2 cells needs a more plastic environment, similarly to what happens after brain injury.

Other studies demonstrated reprogramming in the injured brain of 1) parenchymal astrocytes into NeuN-positive neurons in the striatum, by using a transgenic mouse model, (Torper et al., 2013);

2) glial cells into neurons by Neurog2/Bcl-2 coding retroviruses and additional antioxidants in the cortex (Gascón et al., 2016);

3) dorsal midbrain astrocytes into functional neurons by overexpression of Ascl1 delivered via a GFAP-adeno-associated virus vector (Liu et al., 2015); 4) functional dopamine neurons from mouse astrocytes in a Parkinson's model (Rivetti Di Val Cervo et al., 2017).

Taken together, in vivo reprogramming of reactive glial cells has been successfully employed and might represent a powerful tool for cell replacement in the injured brain. A more detailed overview can be found in Torper and Götz (2017).

b. Direct neuronal reprogramming in vivo under physiological conditions

So far, most studies have made use of an altered, more plastic environment after brain injury to reprogram glial cells into neurons. Thus, it was not clear whether glia-to-neuron conversion was feasible under physiological conditions, which could potentially facilitate future clinical approaches long after the acute injury phase. Recent publications demonstrated that it is indeed possible to reprogram glia cells into neurons in a healthy brain. For example, Brulet et al. (2017) showed neuronal conversion of non-reactive astrocytes in the adult mouse brain.

By injecting an AAV9 virus with an expression cassette for NeuroD1 and a transgenic mouse labelling GFP in astrocytes (GFAP promotor), they specifically targeted astrocytes in the cortex and the striatum. Only astrocytes of the striatum showed NeuN-expression after transfection (around 2%, Brulet et al., 2017). However, there was a possibility that SVZ-stem cells (persisting in the adult brain) could have migrated into the striatum and converted into neurons upon NeuroD1 overexpression, resulting in false-positive results. Pereira et al. (2017) showed reprogramming of NG2 cells in the intact mouse brain. NG2 cells are an interesting target cell-type, as they retain some ability of self-renewal (Simon, Götz and Dimou, 2011), which avoids depletion of an endogenous population upon reprogramming. In their study, they have overexpressed Ascl1, Lmx1a and Nurr1 using AAV vectors and a transgenic mouse line, which expresses GFP upon neuronal identity (genes under neuronspecific synapsin promoter) after transfection (Torper et al., 2015). NG2 glia in the striatum revealed successful conversion into neurons showing parvalbumin-containing interneurons (Pereira et al., 2017). Other approaches sought out to directly reprogram neurons into a distinct cellular type. The advantage is that neuron-to-neuron reprogramming does not need to switch the metabolic state, as it is instead the case for astrocyte-to-neuron conversion (Gascón et al., 2016). On the other hand, reprogramming of neurons will result in a depletion of the targeted cells. In case of conversion of a small population it might be less dramatic, but these alterations have to be further investigated. In the brain, successful direct reprogramming has been reported in the striatum and in the neocortex. Upon overexpression of Sox2 together with dopaminergic-specific TFs, Niu et al. (2018) were able to convert striatal neurons into dopaminergic neuron-like cells in the adult brain. Moreover, electrophysiological investigations of reprogrammed cells revealed functional connections with other neurons (spontaneous postsynaptic currents, Niu et al., (2018)). This revealed an important property, since it not always clear whether reprogrammed neurons can properly integrate into the host system.

Instructing a reprogrammed cell into a precise and reproducible neuronal sub-type in the brain is still in its infancy. Only few studies have been able to show reprogramming to a specific neuronal sub-type (Gascón et al., 2017). One good example comes from the in vivo Fezf2driven reprogramming shown to convert one neuronal sub-type into another one.

In 2010, Rouaux and Arlotta (Rouaux and Arlotta, 2010) In parallel, Rouaux and Arlotta (Rouaux and Arlotta, 2013) showed Fezf2-driven direct reprogramming of callosal projection neurons into CFuPNs in vivo. In their study, they made use of a plasmid, which expressed Fezf2 (with GFP reporter) under a cdk5r-promoter, which is specifically turned on in post-mitotic neurons. By in utero electroporation at E14.5, they were able to target post-mitotic migrating upper layer neurons. Investigation of their molecular identity (P7) revealed that Fezf2-electroporated cells expressed lower layer markers such as ER81, CRYM, TLE4, CTIP2 and FOG2. The upper layer marker CUX1 was also reduced.

Molecular identity of converted cells was maintained at later stages, even if with a lower percentage. No significant cell death was found when GFP-positive neurons were stained for CASPASE-3. Moreover, by using an inducible expression plasmid for Fezf2, they could show that reprogramming at P3 still led to molecular identity changes, even though with very reduced efficiency (Rouaux and Arlotta, 2013). Interestingly, Ctip2, Tle4 and Fog2 expressions could not be observed anymore when Fezf2 was induced at P3. Induction of Fezf2 at later stages than P3 failed to convert upper layer neurons into lower layer ones. In addition, Fezf2electroporated cells projected aberrantly to corticofugal targets, such as thalamus, internal capsule, cerebral peduncle and some axons were also found in the spinal cord. The number of axons crossing the CC, which CPN normally project through, was reduced, but not absent.

When Fezf2 was induced at E17.5, few neurons projected into the internal capsule and the thalamus. Induction of Fezf2 at later stages did not show any aberrant projections anymore.

Taken together, these data demonstrate that post-mitotic upper layer neurons at early developmental stages retain a certain degree of plasticity, being able to be reprogrammed into corticofugal neurons. However, this window of reprogramming capacity vanished after P3, suggesting that with increasing maturity, neurons lose the ability to change cell identity.

c. Data leading to the thesis work

A recent study from our lab demonstrated a de-repressing function of Lmo4 in the SATB2mediated Ctip2 repression (Harb et al., 2016). Furthermore, this study also revealed an interplay between Lmo4 and Ctip2 expression in lower layers of S1. Upon cortical deletion of Lmo4 (cKO), the total number of Ctip2-expressing neurons in lower layers L5 and L6 was reduced in P7 brains. On the other hand, overexpression of Lmo4 at E13.5 showed an increase of CTIP2-positive neurons in lower layers in P7 brains (Harb et al., 2016). These results

III Aims of the thesis

As aforementioned, the brain lacks a significant regeneration capacity and thus, has no or very little capacity for self-repair upon injury. Direct reprogramming has been shown to be a potential tool for restoring lost neurons by converting neighboring cells into the lost cell-type.

Transcription factors, which are known for their role in cell fate specification during development, are good candidates for directly converting one cell-type into another one. The transcription factor FEZF2 is necessary and sufficient to specify L5 subcerebral projection neuron identity. Its ectopic expression in cortical upper layers and in the striatum can specifically convert cells into corticofugal neurons in vivo, revealed by molecular identity and axonal projections. However, these studies lacked efficiency in reprogramming, and conversion in the cortex was restricted to immature neurons.

Our lab has demonstrated that the nuclear co-adaptor Lmo4 can act as a de-repressor of the Ctip2-locus. Ctip2 is a downstream target of Fezf2 and is highly expressed by L5 subcerebral projections neurons. Hence, the aims of the thesis have been the following:

1) Investigating whether Lmo4 can as a potential reprogramming factor in the conversion of upper layer neurons into corticofugal neurons.

2) Examining the combinatory effect of Lmo4 and Fezf2 as a reprogramming cocktail for a more efficient way of converting upper layer neurons into corticofugal neurons.

3) Carefully following axonal projections of reprogrammed neurons in vivo by identifying an improved reporter strategy.

4) Exploring until which postnatal stage upper layer neurons can still be converted into a corticofugal sub-type.

5) Searching for downstream and induced targets of reprogrammed neurons as a strategy

for identifying novel factors and/or pathways useful in direct in vivo reprogramming.

I. Animals

All in utero electroporations were conducted on RjHan:NMRI mice obtained from Janvier.

Pregnant mothers from this outbred strain have a drastically higher number of embryos, when compared with mothers from other strains, they are good breeders and they exhibit strong fitness. Due to these features, this strain has been chosen as the experimental model. Female and male RjHan:NMRI mice were put in matings in the evening, a positive plug on the next day has been considered as pregnant stage of 0.5 days.

II. Plasmids

A. pCdk5r-Lmo4-IRES-GFP, pCdk5r-Fezf2-IRES-GFP and pCdk5r-IRES-GFP

For reprogramming at embryonic stage E14.5, pCdk5r-Lmo4-IRES-GFP, pCdk5r-Fezf2-IRES-GFP and pCdk5r-IRES-GFP plasmids were used. pCdk5r-Fezf2-IRES-GFP and pCdk5r-IRES-GFP were donated by the lab of Paola Arlotta (Rouaux and Arlotta, 2013), while pCdk5r-Lmo4-IRES-GFP has been generated by Kawssar Harb (Harb et al., 2016).

B. Inducible pCAG-Lmo4-IRES-GFP

To induce the expression of Lmo4 at postnatal stages after in utero electroporation, Lmo4 has been subcloned into a vector with a floxed stop codon sequence just before the gene of interest, a subsequent IRES sequence and a Gfp reporter (gift of V. Tarabykin lab).

Insert

For cloning, Lmo4 has been amplified by PCR from the pCdk5r-Lmo4-IRES-GFP plasmid while adding restriction sites on both ends of the strands (EcoRV). The used primers had the following sequence:

1) Forward: ' GTCATGGATATCATGGTGAATCCGGGCAGCAGC '

2) Reverse: ' GTCATGGATATCTCAGCAGACCTTCTGGTCTGG '

And the respective PCR program:

95°C -5 min 95°C -30 s 60°C -30 s 35 cycles 72°C -30 s 72°C -8 min Subsequently, the PCR product has been digested with restriction enzyme EcoRV to create blunt ends:

At the end, the digested PCR product has been purified by gel-electrophoresis (1%) and purified using DNA Gel Extraction Kit (Qiagen, #28705). Incubated for 1h at 37°C

The ligation was incubated at 16°C ON. Note that between each incubation step, the DNA has been purified using QIAquick PCR Purification Kit (Qiagen, #28104). The ligation product has been mechanically transfected in bacteria and plate on LB agar (Amp+) plates. Grown colonies were picked and amplified in 4 ml LB buffer + 1 mg/ml Ampicillin. The plasmid of the resulting was purified using EndoFree Plasmid Maxi Kit (Qiagen, #12362).

Inducible pCAG-Fezf2-IRES-GFP

The same target vector has been used as for inducible Lmo4 described above. The Fezf2 sequence has been amplified from the cdk5r-Fezf2-IRES-GFP plasmid by PCR and flanked with EcoRI and EcoRV restriction cassettes using the following primers: 1) Forward: ' CCGAATTCCATGGCCAGCTCAGCTTC '

2) Reverse: ' CCGATATCTCAGCTCTGAACTGTCC ' Subsequently, the PCR-generated Fezf2-insert and the target vector have been digested with EcoRI and EcoRV and then ligated to finally generate the inducible Fezf2 plasmid:

Inducible pCAG-IRES-GFP

To generate a control plasmid which only expresses GFP upon action of CRE recombinase, the Inducible pCAG-Fezf2-IRES-GFP has been digested with EcoRI and NotI in order to remove the Fezf2 sequence and, sticky ends were filled up and the open plasmid then has been ligated again.

pCAG-ERT2-CRE-ERT2

In order to remove the floxed stop codons from inducible Gfp, Lmo4 or Fezf2 plasmids, we made use of a conditionally regulated plasmid which expresses CRE upon tamoxifen application. The plasmid is commercially available from addgene under the reference number #13777.

pCAG_smFP FLAG

As the GFP reporters of inducible Lmo4 and Fezf2 plasmids were not sufficient to express signal in fine axons and collaterals of electroporated cells, pCAG_smFP FLAG has been used to overcome this hurdle. Adding this plasmid to the reprogramming factors at postnatal stages, we were able to improve the signals in fine axons and collaterals of transfected cells. The plasmid is commercially available from addgene under the reference number #59756.

Inducible pCAG-Ctip2-IRES-GFP

This plasmid has been cloned by removing the Fezf2 sequence from inducible pCAG-Fezf2-IRES-GFP via digestion with EcoRI and NotI and ligation of a Ctip2-insert with EcoRI and NotIrestriction sites. The insert has been cut out from another plasmid containing the Ctip2 sequence which fortunately was flanked by EcoRI and NotI too (kind gift from V. Tarabykin).

Inducible pCAG-hPbx1-IRES-GFP

Also, hPBX1 has been subcloned using inducible Fezf2 as backbone. Briefly, Fezf2 has been cut out from the vector via digestion of EcoRI-HF and NotI-HF. The insert of Pbx1 has been amplified from a plasmid (kind gift of Licia Selleri) via PCR using the following primers:

Forward: ' GACAGAATTCATGGACGAGCAGCCCAGGGC ' Reverse: ' CAGCGGCCGCTACTGTATCCTCCTGTC '
Then, after digestion of the PCR product, both vector and insert were ligated.

III. In utero electroporation

The experimental procedure for in utero electroporations were performed according to the protocol in Saito and Nakatsuji (2001) with several modifications as stated in the following: Endofree-toxin plasmids were diluted in TE-Buffer (Qiagen, #1018499) until it reached a total concentration of 1 µg/µl in a final volume of 20 µl, including 1x Fast Green FCF (Sigma-Aldrich, #F7252). For co-electroporations of multiple plasmids, the concentration of each single plasmid has been lowered until they together reached a final concentration of 1 µg/µl.

For instance, co-electroporation of four plasmids together would mean a concentration of 0.25 µg/µl for each plasmid. After a pregnancy duration of exact 14.5 days (E14.5), mothers were deeply anesthetized via Intraperitoneal (IP) injection of 10µl Ketamin/Xylazine (10mg/ml) per gram body weight. Then, the mice were shaved on the upper abdomen and cleaned with disinfectant (Pierre Fabre, #716431). Afterwards, a lateral laparotomy has been performed and the uterus was exposed by carefully pulling it out with ring forceps. Via microinjection of the DNA mix with capillaries (Harvard-Apparatus, #30-0016), which have been generated by pulling with a micropipette puller (Sutter Instrument, Model P-1000; Parameters: Heat 459, Pull 60, Vel 75, Time/del 100, Pressure 200) and cut at approximately 60 µm away from the tip. The capillary has been inserted into a holder of a microinjector (eppendorf FemtoJet 5274 V2.02; Parameters: Pi[hPA] 100 -300, ti[s] 0.7, Pc[hPA] 7 ) and approximately 1 µl have been injected into one of the two lateral ventricles (see Fig. 17). Then, by placing tweezer electrodes (3 mm, Nepagene, #CUY650P3) onto the uterine wall with the plus node on the premature M1 or S1 area underneath, square electric pulses were applied (Without poring pulse; Transfer pulse with Voltage: 37, Pulse length: 50 ms, Pulse interval: 999 ms, Number of pulses: 4, Decay rate: 5%) via an electroporator device (Nepagene Superelectroporator, Nepa21 TypeII). To finish, uterus with embryos was repositioned into the original location and the incision was sewed up with sutures (Péters Surgical; 6/0 #87002F for peritoneum and 5/0 #87001F for skin). 120 µl Meloxicam (Metacam) and 200 µl Gentamicin (1 mg/ml, Sigma #G1272) were subcutaneously injected to prevent inflammation. Electroporated mice were housed in solitary and in a ventilated black box for 24h after the surgery to recover.

Procedure of in utero electroporation. A. Embryos were exposed by lateral laparotomy. B. DNA

was injected in the lateral ventricles of mouse brains. C. Schema showing how DNA is transfected in cortical progenitors. Taken and modified from (Wang and Mei, 2013).

IV. Tamoxifen injection

To induce the expression of CRE recombinase from pCAG-ERT2-CRE-ERT2 plasmid, tamoxifen has been applied at the desired developmental stage of the mice. Briefly, 10ml of 10 mg/ml tamoxifen has been prepared as followed: 100 mg tamoxifen (Sigma, #T5648-1G) has been taken up in 500 µl Ethanol absolute and transferred in an eppendorf tube. Next, the tube has been put on a rotator for 20 min at RT and then taken up with 9.5 ml of corn oil (Sigma, #C8267). Then, the tube has been thoroughly vortexed and put back on a rotator until the tamoxifen completely dissolved. 1 ml aliquots have been stored at -20°C until usage.

Mice received tamoxifen subcutaneous under the neck depending on their age:

V. Cell Dissociation

All reagents were prepared according to the manufactures protocol (Worthington Papain Dissociation System, Cat. No. LK003150) 48h after in utero electroporation at stage E14.5, pregnant mice were sacrified via cervical dislocation. The embryos have been removed from the uterus in icecold HBSS+Glucose (6g/l) with dissection tools and the brains have been extracted under a fluorescent macroscope.

Only GFP-positive areas were dissected, and 2-3 of them were dissociated together in one Eppendorf tube with 1 ml of papain-mixture for 10 min at 37°C. To mechanically digest the tissue, the suspension has been pipetted up and down 20 times with a P1000 pipette. The suspension has been transferred on a 70 µm cell strainer and filtrated and then centrifuged at 400 g, 4°C for 5 min. Subsequently, the liquid has been removed and the remaining pellet was resuspended in 0.6 ml of Ovo-mixture by pipetting up and down ten times. Next, the suspension was filtrated again with a 70 µm cell strainer and centrifuged at 400 g, 4°C for 5min. The liquid has been discarded and the pellet was resuspended in 1 ml of ice-cold DPBS (1X) by pipetting up and down 5 times and centrifuged again at 400 g, 4°C for 5 min. Finally, liquid has been removed and the pellet was resuspended in 300 µl PBS-FBS (3%) in a tube.

VI. Cell sorting

Sorted cells were resuspended in 100 µl RA1 + 2 µl TCEP (see RNA extraction for further references) and stored for short term at -20°C.

XI. Microscopy

Overview images (mosaics, 10X) of IHC floating slices were taken using Axio Imager 2 (Zeiss) while images for counting cells were taken using a Zeiss 710 confocal or Zeiss 780 NLO confocal. The confocal images have been taken and processed as mosaic and z-stack (2 µm between layers).

XII. Analysis

To process the images taken from microscopes, Fiji2 has been used. Each immunofluorescent slice has been carefully checked for the right neocortical area (M1 or S1) and their respective anatomy using Allen Brain Atlas or Atlas of the Developing Mouse Brain (Paxinos et al., 2007, ISBN 0-12-547622-1). Experiments were given random numbers to ensure bias was avoided.

Four layers of confocal images were stacked (2 µm x 4) and the total number of GFP-positive cells were counted. Then, markers were screened for co-localization with GFP-positive cells using orthogonal view for 3D presentation. Each marker that has been identified as colocalized with GFP-positive has been counted and divided by the number of total GFP-positive cells. The result was represented as a percentage and error bars were calculated as standard error of the mean (SEM). For comparisons between plasmids, 1-way ANOVA has been applied together with a tukey post-hoc test. For grouped comparisons between areas together with plasmids, a 2-way ANOVA has been applied together with a tukey post-hoc test. Lmo4 is expressed in an area-and subtype specific fashion and controls post-mitotic projection neurons diversity in the rostral motor cortex (Cederquist et al., 2013). Furthermore, previous observations found out that Lmo4 over-expression led to an increase of CTIP2 in L5 neurons (Harb et al., 2016). Thus, we sought out whether the nuclear co-adaptor LMO4 has the capacity of reprogramming upper layer neurons, into CFuPNs.

Therefore, we used the Cdk5r-Lmo4-IRES-GFP plasmid or control Cdk5r-GFP to deliver Lmo4 or control GFP into target cells. The plasmid drives gene expression only in migratory, postmitotic neurons thanks to the Cdk5r gene promoter (Tsai et al., 1994;Delalle et al., 1997).

Furthermore, the expression of Lmo4 was revealed by a reporting GFP signal. Mouse embryos at E14.5 were electroporated in utero, to deliver Cdk5r-Lmo4-IRES-GFP (cLmo4) or control

Cdk5r-GFP (cGFP) in cortical progenitors at the position of the presumptive somatosensory area (S1, see Fig. 17A). The developmental stage of E14.5 marks peak generation of upper layer (UL) neurons, while the generation of lower layer neurons is almost at its end (Greig et al., 2013). As a next step, we perfused and extracted GFP-positive brains of pups at P7, when laminarization of the cortex has almost finished. Then, brains were cut with a slicing vibratome into coronal sections. As a last experimental step, we applied immunohistochemistry (IHC) for visualizing proteins, which are specific molecular markers of UL or LL neurons and we analyzed expression of these markers in GFP-positive neurons located in upper layers of S1 (see Fig. 17B andB'). Here, we applied IHC to reveal the expression of CUX1, a protein that is selectively expressed in upper layer CPNs (Molyneaux et al., 2007), CTIP2, a protein specifically expressed in lower layer SCPNs and CThPNs (Molyneaux et al., 2005), FOG2, a protein restricted to lower layer CThPNs (Galazo, Emsley and Macklis, 2016), PCP4, a protein found in SCPNs and CThPNs [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]Watakabe et al., 2012) and DARPP32, a protein restricted to lower layer 6

CThPNs (Ouimet, 1991).

The data were then generated by counting the UL GFP-positive neurons expressing molecular markers (see Fig. 17C). Results were then presented as a percentage of electroporated GFPpositive UL neurons expressing molecular markers (see Fig. 17D).

The examination of cLmo4-electroporated cortices revealed, that the percentage of targeted UL neurons expressing CUX1 was very similar to cGFP-control electroporated cortices (94.13 ± 1.45% vs. 92.57 ± 2.51% in cGfp). Furthermore, cLmo4 did not induce expression of CTIP2 (2.8 ± 0.84 vs. 0.53 ± 0.53% in cGfp) or PCP4 (0.33% ± 0.3% vs. 0% in cGFP). Interestingly, some targeted UL neurons expressed FOG2 (7.86 ± 1.43% vs. 0.37 ± 0.31% in cGFP) and, a significant proportion showed DARPP32 expression (12.43 ± 2.4% vs. 0.97 ± 0.27% in cGfp).

Taken together, the ectopic expression of Lmo4 did not lead to a downregulation of Cux1, nor an upregulation of Ctip2 or Pcp4 gene expression in targeted UL neurons, with a subtle induction of lower layer markers FOG2 and DARPP32 in UL neurons was observed. These results indicate that Lmo4 does not lead to an efficient conversion of molecular identity in UL neurons, especially in regard that UL neurons maintained CUX1 expression. Furthermore, a similar effect of Ctip2 by Lmo4 as reported upon overexpression in post-mitotic LL neurons could not be observed.

B. Ectopic expression of Lmo4 in UL neurons does not alter their axonal projection

The previous results indicated that upon Lmo4 induction in UL neurons, a strong change of molecular identity did not occur. Nevertheless, LL marker FOG2 and DARPP32 were found in a significant higher number upon Lmo4 ectopic expression.

Neuronal identities are defined by a combination of morphology, electrophysiological properties or connectivity features. Under physiological conditions, many upper layer neurons project through the corpus callosum (CC) towards the contralateral hemisphere, some UL neurons in addition project ipsilaterally within the cortex and to the striatum (Str, Fame, MacDonald and Macklis, 2011). In contrast, CFuPNs project outside the cortex, to the striatum, the thalamus (Th) and within the pyramidal tract which includes the internal capsule (IC), the cerebral peduncle (CP) and the spinal cord (SC).

Thus, to further investigate any possible identity changes in targeted UL neurons, we sought out whether axonal projections are changed upon Lmo4 ectopic expression. Therefore, we investigated sequential coronal sections, from rostral to caudal, of electroporated brains and followed GFP-positive axonal projections.

Examination of axonal projections revealed numerous GFP-positive axons in the corpus CC and few in the striatum (Str) in control cGFP and cLmo4-electroporated brains (see Fig. 18A,B).

Projections of targeted UL neurons were not present in subcerebral trajectories and structures (including IC, Th, CP or spinal cord SC).

These results showed axonal projections that are characteristic of CPNs, the most abundant neuronal cell-type in the upper layers of the cortex. Furthermore, no axonal projections were detected in corticofugal structures. Thus, reprogramming of UL neurons upon ectopic expression of Lmo4 is not conclusive regarding alterations of axonal projections.

C. Lmo4 in combination with Fezf2 efficiently induces lower layer markers in UL neurons

Concluding that LMO4 is not inducing efficient reprogramming of UL neurons, we wondered whether LMO4 could improve FEZF2-driven reprogramming of UL neurons into CFuPNs. FEZF2 is a transcription factor which promotes SCPN cell fate in post-mitotic LL neurons, and studies demonstrated that ectopic expression of FEZF2 in UL neurons can change their molecular identity (De La Rossa et al., 2013;Rouaux and Arlotta, 2013). Ctip2 is a direct target of FEZF2

and ectopic expression of FEZF2 in UL neurons showed, among other CFuPN genes, upregulation of Ctip2 (Rouaux and Arlotta, 2013). Based on previous studies which showed that LMO4 is implicated in de-repressing the Ctip2 locus, we wondered whether a possibly more accessible Ctip2 locus could be targeted by FEZF2 to induce CTIP2 expression more efficiently. Furthermore, this could also be the case for other CFuPN identity genes.

Therefore, we co-electroporated Cdk5r-Fezf2-IRES-GFP (cFezf2) together with & cLmo4 in embryo brains at E14.5, targeting UL progenitors (see Fig. 19A). To ensure that both proteins are expressed in GFP-positive cells, we carefully examined co-localization of FEZF2 and LMO4 in electroporated neurons. FEZF2 and LMO4 protein was present in all examined GFP-cells, thus revealing both plasmids are collectively delivered in targeted neurons (see Fig. 19B).

Next, we quantified molecular marker expression of CUX1, CTIP2, FOG2, PCP4 and DARPP32 in cFezf2 & cLmo4-electroporated UL neurons (see Fig. 19C,D). UL neurons electroporated with cFezf2 & cLmo4 revealed a significant decrease of cells expressing CUX1 (3.43 ± 0.29% vs.

92.57 ± 2.51% in cGfp), CTIP2 was present in a significant higher number of GFP-positive cells (64.1 ± 2.28% vs. 0.53 ± 0.33% in cGfp), as well as FOG2 (29.67 ± 2.91% vs. 0.37 ± 0.21% in cGfp), or PCP4 (54.13 ± 7.16% vs. 0% in cGfp) or DARPP32 (47.37 ± 2.67% vs. 0.97 ± 0.42% in cGfp, see Fig. 19E).

As it could not be excluded that the driving factor here was solely FEZF2, we electroporated

E. The motor area is a more instructive environment for CFuPNreprogramming

As a next step, we wanted to investigate whether the cellular environment may have an impact on reprogramming efficiency. Therefore, we chose to electroporate our plasmids into post-mitotic UL neurons of the presumptive motor area (see Fig. 21A,B). The motor area differs in its cytoarchitecture from the somatosensory cortex. Lower layer 5 and 6 are enlarged in the motor area with a broader and denser population of CSMN, compared to the somatosensory area. Accordingly, the thicker lower layer 5 is resembled by a larger CTIP2 expression in the motor area [START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]. Thus, the motor area might be a more

F. Reprogrammed post-mitotic UL neurons maintain stable regarding their altered molecular identity

As reprogrammed post-mitotic Ul neurons might not be able to maintain their altered expression profiles and thus, revert to their original identity, we investigated animal brains electroporated with either control cGFP, cFfezf2 or cFezf2 & cLmo4 at P35 (see Fig. 22A,B).

Then, we compared the quantified percentages of GFP-positive UL neurons in P35 with the results of quantified percentage at P7. Here, we showed that the plasticity of early-postmitotic UL neurons can indeed be exploited to reprogram neurons, which remain stable altered molecular marker expression, even in an environment that is not in favour of their newly-acquired identity. showed low efficient reprogramming in previous studies (De La Rossa et al., 2013;Rouaux and Arlotta, 2013). Thus, we investigated if UL neurons can be reprogrammed at P3 by applying cFezf2 & cLmo4 and, whether this factor cocktail would reveal a higher efficiency compared to single cFezf2.

To test this hypothesis, we subcloned Fezf2 or Lmo4 into an inducible vector pCAG-fl-mutCherry-fl-IRES-EGFP (gift from V. Tarabykin). This plasmid consists of a stop sequence flanked by loxP sites in same directional orientation before the actual gene, a subsequent IRESsequence and a GFP-reporter (see Fig. 23A). The pCAG promoter drives high levels of gene expression in mammalian expression vectors, but is not limited to specific cell types or the developmental stage of a cell (Niwa, Yamamurai and Miyazaki, 1991). All vectors were coelectroporated with pCAG-ER T2 -CRE-ER T2 (Matsuda and Cepko, 2007), a plasmid that expresses CRE-recombinase protein upon administration of 4-hydroxytamoxifen (TAM). The gene expression of inducible vectors is haltered until activity of CRE, which can be induced at any given time by administration of TAM.As single Lmo4 was not sufficient to reprogram UL neurons at earliest post-mitotic stages, it has not been applied for later postnatal stages. Thus, only control pCAG-Ind-GFP, from now on referred to as iGFP, pCAG-Ind-Fezf2, now iFezf2 and pCAG-Ind-Fezf2 together with pCAG-Ind-Lmo4, from now on iFezf2 & iLmo4 were electroporated in the somatosensory area. We made this choice due to the difficulty of targeting the motor area and the observations of a higher embryonic lethality when plasmids are electroporated in presumptive M1. Here, we injected TAM in electroporated mice at P3, collected the brains at P7 and analyzed GFP-positive UL neurons for expression of upper and lower layer markers.

As a first step, we investigated whether iFezf2 & iLmo4 express their respective proteins in GFP-positive cells. Indeed, FEZF2 and LMO4 are co-expressed in iFezf2 & iLmo4-electroporated cells (see Fig. 23B). Furthermore, to ensure that inducible vectors are not expressing their respective proteins without TAM administration, we electroporated embryos with iFezf2 & iLmo4, pCAG-ER T2 -CRE-ER T2 and an mCherry plasmid (to reveal successful electroporation).

Then, at P3, we injected corn oil without TAM into electroporated mice and analyzed their brains by application of IHC. The expression of CHERRY revealed electroporated cells. No GFP signal was found in CHERRY-positive cells, demonstrating that inducible vectors do not express their respective proteins without TAM administration (see Fig. 23C) is a TF, known for its specificity of SCPNs and in addition, known for regulating CST formation during development [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]Chen et al., 2008). Moreover, ectopic expression of Ctip2 in UL progenitors was demonstrated to re-route their axonal projections towards subcerebral targets (Chen et al., 2008).

Next
Thus, we co-electroporated iFezf2 & iLmo4 together with pCAG-ER T2 -CRE-ER T2 and iCtip2 and applied TAM at P3 (see Fig. 25A). This result showed that the addition of Ctip2 to the reprogramming factors Fezf2 & Lmo4 did not increase efficiency of reprogramming UL neurons. Together with the observation that CTIP2 is not expressed in targeted P3 UL neurons, we assume that the role of CTIP2 is limited to early post-mitotic development of CFuPNs.

Another factor we investigated for possible improvement in reprogramming was PBX1. PBX1 is implicated as pioneer factor in adult neurogenesis and as patterning gene for arealization and cortical laminarization during development (Golonzhka et al., 2015;Grebbin et al., 2016).

Pioneer factors are especially well-suited to initiate cell fate changes as they can recognize their target sites in barely accessible chromatin and initiate a cascade of events that allows for later transcriptional activation of the respective genes (Grebbin and Schulte, 2017).

To test this whether hPBX1 could improve reprogramming efficiency, we electroporated an inducible vector carrying a PBX1b sequence (iPBX1) together with iFezf2 & iLmo4 and pCAG-ER T2 -CRE-ER T2 and applied TAM at P3 (see Fig. 25B). The effect of adding PBX1 to the reprogramming cocktail did not increase the number of reprogrammed UL neurons compared

C.1. Postnatal induction of single iFezf2 or iFezf2 & iLmo4 drives UL neurons to change their axonal projections towards corticofugal targets

To further investigate reprogrammed UL neurons, we followed their axonal projections. An issue to overcome was the absence of GFP-positive axons when vectors were electroporated at postnatal stages (data not shown). A recent publication demonstrated that an engineered probe was able to reveal signal in fine axons and collaterals upon electroporation in neurons of the cortex and the hippocampus (Viswanathan et al., 2015). This protein, called "spaghetti monster" pCAG_smFP_FLAG, is equipped with a CAG promoter, the engineered probe attached with 3x polypeptide protein tag. The "spaghetti monster" protein itself does not emit any fluorescence but can be detected by anti-FLAG antibodies and subsequent secondary antibody binding.

Therefore, we co-electroporated either iGfp, iFezf2 or iFezf2 & iLmo4 together with pCAG-ER T2 -CRE-ER T2 and pCAG_smFP_FLAG and induced expression of inducible vectors at P3 (see Fig. 26). Brains were collected at P10. When vectors were induced at P3, FLAG-positive axons could be observed in the CC, in the Str, the Th and the IC in all conditions. Note, that only very faint signal was observed in control iGFP in subcerebral targets, Th and IC. In cortices electroporated with iFezf2, FLAG-axons were found in the CP and in the SC, while more FLAGpositive axons could be observed in cortices electroporated with iFezf2 & iLmo4 in CP and the SC, indicating a stronger reprogramming efficiency specifically into SCPNs.

To conclude, UL neurons can change their axonal projections even at postnatal stages.

Nevertheless, axons in the CC of iFezf2 & iLmo4 induced cortices, reveal that the reprogramming efficiency seems to decline with higher developmental stages of UL neurons.

Furthermore, only very few FLAG-axons were found in Th, suggesting that at P3 the majority of reprogrammed UL neurons turn into SCPNs.

D. Postnatal induction at P7 of iFezf2 & iLmo4 still drives UL neurons to express lower layer marker PCP4

The next step was to investigate whether UL neurons can still be reprogrammed at later developmental stages. Therefore, we induced expression of vectors control iGFP or iFezf2 & iLmo4 at P7 and analysed electroporated brains at P14 (see Fig. 27A,B,C). Quantification of percentage of UL neurons expressing upper and lower layer markers revealed alteration of molecular identity in cortices electroporated with iFezf2 & iLmo4. Upper layer marker CUX1

was expressed in significant fewer numbers of GFP-positive UL neurons (62.8 ± 5.76% vs.

89.97 ± 2.13% in control), while again lower layer marker CTIP2 was not expressed (0% vs. 0% in control). FOG2 expression was not significantly induced upon iFezf2 & iLmo4 electroporation (0.67 ± 0.67% vs. 5.9 ± 2.07% in control), but interestingly PCP4-expression was significantly induced (49.27 ± 9.48% vs. 0.67 ± 0.33% in control).

Thus, UL neurons at P7 be changed regarding their molecular identity towards a CFuPN subtype revealed by fewer number of cells expressing CUX1 and an increased number of cells expressing PCP4 upon induction of Fezf2 & Lmo4. Furthermore, these results support the assumption that PCP4 is involved in late developmental CSMN specification.

D.1. UL neurons receiving expression of single iFezf2 or iFezf2 & Lmo4 at P7 show aberrant projections to corticofugal targets

Again, to test whether P7 UL neurons can also change their axonal projections at this stage, we induced vector iGFP, iFezf2 or iFezf2 & iLmo4 by TAM application and followed their axonal projections.

Induction of vectors at P7 and showed axons of targeted UL neurons in the CC and in the Str in all conditions (see Fig. 28). Moreover, no FLAG-signal was detected in the Th in control cortices, while both iFezf2 or iFezf2 & iLmo4 induction revealed signal in the Th. In all conditions, axons of FLAG-positive UL neurons showed projection into the IC, while only in cortices electroporated with reprogramming factors iFezf2 or iFezf2 & iLmo4 FLAG-positive axons were present in the CP. Interestingly, iFezf2-induction in UL neurons did not show axonal projections in the SC, while iFezf2 & iLmo4 reprogrammed UL neurons reached the SC.

Taken together, UL neurons can still be instructed to change axonal projection towards corticofugal targets. In particular, iFezf2 & iLmo4 induction revealed innervation of the SC, the most caudal target of SCPNs. These results reveal an unknown plasticity of UL neurons at P7, and adds up a time point, where neurons in the cortex can still be converted.

E. Ectopic expression of single iFezf2 or iFezf2 & Lmo4 in late postmitotic UL neurons show corticofugal projections

We next explored whether we can reprogram UL neurons at later developmental stages.

Induction of iFezf2 & Lmo4 at P10, did not show expression of CFuPN markers anymore, thus our examination was limited to connectivity features (data not shown).

At P10, induction of vectors mimicked phenotypes of P3 and P7 regarding projections in CC and Str in all conditions (see Fig. 29A andB). iGfp-induction in UL neurons did not reveal any signal in subcortical targets, whereas iFezf2-induction in UL neurons showed FLAG-positive axons in the CC, Str, Th, IC and CP but not in SC, similar to results obtained when induced at P3. In contrast, iFezf2 & iLmo4 electroporated cortices revealed FLAG-positive axons of UL neurons in CC and Str, but also subcortical structures Th, IC, CP and signal was even detected in the SC. These results indicate that UL neurons can still be reprogrammed at postnatal day P10, showing axonal projections characteristic of CFuPNs. The addition of iLmo4 to iFezf2 seems to facilitate the projections of converted UL neurons to the SC, which could not be observed in single iFezf2 at P7, nor P10.

These data demonstrate for the first time that reprogramming of postnatal P10 cortical neurons is feasible and reveal an unkown plasticity of differentiating cortical neurons.

Thus, we went one step further and induced plasmid expression at later developmental stages in UL neurons. Induction at P21 showed axonal projections in all examined structures of the brains electroporated and induced with iGFP, iFezf2 or iFezf2 & iLmo4, besides absence of FLAG-signal in Th of cortices of control iGfp or iFezf2 & iLmo4 induced vectors (see Fig. 29B).

As subcortical targets such as CP and SC were even innervated by FLAG-positive axons of control iGfp-induced UL neurons, qualitative conclusion of reprogramming was not possible.

Thus, whether UL neurons can be still reprogrammed at P21 is not clear at this stage and further investigations are necessary to gain a better understanding of these results.

Section III.

RNA extraction from UL neurons electroporated with cGFP, cFezf2 or cFezf2 and cLmo4 to screen for downstream targets via RNA-sequencing As a last step, we were interested in examining downstream targets upon reprogramming UL neurons at embryonic stage E14.5. RNA-sequencing is a common technique to screen for genes showing altered expression levels upon comparing test and control conditions. Thus, finding genes which show up-or downregulation could help us to better identify reprogrammed cells and reveal possible downstream targets of FEZF2 or LMO4.

Therefore, we electroporated embryo brains at 14.5 with either cGFP, cFezf2 or cFezf2 & cLmo4 and collected brains two days later at E16.5 (see Fig. 30). Subsequently, GFP-positive brains were dissociated and sorted on a FACS-cell sorter device. As a last step, RNA was extracted from these sorted GFP-positive cells.

Unfortunately, the results of this experiment were not available before the submission of this thesis and therefore, I will discuss possible outcomes in the discussion section.

I. Induction of Lmo4 alone fails to efficiently convert post-mitotic UL neurons

At first, we explored whether Lmo4 alone has the potential to reprogram UL neurons into cells showing characteristic features of CFuPNs.

LMO4 is expressed during early cortical development and is implicated in controlling the diversity of motor cortex projection neuron subpopulations, establishing their area-specific identity and specialized connectivity (Cederquist et al., 2013). Furthermore, it is involved in the delineation of post-mitotic SCPNs and CPNs in L5 of the motor cortex (Azim et al., 2009). SATB2 has been shown to be a transcriptional repressor of CTIP2, thereby inhibiting its expression and promoting CPN molecular identity (Alcamo et al., 2008;Britanova et al., 2008).

However, our lab has recently demonstrated that both proteins, mostly mutually exclusive during pre-natal stages, are co-expressed in a subpopulation of lower layer neurons from birth to adulthood. This co-expression is enabled via mediation of LMO4, whose expression gradually increases in somatosensory cortex after birth and which sequesters histonedeacetylase HDAC1, thereby de-repressing the SATB2-regulated repression of the Ctip2-locus (Harb et al., 2016). Furthermore, overexpression of LMO4 in early post-mitotic L5 neurons increased the number of cells expressing CTIP2, whereas reduction of LMO4 strongly reduced CTIP2-expressing neurons in L5 (Harb et al., 2016). Thus, LMO4 is involved in the epigenetic regulation of Ctip2 in post-mitotic L5 neurons.

As seminal work demonstrated that upper layer early post-mitotic CPNs can be reprogrammed into CFuPNs by Fezf2, including upregulation of CTIP2 expression (Rouaux and Arlotta, 2013), and that Lmo4 was also able to induce CTIP2 expression in lower layer neurons (Harb et al., 2016), we wondered whether Lmo4 alone would allow UL neurons (including CPNs and IT neurons) to subcerebral reprogramming in UL neurons. Overexpression of Lmo4 revealed neither significant changes in expression of UL neurons marker CUX1 nor in expression of CFuPN markers CTIP2 or PCP4 (see Fig. 17). It did however induce FOG2 and DARPP32 in a significant proportion of UL neurons. The number of cells expressing these CFuPN markers was low compared to published data upon Fezf2 ectopic expression (Rouaux and Arlotta, 2013). Thus, Lmo4 showed a slight change in the molecular identity in UL neurons but its reprogramming efficiency was low.

Since LMO4 is not a transcription factor binding directly to target genes, but a co-adaptor acting on the epigenetic regulation of chromatin structure, we hypothesized that its In conclusion, Lmo4 ectopic expression in post-mitotic UL neurons is not sufficient to convert neither UL neurons molecular identity nor their axonal projections.

II. Lmo4 synergize with Fezf2 to trigger efficient reprogramming of postmitotic UL neurons into CFuPNs

As described above, Lmo4 does not possess the capacity of reprogramming UL neurons on its own, probably due to a lack of TFs acting on opened loci of CFuPN determination genes. Thus, we wondered whether the addition of FEZF2 would enable UL neurons conversion, and, whether FEZF2 could take advantage of LMO4-mediated de-repression of corticofugal determination genes. Our electroporation experiments revealed that Lmo4 synergizes with Fezf2 to convert UL neurons into neurons showing molecular identity characteristic of CFuPNs.

Almost none of the Fezf2 & Lmo4-electroporated cells showed expression of the upper layer marker CUX1, whereas the majority of UL neurons expressing Fezf2 & Lmo4 induced expression of CFuPN markers (see Fig. 19). These results clearly reflect the developmental role of FEZF2 during newly post-mitotic PNs specification and are in accordance with reported studies about developmental function of FEZF2 (Shimizu and Hibi, 2009;Shimizu et al., 2010;C. Guo et al., 2013;McKenna et al., 2015). The differentiation of these neurons is regulated via competing molecular programs. In newly post-mitotic PNs, FEZF2 is inhibiting SATB2, a transcription factor that promotes upper layer neurons. These neurons express CUX1 downstream of SATB2 (McKenna et al., 2015). Thus, ectopic expression of Fezf2 & Lmo4 might have led to SATB2 repression, loss of callosal identity, as in the case of Satb2 KO mice (Leone et al., 2015;McKenna et al., 2015) and, consequently, CUX1 downregulation and CTIP2 upregulation. Thus, Fezf2 and Lmo4 ectopic expression in post-mitotic UL neurons allowed CFuPN marker induction. In particular, CTIP2-expression was visible in over 60% of doubleelectroporated UL neurons, whereas overexpression of only Fezf2 led to an induction of around 25% (Rouaux and Arlotta, 2013)). During development, Fezf2 acts upstream of CTIP2, promoting its expression in layer 5 SCPNs [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]Chen et al., 2008). Thus, ectopic expression of FEZF2 and LMO4 leads to an increased induction of CTIP2 in electroporated UL neurons.

Another CFuPN induced marker upon Fezf2 & Lmo4 overexpression in UL neurons was FOG2.

This protein is known to be specifically expressed by L6 CThPNs at early postnatal stages (Galazo, Emsley and Macklis, 2016). This was not unexpected, since FEZF2 not only promotes SCPN differentiation, but also has a role in CThPN specification (Shimizu et al., 2010). FEZF2 is expressed at low levels in CThPNs, contributes to the formation of reciprocal connections between thalamus and cortex but is most probably not highly involved in the specification of CThPNs (Komuta et al., 2007;Chen et al., 2008;Tantirigama et al., 2016) (Rouaux and Arlotta, 2010) most probably as part of its role in axon guidance, as previously reported (Lodato et al., 2014).

Another CFuPN marker we found ectopically induced in Fezf2 & Lmo4 electroporated UL neurons is PCP4. The role of PCP4 in the cortex is still not well understood, but its expression profile has been mentioned in some studies. PCP4 has been described to be expressed in deep layer neurons (Watakabe et al., 2012), and to be a late determination gene of corticospinal motor neurons, a sub-type of SCPNs [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. Interestingly, FEZF2 has been shown do directly bind on the proximal promoter region of Pcp4 (Lodato et al., 2014), suggesting a regulative role of FEZF2 on Pcp4 expression. In this work, we show that upon ectopic expression of Fezf2 and Lmo4, a large number of electroporated UL neurons show PCP4 induction, most probably mediated by the activation of Fezf2, even if this population increases in the presence of high Lmo4 expression. We thus propose that the PCP4-induced neuronal population represents a mixture of SCPN and CthPN subpopulations.

At last, induced DARPP32 expression could be observed in reprogrammed UL neurons upon

Fezf2 & Lmo4 overexpression. As for PCP4, DARPP32 expression and function in the cortex is not extensively described in the literature, whereas it is highly expressed in the striatum (Anderson, 1991). It has been reported to be expressed in lower layer 6 at postnatal stages by a Tbr1-negative CThPN subpopulation in the mouse somatosensory cortex (McKenna et al., 2011) and in the rat cingulate cortex (Ouimet, 1991). In Fezf2 KO, DARPP32 is downregulated in layer 6 (Molyneaux et al., 2005), in agreement with our results in which ectopic Fezf2 et al., 2013;Rouaux and Arlotta, 2013). Interestingly, the number of Fezf2 & Lmo4electroporated UL neurons expressing CTIP2 or PCP4 was significantly higher than in single Fezf2-electroporated UL neurons increasing to an efficiency of over 60% (see Fig. 19) An

IV. Fezf2 & Lmo4 overexpression drive UL neurons to re-route their axonal projections towards corticofugal targets

In addition to the investigation of molecular identity of reprogrammed electroporated UL neurons, it was necessary to follow their axonal projections. After electroporation of the above-mentioned plasmids containing GFP, we could follow their trajectories thanks to the presence of the GFP (see Fig. 18, 20). In accordance with the data obtained with the molecular markers, axonal projections of Lmo4-electroporated cells maintained projections through the CC and the Striatum (Str), similarly to Gfp control-electroporated cortices. This connectivity feature is characteristic of CPNs, the most abundant cell-type of UL neurons. Furthermore, no GFP-positive axons were found in targets of CFuPNs. Thus, Lmo4 did not alter the molecular identity and axonal trajectories of UL neurons.

This situation differs after induction of Fezf2 & Lmo4 electroporated UL neurons, which showed clear projections towards corticofugal targets, with no axons projecting towards the CC. Specifically, reprogrammed UL neurons revealed axonal projections to the Str, the internal capsule (IC), the thalamus (Th), the cerebral peduncle (CP) and the spinal cord (SC). However, we also noticed that some electroporated cells displayed an ectopic location in the lower layers. Since overexpression of Fezf2 & Lmo4 expression occurred in post-mitotic neurons during migration, these cells most probably experienced a migratory deficit. Nevertheless, the majority of electroporated cells properly migrated to their location in the upper layers, revealing that Fezf2 & Lmo4 led to migration deficits in only a subset of cells. This phenotype was also observed in cortices electroporated with single Fezf2 and has been reported in literature (Rouaux and Arlotta, 2013), indicating that high expression of Fezf2 can induce a migratory defect without altering the differentiation process, since abnormally located neurons do express appropriate differentiation markers (Lodato et al., 2011).

Interestingly, the axonal projections of Fezf2 & Lmo4 electroporated UL neurons seemed to split up into two separate populations at caudal positions, where Th and IC are visible. Some GFP-positive axons were directed towards the Th, whereas other axons projected within the IC. Whether these are collaterals of the same neuron, or are independent neurons remains to be elucidated. It is known that SCPN have multiple projection targets, including the Th, the striatum, the brainstem region and the spinal cord (Spheperd, 2013). Since the GFP-signal in even more caudal parts of the brain, at the level of the CP, is similarly strong to the signal in the Th and IC, we propose that they might represent collaterals of GFP-positive axons directed to the Th. This could be examined by lowering the concentration of the plasmids, which would lead to fewer numbers of electroporated cells or combining electroporation with retrograde double tracing in CP and Th. In this way, both populations could be independently labeled and reveal whether projections to the Th are collaterals or independent axons.

Another aspect that should be further investigated is, why there are only few GFP-positive axons reaching the SC. Does the majority of GFP-positive axons stop at a certain level before the SC and if yes, which structure is it? The brainstem is a target of SCPN [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]Shepherd, 2013) and is a structure that lies just rostral to the spinal cord. It is possible that axons of reprogrammed cells stop at the level of the brainstem and only few continue to the SC. This would imply that the majority of reprogrammed UL neurons turn into SCPNs that project to the brainstem, in line with the role of Lmo4 driving layer 5 neurons to the brainstem region (Cederquist et al., 2013). Another possibility is, that the GFP signal diminished by distance and only some GFPs are maintained in the very fine axonal endings of electroporated UL neurons in the spinal cord.

The alteration of axonal projection patterns in UL neurons with high Fezf2 & Lmo4 expression is similar to CPNs with single Fezf2, as also observed by Rouaux and Arlotta (2013)

V. The cellular environment of the motor cortex has an impact on reprogramming efficiency of Fezf2 & Lmo4

As a following step, we wondered whether the cellular environment of the motor cortex would impact the reprogramming efficiency of UL neurons into CFuPNs. The motor cortex has a distinct cytoarchitecture with a thick L5 containing a large number of CSMN, whereas the somatosensory cortex possesses a thick L4 where neurons that receive relayed sensory inputs are located and a thin L5 (Dehay and Kennedy, 2007;[START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]. These differences in the cytoarchitecture can also be seen by the expression of FEZF2 and CTIP2. In the motor cortex, both proteins delineate a much broader and denser L5 than in the somatosensory cortex [START_REF] Tomassy | Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI[END_REF]. Thus, we sought whether the cellular environment of the motor cortex was more instructive for converting UL neurons into CFuPNs. Quantification of the percentage of Fezf2 & Lmo4 electroporated cells in the motor cortex revealed a higher number of GFP-positive cells expressing CTIP2 compared to the somatosensory cortex (see Fig. 21). In addition, also DARPP32 was present in a higher number of Fezf2 & Lmo4 electroporated UL neurons in the motor cortex compared to the somatosensory one. Interestingly, the percentage of CUX1-, FOG2-and PCP4-expressing neurons did not differ between motor and somatosensory cortex upon Fezf2 & Lmo4 electroporated UL neurons. Furthermore, cortices electroporated with single Fezf2 did not show significant changes of marker expression in GFPpositive UL neurons when comparing motor and somatosensory cortex. Also, electroporation of Lmo4 into UL neurons of the motor cortex did not induce any significant CFuPN marker expression as it was the case in the somatosensory cortex. Thus, the cortical environment had only an effect on Fezf2 & Lmo4 mediated reprogramming regarding CTIP2+ and DARPP32+ induced neurons. This result was not confounded by a probable higher number of upper layer cells expressing CTIP2 in the motor cortex, since control GFP electroporated UL neurons did not reveal significant changes in CTIP2 expression.

Taken together, the motor cortex with its thick L5 and its broad CTIP2 expression had a limited, but statistically significant impact on the efficiency of Fezf2 & Lmo4 reprogrammed UL neurons, showing higher numbers of reprogrammed UL neurons expressing CTIP2 or DARPP32, while expressions of other markers were not affected. Thus, it could be that the motor area is more instructive to reprogram UL neurons into CFuPNs, indicating an impact of the cellular environment on reprogramming capacities.

VI. Reprogrammed post-mitotic UL neurons maintain expression of CFuPN markers at juvenile-adult stages

It might be possible that reprogrammed UL neurons were not stable and reverted to their original identity or might have died over time. My results showed that this is most likely not the case, since at P35, reprogrammed single Fezf2 or double Fezf2 & Lmo4 UL neurons revealed stable expression of the majority of CFuPN markers (see Fig. 22). However, DARPP32

was clearly downregulated in both conditions at P35 and maintained its expression in a drastically lower number of converted cells compared to P7. Since other markers remained stably expressed in reprogrammed UL neurons, and since DARPP32 is co-localized with CTIP2

in almost half of the electroporated UL neurons and, since cell death has not been reported

for Fezf2-reprogrammed CPNs (Rouaux and Arlotta, 2013), it is unlikely that the DARPP32expressing neurons died or reverted to their original identity. A possible explanation could be either that Darpp32 was simply downregulated over time, since its function was not needed anymore, or that the axons expressing DARP32 were pruned over time, or that Fezf2 and Lmo4 expression was unable to maintain DARPP32 expression at advanced differentiation stages.

It would also be important to investigate the axonal projections of reprogrammed UL neurons at P35, in order to see whether altered axonal projections can be maintained with time.

Unfortunately, the GFP-signal of reprogrammed UL neurons was faint at P35 and thus, axonal trajectories could not be followed anymore. An additional tracing molecule that is stable for long times could overcome this issue and could reveal whether axonal projections of reprogrammed UL neurons remain stable over time.

VII. Postnatal UL neurons retain the capacity to convert their molecular identity

It became important to understand whether reprogramming of UL neurons was possibly not only at pre-natal stages, when neurons are still immature, but also at postnatal ages when neuronal maturation progressively takes place. Thanks to the use of tamoxifen (TAM)inducible vectors, we were able to overexpress Fezf2 or Fezf2&Lmo4 at different postnatal stages (see Fig. 24).

Our data show that at P3, induction of Fezf2 & Lmo4 in UL neurons of the somatosensory cortex led to expression of FOG2, PCP4 or DARPP32, while CUX1 was downregulated (see Fig. 5B). Surprisingly, at P7, the stage we analyzed the TAM-induced neurons, CTIP2-expression was not induced in electroporated UL neurons. According to the literature, Ctip2 is an early developmental gene in CSMN, a subgroup of SCPN [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF]. Thus, at P3, when UL neurons have positioned in upper layers, the Ctip2-locus might not be more accessible in these cells. Lmo4 has been shown to de-repress SATB2-repressing Ctip2-locus, a mechanism that might be limited to early post-mitotic lineage specifications. This was also observed in the study of Rouaux and Arlotta, 2013, upon Fezf2 overexpression, but the authors did not discuss this issue in their paper. Interestingly, the number of PCP4-positive neurons in Fezf2 & Lmo4 targeted UL neurons resulted even higher than after embryonic induction. As a late developmental gene for CSMN [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF], its mRNA-expression reaches its highest peak at P3. Thus, the induction of PCP4 in P3 UL neurons might be facilitated due to a more favorable gene accessibility.

Moreover, the efficiency of Fezf2 & Lmo4 reprogrammed UL neurons at P3 resulted also higher than single Fezf2-driven reprogramming, as observed by the reduced number of CUX1-positive neurons in electroporated UL neurons, as well as by the increased number of PCP4-positive neurons. On the contrary, FOG2-and DARPP32-induced expression diminished and was only expressed in few neurons. These data suggest, that either UL neurons gradually lose their ability to reprogram their molecular identity or, that at P3, reprogramming of UL neurons is restricted to a more limited subpopulation of CFuPNs. Additional CFuPN molecular identity markers, especially the ones that reveal high expression at early postnatal stages, could provide a broader picture at this stage.

Addition of inducible Ctip2 to inducible Fezf2 & Lmo4

Since reprogrammed UL neurons at P3 failed to show any CTIP2-expression, we wondered whether inducing Ctip2 additionally to Fezf2 & Lmo4 would facilitate reprogramming (see Fig. 25A). Ectopic embryonic CTIP2 expression has been demonstrated to alter axonal projections of upper layer neurons towards corticofugal targets, including Th, CP and pons (Chen et al., 2008). Thus, since CTIP2 ectopic expression showed a similar reprogramming at embryonic stages to Fezf2 & Lmo4, we hypothesized that maintenance of CTIP2 would improve reprogramming. Unfortunately, this was not the case and addition of Ctip2 to the reprogramming cocktail did not lead to a lower number of CUX1-expressing UL neurons, nor to an increased number of UL neurons expressing PCP4 when compared to Fezf2 & Lmo4 reprogramming. It is not known whether other genes are indeed induced, as only a limited number of molecular markers were examined here. Unfortunately, GFP-positive axons were not visible and therefore changes in axonal projections could not be examined. Thus, Ctip2

does not seem to have a facilitating effect on the reprogramming efficiency induced by Fezf2 & Lmo4 at postnatal stages, despite its downregulation.

Addition of inducible PBX1 to inducible Fezf2 & Lmo4

We wondered whether other factors would improve reprogramming efficiency at postnatal stages and tested Pbx1, as a candidate reprogramming gene. PBX1 is implicated in homeostasis of adult neurogenesis in the SVZ (Grebbin et al., 2016), and has been shown to have features of pioneer factors (Grebbin and Schulte, 2017). Pioneer factors can normally access transcriptionally inactive genomic loci. We hypothesized that Pbx1 could transcriptionally open inactive genes, allowing a better chromatin accessibility of FEZF2 & LMO4 in inducing CFuPN related genes. The data indicated that the addition of PBX1 to the reprogramming cocktail did not reveal any drastic changes in expression of molecular markers in electroporated UL neurons (see Fig. 25B). Again, whether other CFuPN markers were induced is not known from these data. Also, GFP-positive axons were not visible and therefore changes in axonal projections could not be examined. Thus, Pbx1 does not seem to increase

VIII. UL neurons retain their ability to change their axonal projections towards corticofugal targets from P3 to P21

To improve axonal trajectories of reprogrammed neurons, we made use of a highperformance tracer, named 'spaghetti monster' fluorescent protein, which has been reported

to be well distributed in neurons and to reveal signals even in few axons (Viswanathan et al., 2015). The protein was encoded by the vector pCAG_smFP FLAG, containing multiple FLAG epitopes, which can be revealed by an anti-FLAG antibody (see Fig. 26, 28, 29). Induction of

Fezf2 & Lmo4 at P3 in upper layer UL neurons revealed at P10 FLAG+ callosal axons as well as FLAG+ signal in axons of subcortical structures. This was the case as well for single Fezf2 induction in P3 UL neurons but with fewer FLAG axons at the level of the IC and CP, indicating a lower efficiency compared to Fezf2 & Lmo4. We noticed that at P3 fewer UL neurons than at E14.5 were re-wired to corticofugal targets. However, it is unclear whether conversion of UL neurons was completed or whether these neurons revealed dual projections, one to the contralateral side and one to corticofugal targets. Throughout development CPNs extend exuberant projections with the maximal number of UL neurons with dual projections occurring at at early to late postnatal stages (Innocenti and Price, 2005;Mitchell and Macklis, 2005).

These dual projections are refined until approximatively P21, when the adult projection pattern for CPNs is established (Fame, MacDonald and Macklis, 2011). It has been reported

for layer 5 CPNs that until completion of refinement, some CPNs still extend a bifurcated axon that projects both to the midline and IC, which is then retracted later during development (approx. P11) (Garcez et al., 2007). Thus, it could be that Fezf2 or Fezf2 & Lmo4-induced UL neurons at early postnatal stages mimic this transient state of L5 dual projection neurons.

Fezf2 & Lmo4 induction at P7 and P10 still enable partial re-routing of UL neuron axons

By inducing Fezf2 & Lmo4 at P7 or at P10, we still observed FLAG signal in the CC and in all examined corticofugal structures at P14 and P21, respectively, as previously described for P3.

Interestingly, single Fezf2 induction at P7 failed to reveal any FLAG signal in the SC, indicating that single Fezf2 overexpression is not sufficient to re-route UL neuron axons to the SC. We noticed that the number of FLAG axons steadily decreased from P3 to P7 and then to P10, suggesting that UL neurons gradually lose their capacity of re-wiring. However, it cannot be excluded that these axons might need longer to reach the SC. Increasing the time window from the onset of TAM administration and brain analysis could resolve this question.

Fezf2 & Lmo4 induction at P21

Induction of Gfp, single Fezf2 or Fezf2 & Lmo4 at P21 revealed a different observation than the previous experiments. In all conditions, FLAG axons were present in almost all corticofugal structures. Presumably, at P35 the complexity of axonal branching is higher and thus, easier to visualize than at earlier postnatal stages. Nevertheless, the FLAG signal in these subcortical structures was clearly higher in single Fezf2 or Fezf2 & Lmo4 conditions. This indicates that at P21, some UL neurons still retain the capacity of re-wiring their axonal projections.

To conclude, until P21, UL neurons still retain the capacity of re-routing their axonal projections.

IX. RNA extraction of early post-mitotic electroporated UL neurons

As a last step, we aim to further investigate the molecular changes that occur in reprogrammed UL neurons at embryonic stages by using high throughput RNA sequencing (see Fig. 30). E14.5 electroporated brains with control GFP, Fezf2, or Fezf2 & Lmo4 were dissociated and FACS-sorted at E16.5 in order to identify early and most probably direct molecular changes upon induction. Bulk RNA-sequencing is the method of choice for analyzing transcriptomes of disease states or of defined biological processes by quantifying gene expression of different biological samples. Thus, mRNA-sequencing of our samples, Gfp, Fezf2

or Fezf2 & Lmo4-electroporated neurons, will give us a more complete picture of changes in gene expression upon reprogramming at this early stage.

I expect to see elevated levels of genes that are specifically expressed by CFuPNs and downregulated gene-levels that are specifically expressed by UL neurons. Furthermore, interesting genes could be discovered, for example, genes that are unknown for their role in CFuPN specification. Additionally, mRNA-sequencing could help us to find downstream targets of Fezf2 in synergy with Lmo4. I would then like to use some interesting genes and try to find out whether induction of these genes would lead to reprogramming of UL neurons. Lastly, it would be interesting to see which genes are differently expressed in Fezf2 & Lmo4 UL neurons compared to single Fezf2. These discoveries could help us to find out why Lmo4 facilitates Fezf2-reprogramming and thus could reveal targets or pathways that might be combined with

I. To further characterize reprogrammed UL neurons

These data showed changes in molecular identity and alterations in axonal projections of reprogrammed UL neurons It would be crucial to further investigate neuronal and molecular properties of these cells to obtain a more thorough characterization of these reprogrammed cells. For instance, analysis of the morphology, electrophysiological properties and functional integration of these neurons could give us more information about their cell identity. L5

neurons possess an apical dendrite that bifurcates in L2/3, a feature which is different from upper layer PNs (Lefort et al., 2009). Thus, investigating whether the morphology of reprogrammed UL neurons resemble such a feature could broaden our identity characterization. Furthermore, investigating a functional input onto reprogrammed UL neurons could reveal whether these neurons are communicating with other neurons. This could be performed by stimulating neighbouring upper layer neurons and recording possible responses in reprogrammed UL neurons.

II. To investigate reprogramming in other cell-types

III. To increase reprogramming efficiency at postnatal stages

It has been reported by other studies that small molecules or chromatin remodelling factors could facilitate reprogramming approaches (Torper and Gotz, 2017). Thus, it would be interesting to examine whether the addition of interesting candidate could improve the reprogramming efficiency of late postnatal or mature UL neurons. Reprogramming of cortical PNs in the adult brain has not been reported so far. However, it is a goal for regenerative medicine, as most patients suffering from e.g. ALS are adult (ALS Association). Thus, reprogramming neurons at adult stages would be a beneficial feature that could possibly serve as an alternative approach in restoring CMNs in patients in the future.

IV. To apply possible downstream targets of FEZF2 revealed by mRNAsequencing

As soon as we will obtain our data from the mRNA-sequencing experiment, possibly interesting candidates could be used for further applications revealing a possible enhancement of UL neuron reprogramming. Furthermore, we could identify other downstream targets of FEZF2 and discover, which genes are particularly targeted by LMO4 in combination with FEZF2. Another possibility could be that we find an interesting molecule that could serve as a potential drug to specifically convert somatic cells into CFuPNs. This would ease the application for studies on patient-derived cells without the necessity of genetically modifying cells. Furthermore, Lmo4 is implicated in cancer formations (Visvader et al., 2001;N. Wang et al., 2004;Singh et al., 2005) and has been reported as an oncogene. Finding a candidate that could replace LMO4 would therefore be beneficial, regarding any possible clinical studies.

  List of Abbreviations ................................................................................................................... INTRODUCTION .......................................................................................................................... I. The Mammalian Neocortex: Cytoarchitecture and Development ..............................................

Figure 1 :

 1 Figure 1: Projection neuron diversity in the cerebral cortex .............................................................. Figure 2: Long-range axonal projections define two classes of corticostriatal projection neuron ....Figure 3: Neocortical projections neurons are generated in an "inside-out" fashion by diverse

Figure 3 :

 3 Figure 1: Projection neuron diversity in the cerebral cortex .............................................................. Figure 2: Long-range axonal projections define two classes of corticostriatal projection neuron ....Figure 3: Neocortical projections neurons are generated in an "inside-out" fashion by diverse progenitor types in the VZ and SVZ .....................................................................................................Figure 4: CPN development and diversity ............................................................................................Figure 5: Mechanisms and time course of mouse corticospinal tract (CST) development ................Figure 6: CST projections to the spinal cord in rodents .......................................................................Figure 7: Transcription factors in the VZ establish an area identity fate map ...................................Figure 8: Competing molecular programs direct differentiation of newly-postmitotic projection neurons into one of three broad subtype identities ...........................................................................Figure 9: IHC of CTIP2 protein expression in M and S1 of WT mice at P8 ..........................................Figure 10: The Fez family of transcription factors. ..............................................................................Figure 11: Fezf1 and Fezf2 expression during embryogenesis. ...........................................................Figure 12: Fezf1 and Fezf2 functions during forebrain neurogenesis. ................................................ Figure 13: Schematic representation of the human LMOs and LDB1. ................................................Figure 14: Schematic representation of LMOs in gene transcriptional regulation. ...........................Figure 15: Schematic model of the putative mechanism by which Lmo4 de-represses Ctip2 expression .............................................................................................................................................Figure 16: The Cook Islands model applied to neuronal reprogramming ..........................................Figure 17: Ectopic expression of Lmo4 induces lower layer markers at low efficiency in post-mitotic upper layer neurons ............................................................................................................................. Figure 18: Ectopic expression of Lmo4 in UL neurons does not alter their axonal projection .......... Figure 19: Ectopic Expression of single Fezf2 or Fezf2 in combination with Lmo4 drives post-mitotic UL neurons to acquire molecular features characteristic of CFuPN. ................................................Figure 20: cFezf2 & cLmo4 or single cFezf2 ectopic expression alter axonal projection of postmitotic UL neurons towards corticofugal targets .............................................................................. Figure 21: Ectopic expression of cFezf2 & cLmo4 in UL neurons of the motor area induces higher levels of corticofugal identity markers CTIP2 and DARPP2 compared to somatosensory area. ..... Figure 22: Reprogrammed post-mitotic UL neurons maintain stable regarding their altered molecular identity............................................................................................................................... Figure 23: A. Verification of iLmo4 and iFezf2 induction ..................................................................Figure 24: Postnatal induction at P3 of single Fezf2 or Fezf2 & Lmo4 alters molecular identity of UL neurons ...............................................................................................................................................Figure 25: Postnatal induction of single iFezf2 or iFezf2 & iLmo4 combined with iCtip2 or iPBX1 does not increase reprogramming efficiency ....................................................................................Figure 26: Postnatal induction of single iFezf2 or iFezf2 & iLmo4 drives UL neurons to change their axonal projections towards corticofugal targets ...............................................................................Figure 27: Postnatal induction at P7 of iFezf2 & iLmo4 still drives UL neurons to express lower layer marker PCP4 ........................................................................................................................................Figure 28: UL neurons receiving expression of single iFezf2 or iFezf2 & Lmo4 at P7 show aberrant projections to corticofugal targets ..................................................................................................... Figure 29: Ectopic expression of single iFezf2 or iFezf2 & Lmo4 in increasingly differentiating UL neurons show corticofugal projections .............................................................................................Figure 30: RNA extraction of UL neurons electroporated with cGFP, cFezf2 or cFezf2 and cLmo4 to screen for downstream targets via RNA-sequencing ........................................................................

Figure 4 :

 4 CPN development and diversity ............................................................................................

Figure 5 :

 5 Mechanisms and time course of mouse corticospinal tract (CST) development ................

Figure 6 :

 6 CST projections to the spinal cord in rodents .......................................................................

Figure 7 :

 7 Transcription factors in the VZ establish an area identity fate map ...................................

Figure 8 :

 8 Competing molecular programs direct differentiation of newly-postmitotic projection neurons into one of three broad subtype identities ...........................................................................

Figure 9 :

 9 IHC of CTIP2 protein expression in M and S1 of WT mice at P8 ..........................................

Figure 10 :

 10 The Fez family of transcription factors. ..............................................................................

Figure 11 :

 11 Fezf1 and Fezf2 expression during embryogenesis. ...........................................................

Figure 12 :

 12 Fezf1 and Fezf2 functions during forebrain neurogenesis. ................................................

Figure 13 :

 13 Schematic representation of the human LMOs and LDB1. ................................................

Figure 14 :

 14 Schematic representation of LMOs in gene transcriptional regulation. ...........................

Figure 15 :

 15 Schematic model of the putative mechanism by which Lmo4 de-represses Ctip2 expression .............................................................................................................................................

Figure 16 :

 16 The Cook Islands model applied to neuronal reprogramming ..........................................

Figure 17 :

 17 Ectopic expression of Lmo4 induces lower layer markers at low efficiency in post-mitotic upper layer neurons .............................................................................................................................

Figure 18 :

 18 Ectopic expression of Lmo4 in UL neurons does not alter their axonal projection ..........

Figure 19 :

 19 Ectopic Expression of single Fezf2 or Fezf2 in combination with Lmo4 drives post-mitotic UL neurons to acquire molecular features characteristic of CFuPN. ................................................

Figure 20 :

 20 cFezf2 & cLmo4 or single cFezf2 ectopic expression alter axonal projection of postmitotic UL neurons towards corticofugal targets .............................................................................. Figure 21: Ectopic expression of cFezf2 & cLmo4 in UL neurons of the motor area induces higher levels of corticofugal identity markers CTIP2 and DARPP2 compared to somatosensory area. ..... Figure 22: Reprogrammed post-mitotic UL neurons maintain stable regarding their altered molecular identity............................................................................................................................... Figure 23: A. Verification of iLmo4 and iFezf2 induction ..................................................................

Figure 24 :

 24 Postnatal induction at P3 of single Fezf2 or Fezf2 & Lmo4 alters molecular identity of UL neurons ...............................................................................................................................................

Figure 25 :

 25 Postnatal induction of single iFezf2 or iFezf2 & iLmo4 combined with iCtip2 or iPBX1 does not increase reprogramming efficiency ....................................................................................

Figure 26 :

 26 Postnatal induction of single iFezf2 or iFezf2 & iLmo4 drives UL neurons to change their axonal projections towards corticofugal targets ...............................................................................

Figure 27 :

 27 Postnatal induction at P7 of iFezf2 & iLmo4 still drives UL neurons to express lower layer marker PCP4 ........................................................................................................................................

Figure 28 :

 28 UL neurons receiving expression of single iFezf2 or iFezf2 & Lmo4 at P7 show aberrant projections to corticofugal targets ..................................................................................................... Figure 29: Ectopic expression of single iFezf2 or iFezf2 & Lmo4 in increasingly differentiating UL neurons show corticofugal projections .............................................................................................

Figure 30 :

 30 RNA extraction of UL neurons electroporated with cGFP, cFezf2 or cFezf2 and cLmo4 to screen for downstream targets via RNA-sequencing ........................................................................

Figure 1 :

 1 Figure 1: Projection neuron diversity in the cerebral cortex Schematic presentation showing origin of PNs and their respective axonal projections. A. Commissural projection neurons project to the contralateral cortical hemisphere. Most cross the midline through the corpus callosum (callosal projection neurons, CPN), while a smaller population crosses through the anterior commissure. CPN reside primarily in upper layer 2 -3, with fewer in lower layers 5 and 6 and extend axons to mirror-image locations in the same functional area of the contralateral hemisphere. B. Associative projection neurons are present in all layers of the neocortex, projecting within a single cortical hemisphere. This populations includes short-distance intrahemispheric projection neurons, which extend axons within a single cortical column or nearby cortical columns and long-distance intrahemispheric projection neurons, which extend axons to adjacent or distant cortical areas (such as forward and backward projection neurons).

Figure 3 :

 3 Figure 3: Neocortical projections neurons are generated in an "inside-out" fashion by diverse progenitor types in the VZ and SVZThis schematic depicts the sequential generation of neocortical projection neuron subtypes and their migration to appropriate layers over the course of mouse embryonic development

  left side of the figure shows the trajectory of the mouse CST from the cortex to the spinal cord. The genes involved in CST development are indicated at the corresponding level. The right side of the figure indicates the time course of CST development. CST, Corticospinal tract; PSPB, pallial/subpallial boundary. Taken from Welniarz, Dusart and Roze (2017) .

Figure 6 :

 6 Figure 6: CST projections to the spinal cord in rodents A The crossed CST (dark blue) is located in the most ventral part of the dorsal funiculus, and the uncrossed CST (red) runs in the ventral funiculus. Taken and modified from Welniarz, Dusart and Roze (2017).

Figure 7 :

 7 Figure 7: Transcription factors in the VZ establish an area identity fate map A. Arealization of the cerebral cortex is initiated by diffusible morphogens and signaling molecules secreted from opposing sides of the neocortical periphery (left panel). These signals induce expression of complementary and orthogonal transcription factor gradients such as Pax6/Emx2 and Sp8/Coup-TFI, seen in a schematized flatmount view of the ventricular zone (VZ)

Figure 11 :

 11 Figure 11: Fezf1 and Fezf2 expression during embryogenesis.

Figure 12 :

 12 Figure 12: Fezf1 and Fezf2 functions during forebrain neurogenesis.A. Loss of both Fezf1 and Fezf2 during early forebrain neurogenesis results in an increase in Hes5 expression. This leads to decreased neurogenesis and intermediate progenitor generation and decreases in the number of lower layer projection neurons at birth. B.Fezf2 is expressed in multipotent RGCs that sequentially generate all major cortical projection neuron subtypes and glia. Taken fromEckler and Chen( 2014).

Figure 13 :

 13 Figure 13: Schematic representation 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 Sang et al. (2014).

Figure 14 :

 14 Figure 14: Schematic representation of LMOs in gene transcriptional regulation. LMOs regulate gene transcription by functioning as 'linker' or 'scaffolding' proteins through their LIM domains and are involved in the formation of multiprotein complexes of DNA-binding factors and transcriptional regulator proteins. 'RNAPII' means RNA polymerase II. Taken from Sang et al. (2014).

  has demonstrated for the first time, that converting fibroblasts into myoblasts was feasible by simple overexpression of MyoD, revealing single-cell-type-specific transcriptional cell fate change. In 2006, a study of Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) demonstrated that overexpression of a four-component-TF-cocktail can de-differentiate somatic cells into pluripotent stem cells. Besides artificial reprogramming approaches, Zuryn et al. (2014), could observe that reprogramming of a mature cell into another cell type is occurring naturally, in this case in C.

  environment of the somatic DNA can reprogram somatic cells. The technique allows biochemical modification of the environment and, therefore, careful monitoring of the outcome of introduction or depletion of reprogramming factors from the extract. Additionally, extract-treated cells may also be used as starting material for induced pluripotency or nuclear transfer experiments (Halley-Stott, Pasque and Gurdon, 2013). Limitations arise from poor efficiency of successfully reprogrammed cells and experiments show a low rate of reproducibility(Liu et al., 2011).

  differentiate and proliferate. Subsequently, these cells then create a new lens vesicle and differentiate into mature lens cells, thereby recovering the lost tissue(Jopling, Boue and Belmonte, 2011). A more recent study in C. elegans demonstrated trans-differentiation occurring during normal development, without any type of injury. A post-mitotic hindgut cell, named Y cell, detaches from the rectal tube and then converts into a motor neuron, named

  demonstrated in the developing striatum conversion of striatal progenitors, normally fated to become medium spiny neurons, into CFuPNs upon Fezf2 overexpression. Via in utero electroporation, a Fezf2 expression vector was delivered into neural progenitors of the lateral ganglionic eminence. Molecular profiles of the electroporated cells revealed expression of Sox5, Tbr1 and Fog2markers of lower layer projection neurons in the cortex. Furthermore, converted neurons showed morphology characteristic of pyramidal neurons and projections to expected corticofugal targets, such as the thalamus and cerebral peduncle. As the striatal niche normally does not host cortical projection neurons, these results demonstrated that cell-autonomous signals that drive neuron type-specific development in the cortex can override extracellular niche restrictions and instructions over progenitor cell fate (Rouaux and Arlotta, 2010). In 2013, two groups independently demonstrated Fezf2-driven reprogramming of upper layer cortical post-mitotic neurons into corticofugal neurons in vivo. De la Rossa et al. (De La Rossa et al., 2013) used a special technique, named iontoporation, to deliver a Fezf2-expressing plasmid in L4 stellate neurons. Iontoporation worked as follows: plasmids are injected into the lateral ventricles of P1 pups followed by an electric pulse allowing transfection of cells. After ectopic expression of Fezf2 in L4 stellate cells, transfected cells showed up-regulation of the lower layer marker Er81 and down-regulation of upper layer markers, such as Satb2 and Cux1.In addition, reprogrammed cells revealed aberrant projection to striatum. L4 stellate cells normally form local neurocircuits and do not project outside the cortex. Electrophysiological examinations further revealed characteristic physiological properties of lower layer projection neurons of reprogrammed cells. Moreover, intracortical inputs were altered upon Fezf2driven conversion of L4 neurons. Normally, these neurons do not receive input from L2-3 neurons, but, in contrast, reprogrammed cells received L2-3 neuronal input. Thus, reprogramming of post-mitotic neurons into corticofugal-like projection neurons in the cortex in vivo had been successfully demonstrated.

  indicated a regulative role of Lmo4 for Ctip2-expression in lower layer neurons of the developing cortex, and therefore implicating Lmo4 in SCPN specification. Thus, we wondered whether Lmo4-ectopic expression in upper layers could instruct SCPN fate conversion of CPNs as it has been demonstrated for Fezf2. Furthermore, the de-repressing mediation of the Ctip2-locus by LMO4 motivated us to investigate whether Fezf2 could possibly take advantage of the opened access of Ctip2 locus and thus, more efficiently convert CPNs into SCPNs in comparison to single Fezf2-reprogramming as shown in Rouaux and Arlotta (2013) and De la Rossa et al. (2013).

  enzyme Filled up to 30 µl with ddH20

  Image analysis has been performed with Fiji (ImageJ 1.51n), graphs and statistical analysis have been performed with Graphpad Prism 7. Scale bars are indicated on the images or in the figure legends. expression of Lmo4 induces lower layer molecular markers in postmitotic upper layer neurons at low efficiency Direct neuronal reprogramming aims at changing cell fate of differentiating or differentiated cells into neurons. In the mammalian brain, significant neuronal generation is limited to early post-mitotic and postnatal stages. Once a neuron is born, it does not change its cell identity until the end of its lifespan. Seminal work has demonstrated that post-mitotic neurons retain a certain plasticity and new-born upper layer CPNs can be converted into lower layer corticofugal projection neurons (CFuPNs) by ectopic expression of one transcription factor in vivo. These reprogrammed cells expressed lower layer markers, including SCPN identity marker CTIP2.

Figure 22 :

 22 Figure 22: Reprogrammed post-mitotic UL neurons maintain stable regarding their altered molecular identity A. Schematic representation of the experimental procedure. This time, brains were collected at P35. Brains were electroporated with vectors cGFP, cFezf2 or cFezf2 & cLmo4. B. Representative images of IHC of upper layer and lower layer markers on brain slices electroporated with the 3 different vectors (left). Filled arrows or triangles indicate expression of marker, empty arrows indicate their absence. C. Quantification of upper and lower layer markers expressed in electroporated UL neurons compared by their expression levels at P7 and P35. Results are expressed as mean ± s.e.m. Scale bars, 20 µm. n = 2 brains for each plasmid.

  transcriptional co-regulation activity was not sufficient to induce an efficient change of molecular identity in post-mitotic UL neurons. Since Lmo4 enabled de-repression of CTIP2locus and, most probably other targets, the system might need a major determinant factor allowing Ctip2 induction. The lack of transcription factors promoting lower layer CFuPNs is indeed the case for upper layer neurons born at E14.5, when for instance, FEZF2 or SOX5 transcription factors are not expressed. This could be a possible explanation of why Lmo4 overexpression in post-mitotic neurons at 13.5, and not at E14.5, leads to an increase of neurons expressing CTIP2. At E13.5 most layer 5 neurons are born and FEZF2 promotes Ctip2 expression in these neurons. Thus, overexpression of Lmo4 leads to de-repression of the Ctip2-locus in a subpopulation of neurons where Satb2 is active. Consequently, FEZF2 might take advantage of these open loci and induce expression of Ctip2. Another explanation could be that an intermediate reprogramming state, molecular changes are not stable until P7 and thus, cells revert to their original molecular identity. To investigate this possibility, sequential IHC analysis after electroporation could reveal whether a transient reprogramming upon ectopic expression of Lmo4 is occurring and at what stage it possibly reverts.

  the reprogramming efficiency induced by Fezf2 & Lmo4.At P7, UL neurons that received Fezf2 & Lmo4 still retained the ability to change their molecular identity (see Fig.6B, C and D). The number of CUX1-expressing neurons in Fezf2 & Lmo4 induced UL neurons was significantly lower compared to the control, whereas PCP4expressing neurons was significantly higher. However, the number of reprogrammed UL neurons was lower compared to Fezf2 & Lmo4 induction at P3, suggesting that the cellular plasticity of UL neurons diminishes over time. Not surprisingly, CTIP2-expression was absent and FOG2 expression almost non-detectable in reprogrammed UL neurons. Overall, UL neurons can alter their molecular identity even at later postnatal stages and reveal lower expression of CUX1 and elevated expression of PCP4.

Fezf2&

  Lmo4 for even higher reprogramming efficiency.The work of my thesis demonstrates that callosal intratelencephalic neurons in cortical upper layers can be reprogrammed into neurons, showing molecular identity, as well as connectivity features, characteristic of corticofugal projection neurons, at both embryonic and early postnatal developmental stages in the mouse. Upon induction of Fezf2, -a transcription factor known for its role in specification of lower layer 5 SCPN and layer 6 CThPNs during cortical development -the number of UL neurons expressing CUX1 is reduced at the expense of neurons expressing lower layer markers. Additionally, at embryonic and early postnatal stages, reprogrammed UL neurons also show axonal projections towards corticofugal targets, such as the thalamus and spinal cord. Addition of Lmo4 -a nuclear co-adaptor that is implicated in the epigenetic regulation of gene transcription and specification of post-mitotic neuronsto Fezf2 drastically increased the number of cells expressing a subset of lower layer markers and re-routing to corticofugal targets. Furthermore, Fezf2 & Lmo4 induction in UL neurons allows these cells to project to the spinal cord until postnatal day P10. This was not the case for single Fezf2-induction, as induction in UL neurons did not lead to axonal projections to the SC after P3. Partial re-wiring of reprogrammed neurons was observed until P21, indicating a higher plasticity of cortical neurons than previously expected.Overall, this work characterized a potent reprogramming cocktail, Fezf2 & Lmo4, that unveiled efficient conversion of UL neurons into CFuPNs and extended the temporal window of UL neuron reprogramming, compared to previous published data. Thus, these results contribute to a deeper understanding of cortical neuron plasticity which can be exploited in direct neuronal reprogramming in vivo.

  It would be interesting to explore whether our factors could possibly reprogram other cell types such as astroglia or fibroblasts. This would give us a hint whether Fezf2 & Lmo4 are able to convert cells from a less related lineage and would open up further possibilities.Reprogramming neurons into other neurons of a specific cell-type is challenging and come with drawbacks. Upon converting a neuron, the subsequent function of the original cell is lost and thus, finding other cell-types such as NG2-cells or astrocytes could circumvent loss of neuronal function upon reprogramming. Moreover, conversion of cells in vitro into CFuPNs could pave the way for subsequent transplantation studies.

  Lmo4 is not required for this defined boundary, as cortex specific KO of Lmo4 does not alter the expression pattern of Bhlhb5 in the medial boundary. But, in the absence of Lmo4 the molecular identity of neurons in the MCr is disrupted and more homogenous. However, Lmo4 is expressed in CPNs and mostly absent of CSMNs in MCr. In contrast, Lmo4 is expressed in MCr SCPNs projecting to the brainstem. Upon deletion of Lmo4, these neurons aberrantly project to the spinal cord,

(MCr) and caudal motor cortex (MCc) by complementary expression patterns of Lmo4 and Bhlhb5. In MCr, Lmo4 is highly expressed while Bhlhb5 expression is absent, vice versa for MCc. indicating a role for Lmo4 in proper development of SCPNs projecting to the brainstem.

  Additionally, a large number of Ctip2-expressing neurons in L5 co-express Lmo4, further indicating that many presumptive SCPNs express Lmo4 during early differentiation. Also, Clim1 is present in both FEZF2 and CTIP2 positive neurons. SATB2, a transcription factor for CPNs, is co-expressed in a subpopulation with both Lmo4 and Clim1, indicating that Lmo4 and Clim1 are expressed in both presumptive CPNs and SCPNs. Lastly, Sox5, a TF expressed in the SP and lower layer 5 and 6, is co-expressed with Lmo4 and Clim1, indicating early expression of Lmo4 in SCPNs. At P0, Lmo4 is expressed in subsets of neurons broadly throughout the cortical layers, whereas Clim1 is restricted to neurons in L5. Both markers are still expressed in L5 but start to be segregated into distinct neuronal subpopulations. Despite some overlapping expression of Fezf2 and Lmo4, Lmo4 becomes partially excluded from SCPN population. At P0, many neurons also co-express Ctip2 and Lmo4, while all Ctip2-expressing neurons also express Clim1. Also, Satb2 and Sox5 are still co-expressed with Lmo4 and Clim1 at this stage. At P6,

when layer 5 SCPNs have more fully differentiated, Lmo4 and Clim1 are expressed in largely distinct L5 neuronal populations with few exceptions. However, Lmo4 expression is merely excluded from CTIP2-positive SCPNs but instead L5 CPNs co-express Satb2 and Lmo4. Vice versa, Satb2 and Clim1 expression in CPNs is mostly mutually exclusive and Clim1 is largely coexpressed in Ctip2 populations. Moreover, retrograde labelling from contralateral cortex at P3 and examination at P6 revealed that nearly all Lmo4-expressing neurons are CPNs, whereas nearly all CPNs do not express Clim1

(Azim et al., 2009)

. Overall, these studies indicate a role for Lmo4 in defining cortical neuron subtypes at embryonic and postnatal stages.

  slices incubated ON at 4°C. Again, slices were washed as described before and 500 µl of PBS with DAPI (Invitrogen, #H3570) was added and incubated at RT for 5 min. Finally, slices were mounted on glass microscope slides with 200 µl of mounting medium and enclosed with a cover slip.

	50 ml Blocking buffer 10% Goat serum 3% BSA 0.03% Triton-X100 Filled up with PBS (1X) Antibody Chicken488 Goat Rabbit488 Goat Rat488 Goat Rabbit594 Goat Rabbit647 Goat each well and Antibody Rat647 Goat	Age P3 P7 P10 P14 P21 Host Mounting Medium 50 ml Antibody buffer µl tamoxifen 50 50 100 100 150 Dilution 3% Goat serum 3% BSA 0.03% Triton-X100 Filled up with PBS (1X) 6g Glycerol 2.4g Mowiol 6 ml ddH20 12 ml 0.2M Tris (pH 8.5) Host Dilution 1:400 1:400 1:400 1:400 1:400 1:400	Reference Reference A-11039 A-11034 A-11006 A-11012 A-21245 A-21247
	GFP	Chicken	1:500	ab13970
	Flag	Rat	1:200	NBP1-0671255
	Cux1	Rabbit	1:200	ABE217
	Ctip2	Rat	1:200	ab18465
	Fog2	Rabbit	1:200	sc-10755
	PCP4	Rabbit	1:200	PA5-52209
	Darp32	Rabbit	1:200	ab40801
	Lmo4	Rat	1:500	Gift from Jane Valsvader
	Fezf2	Rabbit	1:200	JP18997
	RFP	Rabbit	1:200	ab124754

Figure 19: Ectopic Expression of single Fezf2 or Fezf2 in combination with Lmo4 drives post- mitotic UL neurons to acquire molecular features characteristic of CFuPN.

  Schematic representation of the experimental procedure and used vectors. Mouse embryos were electroporated at E14.5 and their brains were collected at P7. Overview of quantification of the percentage of electroporated UL neurons expressing upper layer marker or lower layer markers. n = 3 brains for each plasmid.

	(54.13 ± 7.16% vs. 27.53 ± 1.4% in cFezf2) was expressed by a larger number of cFezf2 &
	cLmo4-electroporated UL neurons (see Fig. 19E, F)
	In conclusion, cFezf2 & cLmo4 ectopic expression in UL neurons triggered CFuPN gene identity For reprogramming approaches cFezf2
	or cFezf2 & cLmo4 were electroporated. cGFP served as control. B. IHC for LMO4 and FEZF2 on slices of expression. Furthermore, the efficiency of cFezf2 & cLmo4 in inducing CTIP2 or PCP4 was cFezf2 & cLmo4-electroporated brains. Full arrowheads indicate protein expression
	significantly higher compared to single cFezf2, while expression of CUX1, FOG2 or DARPP32 C. Immunohistochemistry (IHC) of upper layer CUX1 and lower layer marker CTIP2 on a coronal slice of
	cFezf2 & cLmo4-electroporated brains. White box indicates magnification image on the right side. markers were not significantly different in electroporated UL neurons. D. Representative images of IHC of CUX1, CTIP2, FOG2, PCP4 and DARPP32. Full arrows or triangles
	indicate that the protein is expressed in GFP-positive cells. Empty arrows or triangles indicate absence
	of protein expression in GFP-positive cells. E. Quantification of the percentage of electroporated cells
	expressing upper layer marker CUX1, or lower layer markers CTIP2, FOG2, PCP4 and DARPP32. Results
	are expressed as mean ± s.e.m. One-way ANOVA with multiple comparisons was used for statistical
	analysis, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001, ns = not significant. CUX1, p = 0.58; FOG2, p =
	0.32; DARPP32, p = 0.16. F.
	single Fezf2 in UL neurons and compared quantified marker expression with the data of cFezf2
	& cLmo4. Expression of CUX1 (3.43 ± 0.29% vs. 9.27 ± 2.39% in cFezf2), FOG2 (29.67 ± 2.91%
	vs. 21.63 ± 8.57% in cFezf2) and DARPP32 (47.37 ± 2.67% vs. 57.27 ± 4.55 in cFezf2) was not
	significantly different. Interestingly, CTIP2 (64.1 ± 2.28% vs. 35.97 ± 1.93% in cFezf2) or PCP4

A.

Ectopic expression of cFezf2 & cLmo4 in UL neurons of the motor area induces higher levels of corticofugal identity markers CTIP2 and DARPP2 compared to somatosensory area.

	Figure 21: A. Schematic representation of experimental procedure. Embryos were electroporated with cGFP,
	cLmo4, cFezf2 or cFezf2 & cLmo4 in the motor cortex. B. Representative IHC image of GFP, CUX1 and
	CTIP2 on a brain slice electroporated with cFezf2 & cLmo4. White box indicates the location of the
	detailed image. C. Overview of quantification of the percentage of electroporated cells expressing
	upper and lower layer markers. Results are expressed as mean ± s.e.m. D. Comparisons of quantified
	percentage of markers in electroporated cells by area. Two-way ANOVA with Tukey as post-hoc with
	multiple comparisons was used for statistical analysis, applied to all results together. *p<0.5 **p<0.01
	***p<0.001 ****p<0.0001, ns = not significant. Scale bars 1000 µm in overview image (left), 200 µm in
	detail image (right). n = 3 brains for each plasmid.
	instructive environment for CFuPN reprogramming of UL neurons.
	As a result, CTIP2 and DARPP32 markers were induced in a larger population of cFezf2 &
	cLmo4-targeted UL neurons in the motor area, in comparison to reprogramming in S1 (see
	Fig. 21C). In cFezf2 & cLmo4-electroporated cortices, CTIP2 (79.47 ± 1.01% vs. 64.1 ± 2.28% in
	S1) and DARPP32 (61.87 ± 2.53% vs. 47.37 ± 2.68% in S1) revealed expression in a significantly
	higher number of electroporated UL neurons, while CUX1 (2.6 ± 1.74% vs. 3.43 ± 0.29 in S1),
	FOG2 (23.13 ± 0.95% vs. 29.67 ± 2.91% in S1) or PCP4 (45.73 ± 2.33 vs. 43.97 ± 1.55% in S1)
	were not differently induced in a significant way, in GFP-positive UL neurons of M1 (see
	Fig. 21D. Interestingly, the expression of molecular markers in M1 compared to S1 was not
	significantly different in control, single cLmo4, or cFezf2-electroporated animals,
	To conclude, the cellular environment of M1 had an impact on reprogramming efficiency of
	cFezf2 & cLmo4, while control cGfp, cLmo4 or cFezf2 electroporated UL neurons appeared to
	express molecular markers in a similar number compared to S1. These results suggest, that
	the cellular environment within an organ can indeed affect the reprogramming efficiency and
	thus, selecting the cell niche for in vivo reprogramming approaches, appear to be an important
	factor.

  Overall, reprogrammed UL neurons seem to keep their CFuPN molecular identity over time.DARPP32 is not maintained in reprogrammed UL neurons. An explanation could be, that DARPP32-expression is age-dependent and gets downregulated over time. ISH data from the Allen Brain Atlas however show RNA-signals of Darpp32 in lower layers at adult stages. More insights on the expression profile of DARPP32 in the cortex over time are necessary for a more conclusive explanation.

	Whereas in control cGfp-electroporated brains, expression of CUX1 was present in nearly all
	examined UL neurons (96.75 ± 0.35% vs. 94.5 ± 1.81% at P7, see Fig. 22C), The number of cells
	expressing CUX1 in single cFezf2-(6.75 ± 1.85% vs. 9.27 ± 2.39% at P7) or cFezf2 & cLmo4 -
	electroporated (6.95 ± 1.75% vs. 3.43 ± 0.29% at P7) UL neurons at P35 remained low.
	Furthermore, control cGfp-electroporated brains showed CTIP2 expression in a similarly
	number of UL neurons at P35 (4.05 ± 0.45% vs. 0.53 ± 0.53% at P7), wehereas the number of
	electroporated UL neurons expressing CTIP2 remained almost unaltered in cFezf2
	(33.45 ± 13.95% vs. 35.97 ± 1.93% at P7), with a slight reduction in cFezf2 & cLmo4-
	electroporated UL neurons (42.5 ± 8% vs. 64.1 ± 2.28% at P7). FOG2 expression was almost
	absent in P35 control cGFP-electroporated UL neurons as it was the case when brains were
	collected at P7 (1.95 ± 1.64% vs. 0.37 ± 0.37% at P7). cFezf2-(24 ± 5.4% vs. 21.63 ± 8.57% at
	P7) or cFezf2 & cLmo4-(19.7 ± 8.6% vs. 29.67 ± 2.91% at P7) electroporated brains at P35 show
	almost unaltered number of cells co-expressing FOG2 compared to examination in P7 brains.
	The number of UL neurons expressing PCP4 at P35 did not change for cGfp-electroporated UL
	neurons (2.15 ± 1.14% vs. 0% at P7), and expression pattern at P35 also resembled the
	quantified percentages of P7 brains that were electroporated with cFezf2 (34.85 ± 2.55% vs.
	27.53 ± 1.35% at P7) or cFezf2 & cLmo4 (65.85 ± 4.15% vs. 54.13 ± 7.16% at P7). Interestingly,
	the percentage of DARPP32-expressing UL neurons in cortices electroporated with cGfp
	remained unaltered low (3.95 ± 0.75% vs. 0.97 ± 0.97% at P7), whereas DARPP32-expressing
	UL neurons in both cFezf2 (15.4 ± 1.2% vs. 57.27% ± 4.55% at P7) and cFezf2 & cLmo4
	(11.65 ± 2.65% vs. 47.37 ± 2.68% at P7) dropped drastically at P35 when compared to P7.

Postnatal induction of single iFezf2 or iFezf2 & iLmo4 combined with iCtip2 or iPBX1 does not increase reprogramming efficiency

	B. Next, we sought out whether additional factors could improve the reprogramming efficiency
	of iFezf2 & iLmo4 at this postnatal stage. To test this hypothesis, we subcloned Ctip2 into
	pCAG-fl-mutCherry-fl-IRES-EGFP (iCtip2) to obtain an inducible vector carrying this gene. CTIP2
	, we electroporated embryo brains at E14.5 with either iGfp or iFezf2 or iFezf2 & iLmo4
	and induced expression of these genes at P3 via injection of TAM (see Fig. 24A, B, C), Induction
	of iFezf2 & iLmo4 reduced the number of GFP-positive cells expressing CUX1 (29.89 ± 14.67%
	vs. 93.83 ± 1.33% in control, see Fig. 24D) in an efficient way, compared to single iFezf2
	(71.75 ± 4.75% vs. 93.83 ± 1.33% in control). Interestingly, CTIP2-expression was not induced
	anymore in P3 UL neurons, neither with iFezf2 (0.45 ± 0.45% vs. 0% in control) -confirming
	observations published by Rouaux and Arlotta (2013) -nor with iFezf2 & iLmo4 (0% vs. 0% in
	control). FOG2 was expressed by few numbers of P3 UL neurons induced with iFezf2 & iLmo4
	(10.8 ± 4.96% vs. 5.07 ± 2.46% in control), as it was the case for iFezf2 (10.4 ± 2.4% vs.
	5.07 ± 2.46% in control).
	Very interestingly, GFP-positive UL neurons were expressing PCP4 in high numbers, after
	induction of iFezf2 & iLmo4 (66.67 ± 15.5% vs. 0% in control), whereas iFezf2 (24.05 ± 24.05%
	vs. 0% in control) revealed lower numbers of PCP4-expressing UL neurons., The quantified
	percentage of GFP-positive UL neurons expressing DARPP32 was low in iFezf2 induced cells

  The resulting quantified percentage of UL neurons did not

	reveal drastic higher numbers, besides obvious CTIP2-expression, compared to iFezf2 & iLmo4:
	CUX1 (53.3% vs. 29.89 ± 14.67% in iFezf2 & iLmo4), FOG2 (6.3% vs. 10.8 ± 4.96% in iFezf2 &
	iLmo4), PCP4 (69.5% vs. 66.67 ± 15.5%).

  More specifically, LMO4 is initially expressed in both post-mitotic SCPNs and CPNs during cortical development. CPNs and SCPNs are two different sub-types of projection neurons in the cortex, defined by their axonal projections. CPNs (mainly located in upper layers, but also present in fewer numbers in lower layers) project within the cortex, while SCPNs (located in L5) project outside the cortex to subcerebral targets. During mid to late differentiation, expression of LMO4 is absent in SCPNs but remains expressed in L5 CPNs, indicating a role in specification of PN-subtypes during development. CPNs and SCPNs have distinct molecular identities, which for instance are revealed by expression of SATB2 or CTIP2, respectively.

  . Our data show that Fezf2 induces the CThPN marker FOG2 in electroporated cells, even though in fewer numbers than CTIP2. This observation could be explained by the minor role FEZF2 plays during CThPN development, compared to SCPN specification. It is known that FOG2 controls CThPN molecular differentiation and axonal targeting, but not cell specification, in part by decreasing CTIP2 expression levels in L6(Galazo, Emsley and Macklis, 2016). Thus, induced expression of Fog2 in electroporated cells might counteract Ctip2 induction in upper layers and might explain why not all cells express CTIP2 upon Fezf2 or Fezf2 & Lmo4 misexpression. It is known that ectopic expression of Fezf2 can induce Fog2 expression in striatal neurons

Fezf2 & Lmo4 trigger significant higher numbers of UL neurons to express CTIP2 and PCP4 compared to single Fezf2

  expression induces DARPP32 expression. However, 41% of electroporated UL neurons coexpress CTIP2 and DARPP32 upon Fezf2 & Lmo4 overexpression in upper layers, similarly to what happened in layer 6 of Tbr1 KO mice(McKenna et al., 2011), which acquired a layer 5 SCPN-like fate and suggesting that the presence of Tbr1 might inhibit DARPP32 in CTIP2+ neurons. However, CTIP2 is weakly expressed in layer 6 CThPNs and ectopic expression into upper layer 2-3 neurons results in the extension of axons to the thalamus(Chen et al., 2008).

	Thus, ectopic expression of DARPP32 upon Fezf2 & Lmo4 overexpression might label both
	layer 5 and 6 sub-populations. Moreover, since DARPP32 is highly and specifically expressed
	by MSN in the striatum and some PN in lower layers specifically project to the striatum
	(Shepherd, 2013), we would like to propose that DARPP32 expression might be correlated to
	PNs projecting to the striatum. Further studies on DARPP32 would be beneficial to better
	understand its role in layer 6 development during corticogenesis. For example, retrograde
	labeling of axons in the striatum followed by IHC of DARPP32 in the cortex could give a hint
	on whether DARPP32 is indeed expressed in corticostriatal PNs.
	Ectopic expression of Fezf2 & Lmo4 in postmitotic UL neurons downregulates the upper layer
	marker CUX1 expression, while CFuPN markers CTIP2 and FOG2 are induced in electroporated
	cells. The observations could also be seen in single Fezf2 ectopic expression in UL neurons. In
	this study, single Fezf2 has been used as a positive control, which has been described to induce
	SCPN molecular identity in post-mitotic UL neurons with an efficiency of around 25% (De La
	Rossa

Taken together, Fezf2 & Lmo4 possess the capacity of reprogramming UL neurons into CFuPN as revealed by analysis of the molecular identity of electroporated cells. The resulting molecular identity appeared to be heterogenous, with expression of markers known to be specific for different sub-types of SCPNs and CThPNs.

III.

  Most primary antibodies used in this study were produced in the same host (apart from anti-CTIP2) and thus, IHC of multiple CFuPN was in most cases not feasible. Co-expression studies of Fezf2 & Lmo4 electroporated cells however indicated that CTIP2 & PCP4, as well as CTIP2 & DARPP32 are not homogenously expressed in all electroporated cells. Therefore, it is possible that virtually all electroporated UL neurons underwent reprogramming, resulting in expression of distinct pattern of CFuPN markers and different axonal projection re-wiring.

	. However,
	the presence of high Lmo4 expression definitely increased the efficiency of reprogramming,
	since for example, no contralaterally callosal axons observed in the CC was observed upon
	Fezf2 & Lmo4 electroporation, which is not the case with control GFP-positive and Fezf2-
	electroporated. The above-mentioned results suggest two interpretations. First, Fezf2 & Lmo4
	have a higher efficiency in reprogramming UL neurons than single Fezf2. In line with the
	observations that the induction of certain CFuPN markers are found in higher numbers of
	Fezf2 & Lmo4 reprogrammed UL neurons than single Fezf2, we propose that Lmo4 drastically
	enhances the Fezf2-driven conversion. Second, the finding that no GFP-positive axons were
	found in the CC could possibly indicate that all UL neurons underwent reprogramming or
	partial reprogramming. Possibly, all UL neurons retracted their axonal projections through the

CC, but it does not necessarily mean that all converted into CFuPN, resulting in a partial reprogramming. In my study, I could examine the molecular identity of converted UL neurons by using one or maximum two markers in GFP-positive cells.

Acknowledgements

MATERIAL AND METHODS

Vector

Thanks to the gift of the Tarabykin lab, a plasmid with conditional expression could be used as vector to generate an inducible Lmo4 plasmid.

The plasmid has been digested with EcoRI-HF and EcoRV-HF to remove the Bhlhb5 sequence.

The resulting sticky end of EcoRI has been filled-in using Klenow-Fragment (NEB (M0210):

Then, the plasmid has been dephosphorylated to prepare it for the final ligation step:

As a final step, the vector has been purified on a gel. For the ligation process, 200 ng of the vector have been used, while the quantity of the insert has been calculated according to the following calculation: = ( 200 ng X bp vector X bp insert ) * 4

Ligation occurred according to the following protocol:

VII. RNA Extraction

For RNA extraction of the GFP-positive sorted cells, the NucleoSpin RNA XS kit from MACHEREY-NAGEL (Ref 740902) has been used. The stored sorted cells were thawn up on ice for 20 min and then vortexed thoroughly two times for 5 s. Afterwards, the RNA extraction was done according to the manufacturer's manual. The quality and quantity of extracted RNA has been analyzed using a Bioanalyzer2100 (Agilent). At the end, the extracted RNA has been stored at -80°C.

VIII. Intracardiac Perfusion

At the planned developmental stage, mice underwent intracardiac perfusion for fixation of the brain tissue. For anesthesia without reawakaning, dololethal has been IP injected and the body opened from the sternum to the neck. A butterfly needle was inserted into the left valve of the heart and varying amount of 4% PFA (Sigma-Aldrich, #P6148; were used to perfuse.

Afterwards, the head has been cut and dissected. After checking for successfully electroporated brains (GFP-positive) under a fluorescence-microscope, brains were postfixated in 4% PFA for 2h at RT. Finally, brains were washed one time in PBS and stored in PBS -Azide (0.05%; Sigma-Aldrich, #S8032) at 4°C for long-term storage.

IX. Embedding and Slicing

Successfully electroporated brains were embedded in 4% Select Agar (Sigma-Aldrich, #A5054) in PBS. Therefore, 4 g of Select Agar were dissolved in PBS and heated up with a microwave.

As soon as the agar cooled down to 37°C, the brain has been embedded. and then sliced on a vibratome (Leica VT 1000S; Speed: 7, Frequency 7). All brains were cut coronally in 100 µm thick sections and preserved in PBS -Azide (0.05%) until further processing.

X. Immunofluorescence

Floating slices were incubated in 1 ml blocking buffer per well ON at 4°C, gently rocking. Then primary antibodies were diluted in 500 µl antibody buffer per well for two days at 4°C.

Subsequently, slices were washed three times for 10°min at RT and two times for one hour.

Secondary antibodies were diluted 1:400 in antibody buffer and 500 µl have been added to

RESULTS

D. cFezf2 & cLmo4 or single cFezf2 ectopic expression alter axonal projection of post-mitotic UL neurons towards corticofugal targets

Next, we wanted to investigate whether the induction of CFuPN molecular identity in cFezf2 & cLmo4-electroporated UL neurons also established a CFuPN-like connectivity.

Thus, we followed the trajectory of GFP-positive axons in cFezf2 & cLmo4-electroporated brains (see Fig. 20A,B). No GFP+-axons in the CC of cFezf2 & cLmo4-electroporated cortices were observed, whereas in the Str, the Th and in the pyramidal tract GFP + -axons were present.

These results strongly indicate a loss of UL neuron identity at the expense of a CFuPN-like identity. Interestingly, labelled axons project both to the Th and the IC. This observation is raising the question whether Th-targeting projections of electroporated UL neurons are branches of axons en route towards subcerebral targets, whether these are independent axonal projections or, if CFuPN-like cells resemble a heterogenous population of SCPNs or CThPNs identity.

A similar change in axonal projections was observed in cFezf2-electroporated brains, but GFP +axons were still present in the CC. This might indicate that cLmo4-addition to cFezf2 facilitates Fezf2-driven reprogramming of UL neurons into CFuPNs-like cells.

DISCUSSION

explanation could be the function of LMO4 as de-repressor of the CTIP2 locus (Harb et al., 2016), whereby FEZF2 took advantage and induced expression of CTIP2 more efficiently than when Fezf2 is ectopically induced in the absence of Lmo4. It has not been described whether Lmo4 acts on the PCP4 locus, but our results indicate that LMO4 is implicated in the regulation of PCP4, which could be similar to the de-repressive effect described for CTIP2. This could be examined by chromatin-immunoprecipitation (ChIP) of brain nuclear extracts after electroporation with Fezf2 & Lmo4.

Differently from CTIP2 and PCP4 induction, the number of reprogrammed UL neurons expressing the CThPN markers FOG2 or DARPP32 in Fezf2 & Lmo4-electroporated cortices was similar and not significantly different to electroporation of single Fezf2. A possible explanation could be, that Lmo4 does not have an effect on the induction of factors expressed in CThPNs, in line with its role in driving SCPN from the motor cortex towards the brainstem region (Cederquist et al., 2013). However, the percentages of single Fezf2-reprogrammed UL neurons expressing CUX1, CTIP2 or FOG2 in my study differ from published data, obtained by the same experimental strategy (Rouaux and Arlotta, 2013). In our study, the percentage of Fezf2reprogrammed UL neurons expressing CUX1 was three-times less than in Rouaux and Arlotta (2013), while the percentage of Fezf2-reprogrammed UL neurons expressing CTIP2 and FOG2

were 1.5-times and almost 8-times higher compared to Rouaux and Arlotta (2013). Most probably, this is due to methodological differences and/or differences in analyzing coexpressions of IHC revealed markers in electroporated cells. This is also why we compared our reprogramming efficiency among our experimental conditions.

Taken together, our study shows that the synergistic combination of Fezf2 & Lmo4 triggers CFuPN marker expression in electroporated upper layer post-mitotic UL neurons, revealing conversion of these neurons. In addition, the efficiency of UL neurons inducing CTIP2 or PCP4 was significantly higher compared to single-Fezf2 reprogramming.

CONCLUSION PERSPECTIVES

Abstract

The mammalian neocortex is tangentially organized into functional areas and radially subdivided into six layers of neuronal populations with distinct projections, morphology and expression patterns. While neurons in the upper layers project within the neocortex or towards the contralateral hemisphere, such as callosal projection neurons, most lower layer neurons, named corticofugal projection neurons, innervate subcerebral targets via the corticospinal tract. Injuries or loss of these neurons can lead to severe neurological disorders, such as Amyotrophic Lateral Sclerosis or other diseases and/or lesions implicating the corticospinal tract. Since the adult mammalian brain lacks a significant regenerative strategy and thus, damage or loss of neurons is permanent, methods for restoring neurons are urgently needed.

Recent studies revealed an unexpected plasticity of post-mitotic neurons, which can be converted into cell-types of other neuronal lineages. This approach, called direct neuronal