Julien, Camille R Shao

Romain I Florent

Emeric Merour

Hatem Hmidan

Corentine Marie

Pierre-Henri Helou

Haiyang Lu

Antoine Potel

Adrien Clavairoly

Yi Ping Shih

Sebastien Dussaud

Phillipe Ravassard

Hassan Hafizi

Shu Hao Lo

Bassem Hassan

Carlos Parras
email: carlos.parras@upmc.fr

we were joking about my PhD since the beginning of my studies.

Finally, I must thank my family

First, I would like to thank my supervisor, Dr Carlos PARRAS for all his guidance and dedication all along these four years. It was a great experience for me to be formed by somebody who was both a great scientist and mentor. I would also like to thank Dr Bassem HASSAN who trusted us at the beginning of this project and gave me the opportunity to work in his team. I want to make a special thank to Laetitia Vincensini, who trained me for my first experience in a lab and helped me to find this internship in the first place.

All my recognition goes also to Hatem, who not only started this project but also formed me during my internship and a full year after his PhD defence. A special thanks also to Corentine who helped us a lot with the figures and was always here to give me precious guidance, even when the answers were just in the drive and of course to Pierre-Henry, who was my first trainee and worked so much to help us generate these data, good chance for your future scientific career.

I would like to thank the people working in ICM facilities who helped me a lot throughout this PhD. ICM Quant for the imaging, especially Aymeric Millecamps for saving me an incredible amount of time with his macro, and Dominique Languy for his helpful advice. CELIS for the cell culture, especially David Akbar who helped me so much for the timelapse culture. Histomics, especially Nicolas Raymond, Anick Pringent and Bineta Faye for their help. ICMice for the mice, especially to Joana Droesbeke, Cindy Belson and Melanie Huentz, for their dedication in mice care. iVECTOR for the generation of the virus. And finally, the communication team of the ICM, especially Nicolas Brard and of course Patrica Oliviero.

A special thanks first to Elisa, who already defend her PhD, Jean-Baptiste and Lucas, who are almost done, for all the time spent together discussing about Science or not.

Marlene for her patience when we were discussing my Western Blot data or just chatting about music. Pauline, for her support and our discussions on Xenopus. Rana and Sofia for our pass-by discussions. Natalie and Sandra for their help and advice.

Yanis, for our scientific debates and the discussion we still need to have, and to all the Hassan team and other interesting people I met at the ICM, these years were significantly more pleasant thanks to you!

INTRODUCTION CHAPTER I: Biology of CNS development

From the neural crest to the CNS Neurulation Neurulation is the first step of the central nervous system (CNS) development that starts in the embryo. It consists mainly of the folding of the embryonic neuroectoderm and its closure to form a neural tube from which will originate the whole CNS. Over 80 genes are involved in this well sequenced biological process. Even if it is not homogenous across embryo's regions, neurulation is conventionally divided into two phases [START_REF] Copp | The genetic basis of mammalian neurulation[END_REF].

The primary neurulation is the shaping, folding and the midline fusion of the rostral embryos neuroectoderm area. It will create the primary neural tube from which originates the brain and most of the spinal cord. On the other hand, secondary neurulation occurs in the caudal regions, in the caudal eminence, also called tail bud, and doesn't involve neural folding like primary neurulation. It consists in the condensation and the epithelialization of a resident mesenchymal cell population that will give rise to the secondary neural tube in the continuity of the primary one and form the lowest portion of the spinal cord.

The newly formed neural tube have then to be closed, which occurs in parallel in three different places in a caudal to rostral way. In mice, the first closure (closure 1) starts at E8.5 at the hindbrain/cervical boundary. It then spreads caudally to the posterior neuropore and rostrally to the hindbrain neuropore in a 36h period. A second closure (closure 2) appears independently, usually at the forebrain/midbrain boundary and then spread until the anterior neuropore. Finally, the third neural tube closure (closure 3) appears at the extremity of the forebrain and spreads until it fuse with closure 2 (Fig. 01). The primary neurulation ends with the closing of the posterior neuropore.

Secondary neurulation occurs from the close primary neural tube through the tail bud canalizations. [START_REF] Copp | The genetic basis of mammalian neurulation[END_REF] Contrary to the closure 1 and 3, closure 2 location is polymorphic and varies across mice strains. Some presents a closure 2 in caudal midbrain where others could present a rostral midbrain closure 2 and the proximity with closure 3 makes it hard to differentiate the two events. This could explain why, in the human brain, the closure 2 have not been consistently observed.

Defects in neural tube closing induce a wide range of malformations named open neural tube defects (NTDs). It affects 1 per 1000 pregnancy worldwide and is the second cause of congenital malformation in human pregnancy after heart defects.

Typically, if closure 1 fails, the whole neural tube from the midbrain to the lower spine will remain open (which is called craniorachischisis). Defects in closure 2 induces exencephaly, which mean the development of the midbrain and/or the hindbrain outside the skull. The exposed neural tissues will degenerate and be destroyed during late gestation, converting the exencephaly in anencephaly (the absence of a major portion of the brain, skull, and scalp). Finally, closure 3 deficit induces a forebrainrestricted anencephaly, often combined with split face malformation. Finally, if the closure fails to spread along the spinal region, the spinal cord will remain open, resulting in open spina bifida (also called myelomeningocele).

Just after the onset of neural tube closing, the neural plate is reshaped into an elongated keyhole-shaped structure with broad cranial (rostral) and narrow spinal (caudal) regions. This neural plate shaping is mainly based on convergent extension. Indeed, cells from both the neuroectoderm and the underlying mesoderm will medially move, lengthening and narrowing the neural plate. Studies of mutant mice (loop-tail, crash, cricletail and dishevelled-1 KO ; dishevelled-2 KO ) have shown the importance of planar cell polarity for closure 1, especially the non-canonical Wnt/frizzled signaling pathway. Indeed, the mutation in the gene encoding Celsr1, the binding partner protein of frizzled, or Scrb1 mutation, also required for planar cell polarity, impair closing 1 (Montcouquioi et al., 2003). Dishevelled mutations induce misexpression of Rho kinase 2, downstream of Wnt1 in the Wnt/frizzled planar cell polarity pathway [START_REF] Wallace | Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture[END_REF][START_REF] Marlow | Zebrafish Rho Kinase 2 Acts Downstream of Wnt11 to Mediate Cell Polarity and Effective Convergence and Extension Movements[END_REF]. and MHP (c), and lower spine bending finalisation uses only the two DLHP (d). From [START_REF] Copp | The genetic basis of mammalian neurulation[END_REF] Even if an initial bending of the neural tube (Fig. 02-a) occurs simultaneously, the timing and the positions of the bending points vary along the spinal region. The upper spine (Fig. 02-b) is the first to bend (at E8.5 in mice) and fold at a single bending point named the Median Hinge Point (MHP). The intermediate spine (Fig. 02-c) bent in second (between E9 and 9.5 in mice) and used two bending points, a MHP and a Dorso-Lateral Hinge Point (DLHP) present in each neural fold. Finally, the lowest spinal (Fig. 02-d) region bends last (around E10 in mice) and is solely based on DHLP.

A mesodermal internal structure localized below the neural plate, the notochord, controls this bending through the secretion of Sonic Hedgehog (Shh), which decreases along the spine. High notochordal Shh levels, like in the upper spine, inhibits bending at the DLHP but not at the MHP. Indeed, local exogenous Shh release in the lower spine is sufficient to inhibit DLHP formation in mice [START_REF] Ybot-Gonzalez | Sonic hedgehog and the molecular regulation of mouse neural tube closure[END_REF].

Moreover, Shh KO mice show normal bending at DLHP but Shh overexpression could induce NTDs by inhibiting dorsolateral bending [START_REF] Echelard | Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity[END_REF]. Shh also plays a role in cranial neurulation, as shown in Gli3 mutant mice, a negative regulator of Shh, which present cranial defects [START_REF] Hui | A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene[END_REF]. On the contrary, Gli1 or Gli2 negative mice, two mediators of the Shh signalling, do not develop NTDs [START_REF] Ding | Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice[END_REF]Matisse et al., 1998). In open-brain or Zic2 mutant mice, two mutations where negative regulators of the Shh signalling are disrupted [START_REF] Eggenschwiler | Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway[END_REF][START_REF] Nakata | Xenopus Zic family and its role in neural and neural crest development[END_REF], the neural tube fails to close both in the brain and the lower spine area [START_REF] Günther | Open brain, a new mouse mutant with severe neural tube defects, shows altered gene expression patterns in the developing spinal cord[END_REF][START_REF] Nagai | Zic2 regulates the kinetics of neurulation[END_REF]. Interestingly, the dorsal and dorsolateral cells do not seem to differentiate [START_REF] Eggenschwiler | Dorsal and Lateral Fates in the Mouse Neural Tube Require the Cell-Autonomous Activity of the open brain Gene[END_REF]. One explanation could be that the negative effect of Shh on DLHP formation in the higher spine prevents dorsal or dorsolateral cell differentiation at a localization that will later be needed for bending (DLHP).

After the bending, the two neural folds fuse to finalize the neural tube closing and the neurulation. Apical cells from both neural folds extend their lamellipodial protrusions as they approach from the dorsal midline (Fig. 03-a). When these protrusions come in contact, they interdigitate (Fig. 03-b), fixing the two neural folds together through cellcell recognition [START_REF] Geelen | Ultrastructural observations on closure of the neural tube in the mouse[END_REF]. Indeed, each folds is covered by a 'cell surface coat' of carbohydrate-rich material whose removal impairs neural fold fusion [START_REF] Moran | An ultrastructural examination of the role of cell membrane surface coat material during neurulation[END_REF][START_REF] Rumsby | Microfilament and microtubule organization and dynamics in process extension by central glia-4 oligodendrocytes: Evidence for a microtubule organizing center[END_REF]. Finally, the epithelium is remodelled by apoptosis to ensure the continuity between the surface ectoderm and the neuroepithelium across the midline (Fig. 03-c). to ensure tissue continuity. From [START_REF] Copp | The genetic basis of mammalian neurulation[END_REF] For a long time, the fusion then the separation of the neural tube and the surface ectoderm have been thought to be based solely on cell-cell adhesion and celladhesion proteins. Indeed, the neural tube cells present N-CAM and N-cadherin whereas the surface ectoderm cells express epithelial (E)-cadherin [START_REF] Rumsby | Microfilament and microtubule organization and dynamics in process extension by central glia-4 oligodendrocytes: Evidence for a microtubule organizing center[END_REF]. Moreover, the expression of a dominant negative N-cadherin in Xenopus impairs neural tube formation where dominant negative E-cadherin doesn't [START_REF] Levine | Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant[END_REF]. But since N-CAM expression in the surface ectoderm doesn't inhibit neural tube closure [START_REF] Detrick | The effects of N-cadherin misexpression on morphogenesis in xenopus embryos[END_REF] and N-cadherin KO does not induce any NTD [START_REF] Radice | Developmental Defects in Mouse Embryos Lacking N-Cadherin[END_REF], it is likely that cell-adhesion molecules are crucial for neurulation in mammalians.

Cortical development

After the closure of its most rostral part, the neural tube forms three primary vesicles that will be the future forebrain, the midbrain and the hindbrain. Two of these brain regions will later be subdivided. The hindbrain will be divided into the myelencephalon and the metencephalon and the forebrain will be split into the diencephalon and the telencephalon. This last area is of particular interest for us because its dorsal part will give rise to the cerebral cortex, the most enlarged brain area in humans (Fig. 04-A).

The ventral telencephalon part will give rise to the ganglionic eminences from which some GABAergic interneurons and glial cells will migrate and colonize to the developing cortex (Fig. 04-B).

Figure 04 CNS development and morphogen expression. In embryonic stages, the CNS is divided into four areas (A), the Forebrain (FB), the Midbrain (MB) the Hindbrain (HB) and the spinal cord (SC) that will later complexify to give rise to all the CNS regions. Neurons and Glial cells migrate from the Ganglionic eminence to the cortical areas (B). The telencephalon is regionalised based on gradient expression of several transcription factors such as Wnts, BMP or Shh (C). From [START_REF] Agirman | Cerebral cortex development: an outside-in perspective[END_REF] The cortical neuroepithelium is organised from an outside-in perspective. The first cortical layers created are also the deepest with each layer formed on the top of the previous one. The classical model of cortical development starts with a neurogenic phase, occurring between E10.5 and E18.5 in the rodents, followed by the gliogenesis starting around mid-gestation and continued after birth. Neuroepithelial cells (NECs) create an initial neuroepithelium, on the top of a basement extra-cellular matrix layer, pseudostratified by the apico-basal movement of their nuclei during cell cycle progression . NECs will first undergoes symmetric division, in order to amplify their cell pool, then asymmetrically to give rise to neural stem cells (NSCs) called Radial Glial Cells (RGCs), a pool of progenitor cells whose cell body will stay in the ventricular zone (VZ) [START_REF] Agirman | Cerebral cortex development: an outside-in perspective[END_REF]. Like NECs, RGCs have a bipolar morphology, with a basal cytoplasmic extension to the cortical plate (CP), at the cortex surface, and an apical extension in the VZ. They also first amplify their pool by symmetric cell division before generating other cell types by asymmetric division. Cells (NSCs). RGCs will first give rise to neurons and, latter, to Intermediates progenitors (IPs) producing neurons. Neurons migrate to the Cortical Plate (CP), forming successively 6 layers on the top of the previous ones. From [START_REF] Agirman | Cerebral cortex development: an outside-in perspective[END_REF] At the onset of corticogenesis, RGCs asymmetric divisions predominantly generate projecting neurons, in what is called direct neurogenesis. With time, they stop the direct generation of neurons and start to produce Intermediate Progenitors (IP), thus called indirect neurogenesis. IPs first stay attached to RGCs but after their delamination, they invade the nearest cortical layer: the subventricular zone (SVZ).

There, after an optional unique symmetric division to amplify themselves, they undergo one symmetric cell division giving rise to two identical neurons. Independently of their origin, newly generated neurons by RGCs or IPs migrate to reach the cortical plate, where neurons form the layers. The first neurons generated during corticogenesis go to the CP directly by somal translocation, but the others transits by a multipolar morphology before adopting a bipolar shape. During this stage, they attach to the RGCs surface and move on their basal extension towards the upper layers (Fig. 05). Neurons are produced in waves during corticogenesis, producing six distinct neuronal layers.

The main population of RGCs in the mouse cortex is referred as ventral RGC (vRGC, also called apical, aRGC), given that two minor cortical progenitor cell populations also exist in the cortex: the short neural precursor cells (SNPs, also called truncated RGCs, tRGC), and the outer RGCs (oRGC), also called basal RGC, bRGC). SNPs are generated from RGCs but stay in the VZ and often lack the basal extension attachment. They divide symmetrically only once to give rise to neurons, which helps the neurogenesis of the deepest cortical layers [START_REF] Stancik | Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex[END_REF]. oRGCs are similar to the vRGCs but lack their apical extension attachment and reside in the outer part of the SVZ. The oRGC population is enlarged in the cortex of gyrencephalic mammals and it is involved in the cortex folding [START_REF] Hansen | Neurogenic radial glia in the outer subventricular zone of human neocortex[END_REF].

NECs, SNPs and RGCs all possess at their apical surface a small non-motile primary cilia protruding in the lateral ventricle, in contact with the cerebrospinal fluid (CSF). It allows these cells to probe for extracellular signals and initiate intracellular transduction of specific molecular pathways in response [START_REF] Lepanto | Neuron's little helper: The role of primary cilia in neurogenesis[END_REF] and is required for apico-basal polarity maintenance [START_REF] Higginbotham | Arl13b-regulated activities of primary cilia are essential for the formation of the polarized radial glial scaffold[END_REF]. This cilia had also been described on IPs, immature neurons, and interneurons migrating from the ganglionic eminence (Baudoin JP et al, 2012) Corticogenesis is also controlled by various regulatory cues, including Shh, Wnt, BMP, FGF, and Notch signalling pathways. Through their cilia, apical progenitors (NECs, SNPs and RGCs) are influenced by the CSF, whose composition varies during cortical development. For example, Shh could be secreted from the ventral telencephalon to the CSF but also by choroid plexus cells. It could also be locally secreted by Cajal-Retzius cells at the marginal zone before the cortical plate or the interneurons of the CP. Shh also contributes to the generation of oligodendrocytes and GABAergic neurons in the ventral telencephalon. RGCs exposed to Shh also prolongate their self-renewal cell divisions and the decrease of Shh signalling in RGCs impairs their proliferation, survival and their ability to generate IPs, oRGCs and projecting neurons, inducing a general microcephaly.

The Wnt/b-catenin signalling pathway also plays different stage-dependent roles. At the onset of corticogenesis, it promotes NECs and RGCs self-renewal. Indeed, forced expression of Wnt signalling genes in apical progenitors (APs) increases their differentiation into neurons [START_REF] Munji | Wnt Signaling Regulates Neuronal Differentiation of Cortical Intermediate Progenitors[END_REF]. Interestingly, mouse deficient for Wnt signalling but with a functional β-catenin presents a loss of APs and a reduction of the cortical thickness [START_REF] Draganova | Wnt/β-Catenin Signaling Regulates Sequential Fate Decisions of Murine Cortical Precursor Cells[END_REF], suggesting that Wnt signalling function in brain development could be b-catenin independent. Inhibition of the Wnt/β-catenin signalling pathway in spinal neural progenitors also strikingly increases the production of OPCs, indicating that Wnt/β-catenin pathway inhibits NSCs specification toward the oligodendroglial lineage [START_REF] Shimizu | Wnt signaling controls the timing of oligodendrocyte development in the spinal cord[END_REF]. Furthermore, we will see later that this pathway plays an opposite role on OL differentiation.

BMPs (Bone Morphogenetic Proteins) family cooperate with Wnt proteins to promote the telencephalon dorsomedial patterning [START_REF] Furuta | Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development[END_REF]. They also directly control the specification of the dorsal midline and the generation of the choroid plexus [START_REF] Hébert | BMP Signaling Is Required Locally to Pattern the Dorsal Telencephalic Midline[END_REF]. In the developing cortex, BMP2 and BMP4 supports RGCs neuronal differentiation during cortical neurogenesis [START_REF] Li | Neuronal Differentiation of Precursors in the Neocortical Ventricular Zone Is Triggered by BMP[END_REF] but overexpression of BMP4 during the cortical gliogenesis increase astroglial and decrease oligodendroglial lineage commitment of RGCs [START_REF] Ginhoux | Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment[END_REF]. FGF (Fibroblast Growth Factors) play a general role in RGCs stemness maintenance, therefore controlling brain growth. The knockout of the three FGF receptors in the dorsal telencephalon by E10 in mice induces a reduction of cortical surface area [START_REF] Kang | The Transition from Radial Glial to Intermediate Progenitor Cell Is Inhibited by FGF Signaling during Corticogenesis[END_REF]. FGF also enhances the production of IPs, helping the SVZ expansion [START_REF] Wang | Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex[END_REF][START_REF] Rash | Cortical Gyrification Induced by Fibroblast Growth Factor 2 in the Mouse Brain[END_REF] and promotes RGCs self-renewal (Sahara S and O'Leary., 2016). Finally, this growth factor acts in a Shh-dependent manner on ganglionic eminences interneurons specification [START_REF] Gutin | FGF signalling generates ventral telencephalic cells independently of SHH[END_REF]. Notch signalling also helps in RGCs stemness maintenance [START_REF] Kageyama | Roles of Hes genes in neural development[END_REF] and can even partially compensate for FGFR KO [START_REF] Rash | FGF Signaling Expands Embryonic Cortical Surface Area by Regulating Notch-Dependent Neurogenesis[END_REF]. Notch1 and Notch3 are expressed by RGCs during corticogenesis where their ligands are present at the neighbouring neurons or IPs surface, activating the Notch pathway in RGCs after cellcell contact.

Finally, the integrins play a decisive role in the cortical laminar cytoarchitecture.

Inactivation β1-integrin in RGCs impair their basal process attachment to the basement membrane, disrupting its assembly [START_REF] Graus-Porta | ␤1-Class Integrins Regulate the[END_REF]. On the contrary, β1-integrin KO in migrating neurons does not affect layer patterning, which shows that β1-integrin are mainly required for RGCs basal attachment [START_REF] Belvindrah | β1 Integrins in Radial Glia But Not in Migrating Neurons Are Essential for the Formation of Cell Layers in the Cerebral Cortex[END_REF]. Knocking out integrins in humans or ferrets induce a reduction of oRGCs proliferation but not of IPs, showing again the importance of the basal process for brain development [START_REF] Fietz | OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling[END_REF]. Finally, the high levels of integrins and their ligands in the VZ suggest that integrin signalling also plays a role in RGCs proliferation [START_REF] Lathia | Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K[END_REF].

The glial cells

Historically, the term 'glia' originates from the assumption that these cells were just acting as a 'glue' for the CNS. During the two last decades, extensive proofs showed that glial cells were not only providing valuable support in axonal function but could also play key roles in synaptic plasticity or act as integral mediators for neuronal connectivity [START_REF] Barres | The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease[END_REF]. In addition to development and aging, glial cells play crucial roles in CNS regeneration [START_REF] Gallo | Glial Development: The Crossroads of Regeneration and Repair in the CNS[END_REF] and its remyelination after neurodegeneratives diseases or broader disorders (John [START_REF] Lin | Astrocytes: The missing link in neurological disease?[END_REF][START_REF] Burda | Reactive gliosis and the multicellular response to CNS damage and disease[END_REF]. It is now clear that glial cells play much more functions than just being the CNS "glue".

Across species, the ratio of glial cells to neurons vary a lot. In the human brain 50% neuronal and 50% non-neuronal cells were found [START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF] whereas rodent brains contain significantly less non-neuronal cells. For example the capybra's whole brain, the closest model to the human brain in rodents based on the size, only contains 35% of non-neuronal cells [START_REF] Herculano-Houzel | Cellular scaling rules for rodent brains[END_REF]. Despite these variations in the total number of cells, the ratio varies also depending on the considered brain area. For example, there are 85.6% of glial cells in the capaybara's cerebral cortex but only 32.9% in its cerebellum (Herculano-Houzel, Glia, 2014).

Finally, the glial cells subtypes are not equally distributed. Among the glial cells of the human cortex, oligodendroglia represent 75% of glia, astrocytes represents only 20%, and the last 5% consist of microglia, the resident immunitary cells of the CNS.

Astrocytes

Astrocytes were first described by Rudolf Virchow in 1846 as neuroglial cells supporting neuronal function but the term «astrocyte» was proposed by Mihály Lenhossék, in 1893 referring to their typical star shape [START_REF] Molofsky | Astrocyte development: A Guide for the Perplexed[END_REF].

They are classified based on their morphology and localization as protoplasmic, for gray matter astrocytes, or fibrous, for white matter astrocytes. For historical reasons, astrocytes are called Müller glia in the retina and Bergmann glia in the cerebellum.

Fibrous astrocytes presents fewer but thicker processes and express higher levels of astrocyte intermediate filament protein GFAP (Middelorp and Hol, 2011) where protoplasmic astrocytes are spatially segregated and exhibits hundreds of fine processes [START_REF] Tong | Genetically encoded calcium indicators and astrocyte calcium microdomains[END_REF], delimiting their astrocytic domain [START_REF] Bushong | Examination of the relationship between astrocyte morphology and laminar boundaries in the molecular layer of adult dentate gyrus[END_REF]. This diversity reflects their numerous crucial functions in the CNS physiology, including blood-brain barrier formation or maintenance, synaptogenesis, neurotransmission, metabolic regulation, and support of synaptic transmission [START_REF] Allen | Gliamore than just brain glue[END_REF][START_REF] Matthews | A quantitative study of morphological changes accompanying the initiation and progress of myelin production in the dorsal funiculus of the rat spinal cord[END_REF].

In rodents, the astrocytogenesis happens around E12.5 in the spinal cord, and between E16 and E18 [START_REF] Ge | Local generation of glia is a major astrocyte source in postnatal cortex[END_REF] and start by the specification of neural stem cells in astrocyte progenitor cells (APCs) that will then mature into astrocytes. NSCs specification toward the astroglial lineage is regulated by various factors. For example, Nuclear Factor I-A (NFI-A) and Sox9 have been shown to be both required and sufficient to induce astrocytogenesis by demethylating astrocyte specific genes [START_REF] Deneen | The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord[END_REF][START_REF] Stolt | The Sox9 transcription factor determines glial fate choice in the developing spinal cord[END_REF]. Other factors also are involved in NSCs specification into the astrocytic lineage. Notch ligands are expressed both on committed neuronal precursors (NPCs) and young neurons and activation of their receptors in NPCs induce NFI-A expression (Namihira et al., 2008). Embryonic cortical progenitors from N-CoR KO mice, a transcription repressor, present defects in selfrenewal and differentiation into astrocytes [START_REF] Hermanson | N-CoR controls differentiation of neural stem cells into astrocytes[END_REF]. Finally, the KO in mice of Dnmt1, a methylase controlling the JAK-STAT pathway, also shows astrocyte generation defects. More generally, the demethylation of JAK-STAT signalling genes enhances STATs activation, triggering astrocytes differentiation [START_REF] Fan | DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling[END_REF], revealing the involvement of the JAK-STAT pathway in astrocytogenesis.

Figure 06

Astrocytogenesis typical steps. NSCs first specify toward the astroglial lineage due to pro-Astrocytes Transcription Factors [START_REF] Aggarwal | A Size Barrier Limits Protein Diffusion at the Cell Surface to Generate Lipid-Rich Myelin-Membrane Sheets[END_REF]. The astrocytes' precursors (APs) then migrate to specific CNS regions [START_REF] Agirman | Cerebral cortex development: an outside-in perspective[END_REF], proliferate [START_REF] Aguzzi | Microglia: Scapegoat, Saboteur, or Something Else?[END_REF] and finally differentiate into mature astrocytes [START_REF] Al-Abdi | CNP deficiency causes severe hypomyelinating leukodystrophy in humans[END_REF]. From [START_REF] Molofsky | Astrocyte development: A Guide for the Perplexed[END_REF] Once specified, intermediate astrocytes' precursors migrate to colonize the CNS. A first wave of APCs migrates along RGCs processes but a second one occurs after birth, after the RGCs lose their processes, which indicates that astrocytes could also move on their own (Fig. 06). Interestingly, mature astrocytes have been shown to be tethered to their origin site in the VZ, suggesting a limited migration during development [START_REF] Jacob | HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells[END_REF] After finding their final destination, APCs start to express the canonical astrocytes markers in order to initiate their terminal differentiation. Even if an universal astrocyte marker has not yet been identified, some genes are recurrent in astrocyte subtypes.

GFAP have been broadly used to characterise mature astrocytes and its gene has been extensively studied [START_REF] Michalski | Oligodendrocytes in a Nutshell[END_REF]. Despite this broad use, GFAP expression has been shown to be weak or absent in many protoplasmic astrocytes (Alen brain database; [START_REF] Molofsky | Astrocyte development: A Guide for the Perplexed[END_REF]. New complementary markers for mature astrocytes have been identified, like S100β, Aldh1L1, AldoC, Ascgb1, Glt1 and Aqp4 [START_REF] Molofsky | Astrocytes and disease: a neurodevelopmental perspective[END_REF] but none of them seems to be specific to the whole astrocyte population, which gives insights on how diverse astrocytes could be.

Astrocytes play a broad and diverse function. First they have a crucial function in neuron support. An in vivo loss of astrocytes induce neuronal death and most neuron cultures model required to be co-cultivated with astrocytes to survive. They are in the interface between neurons and the pericyte constituting the brain vasculature and use this dual interaction to provide metabolic support to neurons [START_REF] Pellerin | Activity-dependent regulation of energy metabolism by astrocytes: An update[END_REF].

Interestingly, astrocytes have also shown to influence blood flow through neurotransmitter-mediated signalling to favorise angiogenesis in neuronal active brain regions [START_REF] Attwell | Glial and neuronal control of brain blood flow[END_REF].

Three decades ago, the discovery of the astrocytes' ability to adjust their Ca 2+ levels in response to chemical stimuli, like glutamate [START_REF] Finkbeiner | Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling[END_REF], raised the hypothesis that astrocytes could use Ca 2+ as an extra-neuronal signalling system in the CNS [START_REF] Newman | Calcium Waves in Retinal Glial Cells[END_REF], especially when the Ca 2+ levels from neurons have shown to increase in response to the elevation of Ca 2+ concentration in adjacent astrocytes [START_REF] Parpura | Glutamate-mediated astrocyteneuron signalling[END_REF][START_REF] Nedergaard | Direct signaling from astrocytes to neurons in cultures of mammalian brain cells[END_REF].

Their contribution to the analog of the lymphatic system in the CNS, the glymphatic system, have been recently documented. It is composed of astroglial cells that ensure a pseudo lymphatic role by eliminating macroscopic wastes from the brain. The glymphatic system could also play a role in neuronal trophic support by carrying and distributing non waste molecules, such as nutrient or neurotransmitters, all around the brain [START_REF] Jessen | The Glymphatic System -A Beginner's Guide[END_REF]. From the discovery of this new brain-clearance system have emerged hopes for new therapies to drain waste deposits in neurodegenerative disorders. But even if evidences of deposits clearances have been observed, like tau CSF clearance or b-amyloid carrying, the effects of the glymphatic systems on neurodegeneratives disorders are still highly controversy as ventricular tau CSF clearance was inversely correlated with amyloid deposition [START_REF] Benveniste | The glymphatic system and waste clearance with brain aging[END_REF].

The interest in glymphatic clearance mostly originates from the difficulties encountered by the anti-amyloid-β clinical trials and a better understanding of the natural brain clearance mechanisms could represent a new therapeutic approach to treat neurodegenerative disorders [START_REF] Mestre | The brain's glymphatic system: current controversies[END_REF] Finally, astrocytes are extensively involved in synapse formation, survival and activity modulation. For a long time, astrocytes have been known to improve the fidelity of the synaptic transmission. The smallest processes of protoplasmic astrocytes typically enwrap neuronal synapses, forming what we now call the "tripartite synapse" [START_REF] Reichenbach | Morphology and dynamics of perisynaptic glia[END_REF]. There, they remove neurotransmitters at the synaptic cleft, recycle them, and maintain extracellular ion concentration by buffering potassium [START_REF] Frizzo | 3-Hydroxyglutaric acid enhances glutamate uptake into astrocytes from cerebral cortex of young rats[END_REF]; [START_REF] Emmi | Do Glia Have Heart? Expression and Functional Role forEther-A-Go-Go Currents in Hippocampal Astrocytes[END_REF]. But they are also required for the formation of functional synapses, like in retinal ganglion cells (Pfrieger FW and Barres B, 1997). They also control excitatory synapse formation and activation by releasing proteins (reviewed by [START_REF] Clarke | Emerging roles of astrocytes in neural circuit development[END_REF][START_REF] Allen | Astrocyte regulation of synaptic behavior[END_REF].

Microglia

When Ramon y Cajal first characterised the brain cells, he described three components: the neurons, the astrocytes and the third component. Pio del Rio Hortega later discovered that this third component was instead two cell types: the microglia and the oligodendroglia [START_REF] Pérez-Cerdá | Pío del Río Hortega and the discovery of the oligodendrocytes[END_REF]. Microglia represents about 5-12% of CNS cells and are the resident immune cells of the brain. Indeed, brain cells are insulated from the rest of the body by the Blood Brain Barrier (BBB), which can be passed only by a restricted number of cells. Contrary to the other glial cells, microglia have a mesodermal origin and migrates from the yolk sac entering the CNS during embryogenesis, at E7.5 for the mouse, before the BBB formation [START_REF] Aguzzi | Microglia: Scapegoat, Saboteur, or Something Else?[END_REF][START_REF] Ginhoux | Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment[END_REF][START_REF] Casano | Microglia: Multitasking Specialists of the Brain[END_REF]. First, hematopoietic stem cells in the yolk sac became primitive macrophages that will migrate to the developing CNS to become microglia. After this time window, the BBB will develop, preventing any cell migration to the brain. Other CNS macrophages are non-parenchymal, these are derived from blood monocytes.

Microglia plays different functions in the brain [START_REF] Hickman | Analysis of the Microglial Sensome[END_REF]. First, they constantly scan their environment, by the rapid extension-retraction of their processes, to sense every environmental change. This way, microglia form a whole network which could scan their immediate surroundings every hour and react if any injury occurs. More than 100 genes have been described in what is called the microglial sensome [START_REF] Hickman | The Microglial Sensome Revealed by Direct RNA Sequencing[END_REF]. Then, they ensure a housekeeping function by actively remodeling synapses [START_REF] Zhan | Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior[END_REF], phagocyte brain debris and dead neurons in neuropathological context [START_REF] Fuhrmann | Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease[END_REF] or even getting rid of the myelin to help the tissue to repair in disease contexts like MS [START_REF] Healy | MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells[END_REF]. Finally Microglia are able to be activated to defend the CNS, by exemple from injuries. In response to their receptors, such as Fc receptors or Toll-like receptors, microglia are able to initiate a neuroinflammatory response. Similarly to the inflammatory response observed outside of the CNS, the neuroinflammation is based on cytokine production and aimed to recruit cells to clear pathogens or antigens and maintain the brain homeostasis. Unfortunately, neuroinflammation could also damage or even kill neurons in neurodegenerative disorders such as Multiple Sclerosis, Alzeimer's disease, Huntington's disease or Amyotrophic Lateral Sclerosis. All of these involved a dysregulation of the microglia neuroinflammation controller genes pathways such as Trem2, Cx3cr1 or the progranulin pathways.

Oligodendrocytes

The last but not least of the glial cell types are the oligodendrocyte (OLs). OLs are the myelinating cells of the central nervous system, which mean that they enwrap neuronal axons with their cytoplasmic extensions, forming an isolating sheath: the myelin. In the peripheral nervous system, this function is ensured by the Schwann Cells in a similar manner with the exception that they enwrap only one axon, where OLs could myelinate up to 80 different neurons. All PNS glial cells (Schwann cell precursors and satellite glia) are generated by neural crest cells which start their gliogenesis around E11 in rodents. Like NSCs that could give rise to several cell types, Schwann cells' precursors are able to differentiate into neurons of the parasympathetic system, melanocytes, fibroblasts, or even mesenchymal stem cells (reviewed by [START_REF] Imgrund | Adult Ceramide Synthase 2 (CERS2)-deficient Mice Exhibit Myelin Sheath Defects, Cerebellar Degeneration, and Hepatocarcinomas[END_REF]. Many transcriptional and signalling pathways involved in Schwann cells PNS myelination are also present during CNS myelination by OLs that we will later describe in this manuscript. Several important differences between the two myelination processes have still been observed [START_REF] Salzer | Oligodendrocytes use lactate as a source of energy and as a precursor of lipids[END_REF] Like the other neural cell type (except for the microglia) oligodendrocytes have a neuroectodermal origin. Oligodendrogenesis, from NSCs to myelinating OLs, is tightly regulated by the serial expression of intrinsic (transcription factors, signaling pathways, cytoskeleton remodeling, ...) and extrinsic cues (growth factor, extracellular matrix, ...). The gliogenesis from NSCs is controlled by Sox9, NFI-A and serum response factor [START_REF] Selvaraj | Neurotrophic factor-α1: A key Wnt-β-catenin dependent antiproliferation factor and ERK-Sox9 activated inducer of embryonic neural stem cell differentiation to astrocytes in neurodevelopment[END_REF]. Growth factors also play key roles, like FGF, as FGFR KO in OPCs greatly impair myelin generation without affecting their proliferation or differentiation [START_REF] Furusho | Fibroblast Growth Factor Receptor Signaling in Oligodendrocytes Regulates Myelin Sheath Thickness[END_REF]2011). Platelet-Derived Growth Factor Receptor alpha (PDGFRa) is required in the developing forebrain both for oligodendrogenesis and myelin formation. Indeed, Nestin driven Pdgfra KO in the forebrain induce both a hypomyelination and a reduction in the Olig2 positive cells population in postnatal mice forebrain [START_REF] Hamashima | Oligodendrogenesis and Myelin Formation in the Forebrain Require Platelet-derived Growth Factor Receptor-alpha[END_REF]2020). Olig1 and Olig2 are also crucial for NSCs specification toward the oligodendroglial lineage (Maire C L et al, 2010). Until the initiation of their differentiation, cells stay actively blocked in the OPC stage by transcription factors such as Hes5 [START_REF] Xiao | Differential Inhibition of Sox10 Functions by Notch-Hes Pathway[END_REF] and Id2/4 (Plemmel J R et al,

2013).

OPCs are generated during temporally distinct waves both in the rodent and human developing forebrain. The first wave occurs in medial ganglionic eminence (at E12.5 in rodents) from Nkx2.1 expressing progenitors. These cells will gradually spread throughout the telencephalon, from the ventral part to the dorsal areas. At E15.5 in rodents, a second wave occurs from the lateral ganglionic eminence from Gsh2 expressing progenitors that will migrate and colonise the dorsal areas. Finally the third wave (around P0 in mice) generates OLs from the cortical V-SVZ progenitors, which expressed Emx1 (Kessaris N et al, 2006). Despite obvious technical issues compared to rodent models, it appears that human oligodendrocyte also first originates from ganglionic eminences and then switches to more dorsal origins [START_REF] Mo | Human fetal Radial glia cells generate oligodendrocytes in vitro[END_REF]. OPCs form a stable population of cells in the brain (3~7% of all the brain cells in the adult brain, depending on the area) [START_REF] Bribián | Functional Heterogeneity of Mouse and Human Brain OPCs: Relevance for Preclinical Studies in Multiple Sclerosis[END_REF]. Interestingly, they actively maintain their population even in the adult brain, both by mitosis and apoptosis (Dawson M L R et al, 2003;Richardson W D et al, 2011), but could also differentiate into OLs, especially after OL death or demyelination. These newly formed OLs have been observed around lesions of demyelinating diseases such as Multiple Sclerosis (MS) [START_REF] Macchi | Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production[END_REF]. However, this natural remyelination decreases with age [START_REF] Williamson | G. Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis[END_REF]. A better understanding of the resting OPCs differentiation into new OLs could help us to design new supporting remyelination therapies.

Growing evidence suggests the existence of distinct OPC subpopulations.

Oligodendrocytes originating from ventral or dorsal OPCs myelinate different kinds of axon tracts, despite having the same electrical properties [START_REF] Tripathi | Dorsally and Ventrally Derived Oligodendrocytes Have Similar Electrical Properties but Myelinate Preferred Tracts[END_REF]2011).

These variations may result from different developmental origins, specificity in the myelinated neurons population or just unique local cues. PDGFRa and NG2 are the two most common markers for migrating and proliferative OPCs, despite the fact that NG2 is not expressed in the whole population [START_REF] Nishiyama | Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity[END_REF]. More generally, OPCs are sensitive to EGF, FGF and PDGF but lose these receptors when differentiate into immature OLs. It is possible to induce in vitro OPCs differentiation by switching NSCs in culture from a EGF/FGF/PDGF rich to a GF null growth medium [START_REF] Galli | Adult neural stem cells[END_REF]. In the adult stage, each stock OPC controls an area and avoids the other adult OPCs when they enter in contact. In humans, cortical OLs seem to be mainly generated from radial glial cells from the outer SVZ (oRGCs). An intermediate progenitor stage between RGCs (expressing notably Pax6, GFAP, NES and Vimentin) and OPCs (expressing notably Pdgfra, Nkx2.2, Sox10 and PCDH15) have recently been described and named pre-OPCs (figure 07). These pre-OPCs express high levels of EGFR and undergo additional rounds of amplification after EGF exposition to amplify the OPC pool (Huang W et al;2020).

In the spinal cord, OPCs originate from the pMN domain of the neural tube, which under the effect of Shh signaling, initially produces motoneurons and later switchs to OLs production. This production is also regulated by two homeobox transcription factors: Nkx6.1 and Nkx6.2. Indeed, knocking-out Shh, Nkx6.1 or Nkx6.2 in mice suppress OL generation by the pMN, even if some Olig2 or PDGFRa positive OPCs originating from the dorsal spinal cord could be still found after the typical OPC generation time window (Cai, J. et al;2005).

CHAPTER II: Oligodendrogenesis regulation

To differentiate into OLs, OPCs undergo a series of morphological and biochemical modifications, tightly controlled in time by a broad variety of markers. Here I will list non-exhaustively some of the most well-known.

DNA binding and chromatin remodelling

Chromatin remodelling is extensively involved in oligodendrogenesis (Parras C et al, 2020). Indeed, genes present in compacted chromatin are less accessible and therefore less expressed that those on open accessible chromatin. There are several kinds of chromatin remodelers and transcription factors, these later ones characterized for being able to directly bind DNA through a specific binding domain (DBD) and are classified considering their type of DBD. After binding to a precise motive of 4 to 10 nucleotides on the DNA, they could recruit other the proteins of the transcriptional machinery, including chromatin remodeler, and link enhancer sequences to the promoter to enhance transcription efficiency [START_REF] Panne | The enhanceosome[END_REF] or, on the contrary, repress the gene expression by physically blocking the binding of other transcription factors.

Basic Helix-Loop-Helix transcription factors

The basic Helix-Loop-Helix transcription factor family [CP1] [START_REF] Bertrand | Proneural genes and the specification of neural cell types[END_REF] is one of largest DNA-binding transcription factors subgroups, containing many members already implicated in oligodendrogenesis, including Ascl1, Olig1 and Olig2. Ascl1 (Achaete-Scute Complex-Like 1), also called Mash1, is a gene encoding a transcription factor expressed in neural progenitors cells both in the CNS and PNS [START_REF] Guillemot | Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system[END_REF][START_REF] Parras | Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity[END_REF][START_REF] Parras | Mash1 specifies neurons and oligodendrocytes in the postnatal brain[END_REF], including in many neurogenic progenitors and OPCs in the CNS. Interestingly, Ascl1 function in NSCs seems to be dependent on its modality of expression. A sustained overexpression of Ascl1 is associated with neuronal differentiation whereas its oscillating expression seems to favour NSCs differentiation into OPCs and OPC proliferation (Nakatani et al, 2013;[START_REF] Sueda | Oscillatory expression of Ascl1 in oligodendrogenesis[END_REF]. Olig2 expression is also not restricted to oligodendroglial lineage, as it is also expressed in motoneuron progenitors and other neuronal progenitors (Zhou Q et al., 2000;Lu RQ et al., 2000). Repression of both Olig1 and Olig2 expression in the spinal cord totally suppress the oligodendrocyte differentiation and greatly decreases the motoneuron population. Interestingly, the Knockout of both Olig1 and Olig2 in pMN progenitors change their differentiation fates.

Instead of producing motoneurons then oligodendrocytes they generate respectively V2 interneurons and astrocytes (Zhou Q and Anderson DJ, 2002). Therefore Olig1 has already been used in combination with other oligodendroglial markers to characterise oligodendrogenesis stages as its expression and subcellular localisation varies over oligodendrocytes differentiation (Nakatani et al, 2013;Marie et al, 2018) The Sox family Sox proteins contain a high mobility group box (HMG; [START_REF] Malarkey | The high mobility group box: the ultimate utility player of a cell[END_REF], allowing their direct fixation to DNA. They are separated into several subfamilies like the SoxE, composed of Sox8, Sox9 and Sox10, or the SoxD, including Sox5 and Sox6.

The most important Sox factor during oligodendrogenesis is Sox10, as OPCs do not manage to differentiate into OLs in Sox10 KO (Stolt CC et al., 2002). Overexpression of Sox10 in human pluripotent stem cells is sufficient to induce the generation of O4 and MBP expressing OLs in culture (García-León J A et al, 2018). Sox10 also plays a key role during remyelination. In a cuprizone-induced hippocampal demyelination, the overexpression of Sox10 is sufficient to restore a normal phenotype, the myelin ultrastructure and increase the NG2 expression [START_REF] Shao | Effect of Sox10 on remyelination of the hippocampus in cuprizoneinduced demyelinated mice[END_REF]. The overactivation of Notch suppresses Sox10 induced myelin gene expression, indicating that the Notch pathway inhibits Sox10 positive effect on OLs maturation [START_REF] Xiao | Differential Inhibition of Sox10 Functions by Notch-Hes Pathway[END_REF].

Sox9 and Sox8 are mostly coexpressed in oligodendrocyte lineage with Sox10. In this manuscript, we already described the Sox9 requirement for NSCs specification toward the glial, both astrocytic and oligodendroglial, lineage, but it appears that Sox9 and Sox8, contrary to Sox10, are downregulated in the terminal oligodendroglial differentiation stages for OL maturation [START_REF] Stolt | The Sox9 transcription factor determines glial fate choice in the developing spinal cord[END_REF]. Moreover, Sox9 KO in spinal cord neural progenitors does not impair OPCs differentiation into OLs [START_REF] Finzsch | Sox9 and Sox10 influence survival and migration of oligodendrocyte precursors in the spinal cord by regulating PDGF receptor αexpression[END_REF], suggesting a requirement of Sox9 for NSCs specification toward the 29 glial lineage, especially for NSCs PDGFRa expression during their migration, but not for the latter stages of OL maturation (Weider M and Wegner M, 2017). Sox8, which is also expressed mostly in early gliogenic stages and downregulated in differentiating OL, is likely to have a similar function but its implication remains poorly documented (Weider M and Wegner M, 2017). More generally, it appear that SoxD proteins, especially Sox5 and Sox6, are able to interfere with SoxE to regulates oligodendrogenesis (Stolt et al, 2006)

Chd proteins chromatin remodeling

Chromodomain helicase DNA binding protein (Chd) contains 9 family members. Chd8 has been associated with Autism Spectrum Disorders (O'Roak BJ et al, 2012) and Chd7, with CHARGE syndrome (Vissers LELM et al, 2004). Previous work from our team and collaborators also discovered that they were both involved in oligodendrogenesis. Chd7 is bound both by Brg1 and Olig2 and its loss in neonates rodent OPCs led to a striking 75% loss in the differentiating oligodendrocytes cortical population and a decreased expression of oligodendroglial factors such as Sox10 or Myrf. Co-Immunoprecipitation assays revealed a direct bound between Sox10 and Chd7, indicating that Chd7 and Sox10 could collaborate to initiate OPCs oligodendrogenesis (He D et al, 2016). Interestingly, analyses of chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that Chd7 has also a protective effect on OPCs by closing their chromatin to repress p53 expression. On the contrary, it opens the chromatin and activates some key oligodendroglial factors like Sox10, Gpr17 and Nkx2.2. Chd8, a Chd7 paralog, which interacts with Chd7 [START_REF] Batsukh | CHD8 interacts with CHD7, a protein which is mutated in CHARGE syndrome[END_REF], presents a similar binding pattern. These data suggest that, in OPCs, the pioneer factor Olig2 recruits both Chd7 and Chd8 on oligodendrocyte differentiation genes, opening the chromatin and facilitates Sox10 binding and the clustering of other regulators (Marie C et al, 2018).

Nkx family

Nkx family shares a DNA binding homeodomain. Nkx2.2 is a marker of immature oligodendrogenesis stages. Nkx2.2 KO induces a strong decrease in the OL population and an accumulation of Olig1+, Olig2+ and PDGFRa+ progenitors without affecting the astrogliogenesis (Qi Y et al, 2001). Nkx2.2 could also recruit Groucho 3, the histone deacetylase 1 and the DNA methyltransferase 3 α with its Nter Tinman domain and Cter domain. Their recruitment will suppress the expression of oligodendrocyte repressor genes and promote oligodendrogenesis [START_REF] Zhang | The transcription factor NKX2-2 regulates oligodendrocyte differentiation through domain-specific interactions with transcriptional corepressors[END_REF]. Nkx6 proteins play opposite roles in the spinal cord and the hindbrain oligodendrogenesis.

As we mentioned before, Nkx6.1 and Nkx6.2 knockout strongly decrease OL generation from spinal cord pMN progenitors [START_REF] Cai | Generation of Oligodendrocyte Precursor Cells from Mouse Dorsal Spinal Cord Independent of Nkx6 Regulation and Shh Signaling[END_REF] whereas their expression in anterior midbrain progenitors suppress their differentiation into OLs, revealing differences in OL generation mechanisms from spinal cord or hindbrain progenitors [START_REF] Vallstedt | ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genome-wide association study[END_REF].

Histone post-traductional regulation

To affect gene expression without binding directly to the DNA, some proteins could interact with histones in order to regulate their chromatin compaction activity. Histone deacetylation by histone deacetylases (HDACs) proteins will induce local chromatin compaction, resulting in gene silencing by reducing their accessibility and by the histone-complex physical obstruction [START_REF] Jacob | HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells[END_REF]2011). HDAC1 and HDAC2 are required for oligodendrogenesis by controlling Wnt signalling, known to inhibit OL differentiation (Marin-Hustege M et al;2002). Shh also favors OL differentiation by inducing the histones deacetylation [START_REF] Wu | Differential Modulation of the Oligodendrocyte Transcriptome by Sonic Hedgehog and Bone Morphogenetic Protein 4 via Opposing Effects on Histone Acetylation[END_REF]. On the contrary, histone acetylation tends to open the chromatin and favour gene expression by facilitating the recruitment of the transcription machinery. BMP4 induces histone acetylation, which favors astrogliogenesis but blocks oligodendrogenesis and in a HDAC-independent manner [START_REF] Wu | Differential Modulation of the Oligodendrocyte Transcriptome by Sonic Hedgehog and Bone Morphogenetic Protein 4 via Opposing Effects on Histone Acetylation[END_REF].

Brg1 and Brm transcription factors

Brg1 (or Smarca4) and Brm (or Smarca2) are two ATP-dependent SWI/SWF ATPdependent enzymes containing a bromodomain by which they bind acetylated histones. Brg1 is recruited at the level of Olig2 in OPCs and iOLs (Yu Y et al, 2013).

Brg1 deletion in spinals pMN NSCs and whole brain OPCs does not affect OPCs generation but greatly impair mature OL generation and myelination, with severe behavioural phenotypes (Yu et al., 2013). Even Brg1 deletions driven by the NG2 or CNP promoters induced a partial impairment of OLs generation without affecting OPCs population. Therefore, it appears that Brg1 is critical for OPC differentiation into OLs but not for their generation or proliferation. Interestingly, even if Brm and Brg1 are mostly coexpressed during oligodendrogenesis, ChIP analysis of Brm KO suggests that Brm does not compensate for Brg1 loss in Brg1 KO [START_REF] Bischof | Brg1-Dependent Chromatin Remodelling Is Not Essentially Required during Oligodendroglial Differentiation[END_REF].

Other regulators of oligodendrogenesis

The Wnt/β-catenin pathway I previously described the Wnt/β-catenin pathways inhibition of OPCs production from NSCs [START_REF] Shimizu | Wnt signaling controls the timing of oligodendrocyte development in the spinal cord[END_REF]. But, even if the activation of β-catenin in neural progenitor cells before gliogenesis does inhibit OPC differentiation, it is also critical for OL maturation and differentiation. Indeed, β-catenin KO after OPCs formation delays the OLs formation (Dai ZM et al, 2014). Wnt/β-catenin KO impairs myelin protein genes expression and proteolipids synthesis, both in Schwann cells and oligodendrocytes [START_REF] Tawk | Wnt/β-Catenin Signaling Is an Essential and Direct Driver of Myelin Gene Expression and Myelinogenesis[END_REF]. All of these data indicate a dual role of the Wnt/β-catenin pathway during oligodendrogenesis. In early stages, it represses NSCs specification in OPCs, but in later stages, it is also required for proper OLs differentiation and maturation.

Itpr2

Inositol 1,4,5-Trisphosphate Receptor Type 2 (IP3 R2 or Itpr2) is a receptor whose activation increases the intracellular Ca 2+ levels. Itpr2 expression is involved in cell migration, cell division, smooth muscle contraction, and neuronal signaling. In the brain, Itpr2 is expressed in immature oligodendrocytes (Zeisel A et al, 2015;Marques et al., 2016). Little is known about the role of Itpr2 in immature OLs. Itpr2 KO mice present a reduced number of OLs and an increased OPCs population between P7 and P14, suggesting an involvement of Itpr2 in the initiation of OL differentiation, very likely by acting on the endoplasmic reticulum Ca 2+ release [START_REF] Zhang | Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain[END_REF].

Interestingly, Itpr2 is expressed in Schwann cells but also in axon rough endoplasmic reticulum, suggesting a different function in these cells [START_REF] Toews | Signaling proteins in the axoglial apparatus of sciatic nerve nodes of Ranvier[END_REF].

As it controls Ca2+ release from the endoplasmic reticulum, Itpr2 is also involved in astrocyte Calcium signalling. Itpr2 KO mice have been used to suppress calcium signaling in astrocytes. But, interestingly, calcium modulations could still be found in edges of Itpr2 KO astrocytes processes, suggesting a soma restricted action of Itpr2 in astrocytes [START_REF] Srinivasan | Ca2+ signaling in astrocytes from IP3R2-/-mice in brain slices and during startle responses in vivo[END_REF]. These mice also show an impairment for their astrocytes to prune inefficient synapses, which is rescued by the intracerebral administration of ATP [START_REF] Yang | Astrocytes contribute to synapse elimination via type 2 inositol 1,4,5trisphosphate receptor-dependent release of ATP[END_REF]. Finally, genome-wide analysis in human patients revealed that Itpr2 mutations were associated with an increased risk for sporadic ALS, very likely due to a dysregulation of the Calcium-dependent ATP release from astrocytes (Van Es M A et al, 2007).

CNP

The 2',3'-Cyclic Nucleotide 3' Phosphodiesterase (CNP) is an enzyme mostly expressed in OL cytoplasm and excluded from compact myelin. Furthermore, CNP overexpression induces aberrant OLs membrane formation, which are surprisingly highly enriched for CNP but deficient for MBP. This suggests that CNP favours oligodendrogenesis by targeting MBP to the myelin sheath [START_REF] Yin | CNP overexpression induces aberrant oligodendrocyte membranes and inhibits MBP accumulation and myelin compaction[END_REF]. On the contrary, CNP disruption is involved in hypomyelinating leukodystrophy [START_REF] Al-Abdi | CNP deficiency causes severe hypomyelinating leukodystrophy in humans[END_REF], indicating that CNP is a crucial enzyme for OLs differentiation.

In addition to its enzymatic activity, CNP is also involved in cytoskeletal remodelling. Indeed, CNP is able to bind to microtubules [START_REF] Myllykoski | Structural and functional evolution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase[END_REF] by its C Terminal part and is involved in actin cytoskeleton reorganisation [START_REF] Lee | Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein[END_REF]. This C Terminus also contains a Caax motif, allowing it to bind to membranes after a mandatory post-traductional modification [START_REF] De Angelis | Isoprenylation of brain 2′,3′-cyclic nucleotide 3′-phosphodiesterase modulates cell morphology[END_REF]. Moreover, CNP has been shown to bind to membranes in rat myelin sheath [START_REF] Kim | Myelin glycosphingolipid/cholesterol-enriched microdomains selectively sequester the non-compact myelin proteins CNP and MOG[END_REF]. It therefore is very likely that CNP involvement in myelination is explained by a combination of all its effects.

APC and CC1

Adenomatous Polyposis Coli (APC) is a tumour suppressor gene whose suppression induces adenomatous polyposis (the formation of a polyp in the intestinal tract).

Knockout models have demonstrated that APC was involved in oligodendrogenesis.

APC regulates OL differentiation by regulating the Wnt/β-catenin pathway but also through OL cytoskeleton remodelling [START_REF] Lang | Adenomatous Polyposis Coli Regulates Oligodendroglial Development[END_REF]. Interestingly, even if APC mRNA is mostly found in CNS neurons (Bhat RV et al, 1994), immunofluorescence stainings based on anti-APC antibodies, especially one recognising the CC1 epitope, have been used for more than 20 years to characterise the oligodendroglial lineage in the brain. For a long time, this difference was known to be due to the recognition of another epitope by APC-CC1 antibody but this target remains unknown for a long time (Brakeman et al., 1999). Four years ago, this oligodendroglial-specific other target was identified. The APC-CC1 antibody recognizes Quacking 7, an RNA-binding protein highly expressed in CNS myelinating oligodendrocytes (Bin JM et al, 2017). Recent data shows that the CC1 antibody was also able to recognise astrocytes stained by a GFAP-driven GFP expression in mice [START_REF] Behrangi | Oligodendrocyte Lineage Marker Expression in eGFP-GFAP Transgenic Mice[END_REF], suggesting that it could recognise a broader population than oligodendrocytes.

Opalin

Opalin, or TMEM10, is a brain-specific small transmembrane protein first characterized in temporal lobe epilepsy and spastic paraplegia [START_REF] Nobile | Identification and characterization of a novel human brain-specific gene, homologous to S. scrofa tmp83.5 , in the chromosome 10q24 critical region for temporal lobe epilepsy and spastic paraplegia[END_REF].

Opalin starts to be upregulated at the onset of OL differentiation and is more and more expressed as oligodendrogenesis progresses, with a maximum expression in late stages of OLs differentiation/maturation. Its first intron contains an enhancer directly bound by Myt1 and CREB transcription factors. Overexpression of one of these two proteins activates this region to enhance Opalin expression which promotes oligodendroglial differentiation [START_REF] Aruga | An oligodendrocyte enhancer in a phylogenetically conserved intron region of the mammalian myelin gene Opalin[END_REF]. Moreover, the overexpression of Opalin induces an increased expression of myelin genes, such as CNP or MAG, and Opalin KO in primary cultures induce a dramatic increase of OPCs differentiation and MBP mRNA expression [START_REF] De Faria | TMEM10 Promotes Oligodendrocyte Differentiation and is Expressed by Oligodendrocytes in Human Remyelinating Multiple Sclerosis Plaques[END_REF]. Finally, Opalin plays a crucial role in OL differentiation during MS-related demyelination as leukemia inhibitory factor (LIF) administration in cuprizon MS mice model induces an increase of both Opalin and MOG compared to Sham mice [START_REF] Mashayekhi | Administration of leukemia inhibitory factor increases Opalin and myelin oligodendrocyte glycoprotein expression in the cerebral cortex in a cuprizone-induced model of demyelination[END_REF].

CHAPTER III: Myelination and Remyelination

Myelin generation and function

During the development of the brain and all along life, OLs synthesize large amounts of plasma membrane to enwrap axons called myelin sheaths. The term «myelin» was invented by Rudolf Virchow in 1864 in reference to the Greek term for «marrow» (myelos) when he observed a high concentration of myelin in the brain core, also called the brain marrow. Virchow hypothesized that this substance was secreted by the neurons but, about one century later, Pio del Rio Hortega proved it was indeed synthesized by the OLs, a new kind of brain cells he identified with the improvements of its own staining protocol.

In rodents, myelination first occurs in the spinal cord at embryonic stages, then in the brain and optic nerve (ON) during the first few postnatal weeks. In humans, myelination starts also before birth but occurs mainly during a long postnatal period. The study of the human optic nerve postnatal growth revealed that the ON elongates by 80% during the 3 firsts postnatal years and reaches its final length only around 15 years old [START_REF] Bernstein | Postnatal growth of the human optic nerve[END_REF].

In the CNS, study of the white matter progression from birth to 4,5 years in children revealed that the human brain is almost totally unmyelinated at birth [START_REF] Fonov | Human brain myelination from birth to 4.5 years[END_REF]. In the 9th first months, myelination quickly spread from the most central parts of the brains to the superficial areas and from the occipital and parietal lobes to the frontal and temporal lobes [START_REF] Williamson | G. Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis[END_REF]. Myelination continues during childhood then during adolescence and is associated with cognitive development (Mabott et al, 2006) and increased processing speed [START_REF] Scantlebury | Relations between white matter maturation and reaction time in childhood[END_REF]. Throughout adult life, myelination keeps occuring in the adult brain at a lower level and is the base of cerebral plasticity, which gives the brain the ability to adapt itself all along life, and starts declining with aging.

Myelin composition

Myelin is a multilayered stack of membranes tightly attached together both at their cytosolic and extracellular surfaces. A myelinated segment (lamellae) usually covers 150 nm of axonal surface. Myelin is an exceptionally stable structure, maintained both by its simple molecular composition and its specific architecture. When observed in electron microscopy, myelin shows a characteristic alternating structure between electron-dense and electron-light layers. These electron-dense lines come from the apposition of the inner plasma membrane whereas electron-light lines are created by the small (~12nm) outer space between two wraps (Figure 08). Myelin composition was first analysed by mass spectrometry or by dyes injection in OLs. The firsts electronic imaging attempts were impaired by the shrinkage or collapsing of the intracellular spaces. Tissue architecture preservation was enhanced in the 1950's with high-pressure freezing EM imaging, which is the standard tool of myelin structure evaluation nowadays [START_REF] Bunge | ELECTRON MICROSCOPIC DEMONSTRATION OF CONNECTIONS BETWEEN GLIA AND MYELIN SHEATHS IN THE DEVELOPING MAMMALIAN CENTRAL NERVOUS SYSTEM[END_REF][START_REF] Bunge | AN ELECTRON MICROSCOPE STUDY OF CULTURED RAT SPINAL CORD[END_REF][START_REF] Mashayekhi | Administration of leukemia inhibitory factor increases Opalin and myelin oligodendrocyte glycoprotein expression in the cerebral cortex in a cuprizone-induced model of demyelination[END_REF].

This structure is so close to its thermodynamic equilibrium state that it has been shown to remain stable several weeks after OL depletion [START_REF] Pohl | Genetically Induced Adult Oligodendrocyte Cell Death Is Associated with Poor Myelin Clearance, Reduced Remyelination, and Axonal Damage[END_REF]. Even 5000-yearold myelin samples dissected from the Tyrolean IceMan were still presenting an intact molecular configuration and retained fine structure [START_REF] Hess | 5000-year-old myelin: uniquely intact in molecular configuration and fine structure[END_REF]. Evidence based on carbon dating shows that mOLs themselves remain quite stable, with a turnover rate in the human adult brain of only 0,3% after 5 years [START_REF] Bercury | Dynamics and Mechanisms of CNS Myelination[END_REF].

All myelin components have half-lives of several weeks to months. The strong forces that bring opposite myelin bilayers together are based on a combination of electrostatic and hydrophobic forces. Every mismatch in the positive to negative charges ratio results in repulsion, giving rise to membrane swelling and demyelination.

Lipids are the main component of the myelin (between 73% and 81% of the total dry weight). Glycosphingolipids (~27%) and plasmalogens (~17%) are the most abundant in myelin sheaths. Glycosphingolipids (galactosylceramide (GalC) and sulfatide) present notable differences in myelin sheaths compared to other plasma membranes, with high levels of fatty acid hydroxylation and a headgroup based on galactose instead of glucose like in regular plasma membranes. Myelin glycosphingolipids also incorporate very-long-chain fatty acids with chain lengths up to 26 carbons. Finally, plasmalogens represent 70% of phosphatidylethanolamine, instead of their diacyl form found in other membranes.

Proteins are much less represented in myelin and no genetic building plan for its spatial organisation has yet been identified, suggesting an epigenetic control of myelination. In a regular plasma membrane, the protein-to-lipid dry weight ratio varies from 1/1 to 4/1 compared to ~1/4 in myelin. Interestingly, the most represented proteins in myelin sheaths, like the Myelin Basic Protein (MBP), the Proteolipid protein (PLP) or the Myelin Oligodendrocytes Glycoprotein (MOG), are mostly dispensable for myelination itself but are required in OLs or Schwann cells for axonal health. Indeed, several mice models have been studied to understand protein requirements in myelin but it seems clear that myelin biogenesis in the CNS is indeed relatively insensitive to perturbations, with a strong capacity to maintain its spatial organization (Zoller et al., 2008). Moreover, it is a typical feature of self-organizing systems that are organized in a way that allows redundant control. A lack of UDP-galactose:ceramide galactosyltransferase (CGT), by example, does not affect myelin formation but induces a clear reduction of its thickness. Interestingly, FA2H-deficient mice, that lack 2hydroxylated galactosylceramide and sulfatide, present normal myelin sheaths at the beginning but they tend to degenerate in old age [START_REF] Potter | Central nervous system dysfunction in a mouse model of FA2H deficiency[END_REF].

Interestingly, PLP KO OLs keep their ability to myelinate axons in mice but also generate abnormally condensed extracellular leaflets of adjacent myelin membranes, which are much less stable than regular myelin [START_REF] Klugmann | Assembly of CNS Myelin in the Absence of Proteolipid Protein[END_REF]. Therefore, it is clear that unknown interactions could partially compensate for the loss of PLP. In premyelinating OLs, a PLP pool accumulates in late endolysosomes, which quickly disappears once myelination starts [START_REF] Trajkovic | Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites[END_REF]. As OL have shown to secrete exosomes/microvesicles, it is possible they use these vesicles to get rid of their wastes [START_REF] Fitzner | Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis[END_REF].

All myelin components help the structure to stay stable and repulse all foreign molecules. For example, most of myelin proteins are hydrophobic to minimize the amount of cytoplasm inside the membrane. The Myelin Basic Protein (MBP) is also known to tightly bind the inner membranes together. Strong hydrogen bonds between galactosylceramide and sulfatide, reinforced by their high fatty acid hydroxylation, represents one of the most attractive force binding myelin membranes together.

Impairment of MBP mRNA transport into the myelin sheath (by kif1b KO ) induces an accumulation of MBP in the cell body and the formation of stable complexes at inappropriate cellular locations (Lyons et al., 2010).

Finally, when two lipid molecules are close, their methylene group interacts with each other and generates Van der Waals forces, stabilizing the whole structure.

Interestingly, models with an impaired lipid metabolism show relatively mild phenotypes at the myelin sheath level. CerS2 null mice, by example, form less but relatively normal myelin, with reduced levels of MBP, and present only mild structural defects [START_REF] Imgrund | Adult Ceramide Synthase 2 (CERS2)-deficient Mice Exhibit Myelin Sheath Defects, Cerebellar Degeneration, and Hepatocarcinomas[END_REF]. DAPAT (a substrate of plasmalogen biogenesis) null mice that lack ethanolamine plasmalogens produce normal but reduced amounts of myelin.

Axon wrapping

Myelination varies a lot in the different brain regions and across the CNS. OLs from the spinal cord could create up to 150 lamellae on a length of 1500 μm [START_REF] Remahl | Relations between axons and oligodendroglial cells during initial myelination. II. The individual axon[END_REF], where those from the corpus callosum and the cortex usually form 30 to 80 on a length of 20 to 200 mm with up to 60 lamellae [START_REF] Matthews | A quantitative study of morphological changes accompanying the initiation and progress of myelin production in the dorsal funiculus of the rat spinal cord[END_REF][START_REF] Chong | Neurite outgrowth inhibitor Nogo-A establishes spatial segregation and extent of oligodendrocyte myelination[END_REF] or those from the ON generate 40 internodes.

A strong relationship links the axon diameter, the number of lamellae and the internode length. By example, the increase from 1 to 15 mm of the axon diameter induces the extension of the internode length from 100 to 1500 mm [START_REF] Murray | The relationship between internodal length and fibre diameter in the spinal cord of the cat[END_REF][START_REF] Hildebrand | Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species[END_REF]. OLs produce more myelin wraps around larger caliber axons and a relatively consistent g-ratio (the ratio of the axon diameter and the myelinated fiber diameter) have been described using axons of different diameters [START_REF] Hildebrand | Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species[END_REF][START_REF] Friede | How are sheath dimensions affected by axon caliber and internode length?[END_REF]. More recent works even demonstrated that artificial nanofibers could be myelinated by oligodendrocytes and are even sufficient to induce the differentiation of OPC into OL in neuron free condition In Vitro cultures [START_REF] Li | Nanofibers Support Oligodendrocyte Precursor Cell Growth and Function as a Neuron-Free Model for Myelination Study[END_REF].

Myelin sheaths are formed from the inner turn of the oligodendrocyte plasma membrane extending in a concentric and lateral movement down the axon (Fig. 09-A,B). First, the OL growth cone contacts and binds to an axon using cytoplasmic channels(Fig. 09-C,a). Then, this growth cone shapes into a triangle (Fig. 09-C,b), with its inner part starting to form compact myelin. The outer part of the sheath, on the contrary, stays relaxed and will turn around the axon and the basal part will extend laterally to cover the whole internode length (Fig. 09-C,c). After a precise number of turn (or lamellae), the outer part of the former growth cone also extends laterally and all the cytoplasmic channels closed themselves, forming a ultra-stable rectangular shaped sheath (Fig. 09-C,d) tightly wrapped around the axon [START_REF] Sobottka | CNS live imaging reveals a new mechanism of myelination: The liquid croissant model[END_REF]. -b). This structure will progress around the axon, producing compact myelin at its base where its edges are mostly uncompacted (C-c). When the wrapping end, the edges extend laterally to form a stable rectangular structure of compacted myelin (C-d) From Aggarwal, Yurlova and Simons, 2011 Myelination is a strictly ordered but quick process. In Zebrafish, 5 hour are sufficient for OLs to undergo the full myelination process from their first to their final myelin sheath production [START_REF] Czopka | Individual Oligodendrocytes Have Only a Few Hours in which to Generate New Myelin Sheaths In Vivo[END_REF]. OLs from other parts of the zebrafish spinal cord start their myelination at different time points but always within this 5hr time window.

Furthermore, myelination could be regulated in various ways. More myelin is found in the 5 th and the 6 th cortical layers compared to the 2 nd and the 3 rd . Moreover, these deeper layers are more uniformly myelinated where upper layers present unmyelinated segments [START_REF] Tomassy | Distinct profiles of myelin distribution along single axons of pyramidal neurons in the neocortex[END_REF]. This could be explained by the neuronal diversity with each separate class of cortical neurons having different signalling patterns locally modulating myelination. These neuron-OL interactions could therefore play a critical role in developing adult brain plasticity.

In medial prefrontal or the barrel cortex, a clear impact between myelination and neuronal activity have been demonstrated, by whisker trimming [START_REF] Barrera | Organization of Myelin in the Mouse Somatosensory Barrel Cortex and the Effects of Sensory Deprivation[END_REF], social isolation [START_REF] Makinodan | A Critical Period for Social Experience-Dependent Oligodendrocyte Maturation and Myelination[END_REF] or motor learning experiments. Moreover, a significant increase in the white matter volume can be observed after only several weeks of practicing piano [START_REF] Bengtsson | Extensive piano practicing has regionally specific effects on white matter development[END_REF], learning to juggle [START_REF] Scholz | United we standintegrating the actin cytoskeleton and cell-matrix adhesions in cellular mechanotransduction[END_REF] or even the learning of a new language [START_REF] Schlegel | White Matter Structure Changes as Adults Learn a Second Language[END_REF]. Neuronal activity impacts myelin plasticity which in turn modulates neural electrical activity and behaviour. For example, the release of neurotransmitters impacts OLs calcium levels, which modify the local translation of MBP [START_REF] Wake | Control of Local Protein Synthesis and Initial Events in Myelination by Action Potentials[END_REF]. Myelination is impaired by the action potential inhibition using neurotoxins, whereas increasing neuronal electrical activity promotes myelination [START_REF] Demerens | Induction of myelination in the central nervous system by electrical activity[END_REF].

In myelinating co-cultures, the OL response to glutamate could be altered by neuregulin or other signalling molecules. Interestingly, blocking the differentiation of OPCs into OLs, or even the expression of myelin proteins in OLs, severely inhibits motor learning [START_REF] Gibson | Neuronal Activity Promotes Oligodendrogenesis and Adaptive Myelination in the Mammalian Brain[END_REF]McKenzie et al., 2014), suggesting that myelination is also a key regulator for brain learning and plasticity. More generally, the amount of generated myelin and the OL response to axons of various diameters could be altered by localized cues.

In the uninjured, healthy adult brain, OPCs keep producing OLs and new myelin is continually generated [START_REF] Yeung | Dynamics of Oligodendrocyte Generation and Myelination in the Human Brain[END_REF][START_REF] Young | Oligodendrocyte Dynamics in the Healthy Adult CNS: Evidence for Myelin Remodeling[END_REF]. Furthermore, numerous axons remain unmyelinated, especially in the adult grey matter. In neuronfree cultures, OLs still generate highly asymmetric cell surfaces and form large myelin sheaths. Even if no unique myelination signal has been identified in both neurons and OLs, MBP seems to play a crucial role. Its mRNA is transported from OLs perikaryon to their growth cone by Kif1b, a motor protein, inside granules (Lyons et al, 2009), in response to various stimuli like the fyn [START_REF] White | Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2-dependent RNA granules[END_REF] or the a6β1 integrin activation [START_REF] Lathia | Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K[END_REF]. MBP is strongly charged positively and interacts with the negatively charged cell membrane, which restricts the entry of large cytoplasmic domains' protein in myelin sheaths [START_REF] Aggarwal | A Size Barrier Limits Protein Diffusion at the Cell Surface to Generate Lipid-Rich Myelin-Membrane Sheets[END_REF].

Actin cytoskeleton remodelling

During oligodendrogenesis and myelination, OLs change their shape multiple times [START_REF] Bauer | Role of the oligodendroglial cytoskeleton in differentiation and myelination[END_REF]. These shapes modification is based on cytoskeletal reorganisation, which is composed of two major components: β-tubulin (or microtubules) and F-actin, also called microfilaments (Brown and Macklin, 2020).

At the onset of their differentiation, iOLs first extend their processes, forming lamellipodia that will enwrap axons when they find one. The lamellipodia's tip forms a structure highly similar to a neuronal growth cone [START_REF] Rumsby | Microfilament and microtubule organization and dynamics in process extension by central glia-4 oligodendrocytes: Evidence for a microtubule organizing center[END_REF][START_REF] Fox | Growth conelike sensorimotor structures are characteristic features of postmigratory, premyelinating oligodendrocytes[END_REF]. F-actin is mainly concentrated in these edges whereas microtubules are distributed all along the cell in more stable processes [START_REF] Lunn | Microtubule organization and stability in the oligodendrocyte[END_REF]. More generally, F-actin is localised in dynamically active membrane areas whereas β-tubulin is found in stable parts, away from the moving leading edge (Fig. 010).

Figure 010

Oligodendroglial cytoskeleton dynamic. In oligodendrocytes processes are stabilised by a microtubule network. On the contrary, the motile oligodendrocytes growth cones contained high levels of actin (A). The outgrowth of these structures is ensured by actin-associated proteins such as N-WASP, WAVE1. In parallel, the microtubule polymerisation is inhibited by RhoA (B) From [START_REF] Michalski | Oligodendrocytes in a Nutshell[END_REF] As the growth cone progresses, the easily remodelled F-actin, required for the filopodia progression, is replaced by β-tubulin, which is more stable and will therefore compact the cytoskeleton in stable straight processes (reviewed in [START_REF] Michalski | Oligodendrocytes in a Nutshell[END_REF]. The same phenomenon happen during axon enwrapment: the inner tongue that will progress all around the axon is mainly composed of F-actin and is progressively replaced by microtubules to compact the structure, forming a dense pattern of tendrils snaking between larger pockets of compact membrane (Dyer and Benjamins, 1989, I and II;[START_REF] Boggs | Effect of liposomes containing cerebroside and cerebroside sulfate on cytoskeleton of cultured oligodendrocytes[END_REF].

Maturating OLs acquire a more complex morphology. Their microtubules display increased levels of acetylated α-tubulin, indicating long-term stability both for microtubules and their process [START_REF] Lunn | Microtubule organization and stability in the oligodendrocyte[END_REF][START_REF] Song | Cytoskeletal reorganization during the formation of oligodendrocyte processes and branches[END_REF][START_REF] Lee | Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein[END_REF].

But even if microtubules are a long lasting stabilizing structure, both of the cytoskeletal components are missing in the final myelin sheath, indicating that the microtubules are also actively removed during myelin maturation (Dyer and Benjamins, 1989 I and II;Boggs andWang, 2001, 2004;[START_REF] Bauer | Role of the oligodendroglial cytoskeleton in differentiation and myelination[END_REF][START_REF] Aggarwal | A Size Barrier Limits Protein Diffusion at the Cell Surface to Generate Lipid-Rich Myelin-Membrane Sheets[END_REF] Several key regulating proteins for cytoskeleton remodelling are found at OLs leading edges [START_REF] Fox | Growth conelike sensorimotor structures are characteristic features of postmigratory, premyelinating oligodendrocytes[END_REF]. Among them, the Arp2/3 complex, one of the initiators of the growth cone formation, stays present all along OL differentiation at the edges. Arp2/3 inhibition in OL cell culture impairs the formation of their growth cone and therefore reduces OL branching (Zucchero et al, 2015 II). More generally, the OL growth cone and its actin cytoskeleton could be considered as the driven of all the morphological changes undergone by OLs during their differentiation (Thomason E.J, 2020) WASP family proteins also play a key role in OL cytoskeleton remodelling. In the optic nerve, a chemical inhibition of N-WASP in the optical tract impairs the process extension and filopodia retraction of OPCs. N-WASP inhibition also reduces axon ensheathment [START_REF] Bacon | N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination[END_REF]. Interestingly, N-Wasp KO mice present both a white matter general hypomyelination and a focal hypermyelination of unmyelinated axons or neurons' cell body, indicating that N-WASP plays controls the inner lip of the OL growth cone extension [START_REF] Katanov | N-Wasp Regulates Oligodendrocyte Myelination[END_REF]. WAVE1 KO induces a decreased number of processes produced by OLs without affecting the neuron or astrocyte morphology. These mice also present myelination defects in their optic and a hypomyelination of their corpus callosum (Kim HJ et al., 2006). More generally, it appears that N-WASP, WAVE1 and the Arp 2/3 complex interact together to control the assembly of the microfilament network at the growth cone level (Ridley, 2011).

The involvement of the small Rho GTPases Rac1, Cdc42, and RhoA in oligodendrogenesis are unclear even if they can be found in OLs growth cones (Kim HJ et al., 2006;[START_REF] Bacon | N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination[END_REF]. Indeed, the OL-specific ablation of Cdc42 or Rac1, contrary to the WASP protein inhibition, did not affect OPC proliferation, directed migration, or their differentiation In Vitro. Furthermore, Cdc42 KO or Rac1 KO OLs shows an abnormal cytoplasmic accumulation in their inner tongue [START_REF] Thurnherr | Cdc42 and Rac1 Signaling Are Both Required for and Act Synergistically in the Correct Formation of Myelin Sheaths in the CNS[END_REF], suggesting that Cdc42 and Rac1 synergistically regulate myelination in myelinating OLs [START_REF] Brown | The actin cytoskeleton in myelinating cells[END_REF].

P21-activated kinases (PAK) are also critical regulators of OL differentiation and of their cytoskeletal remodelling. PAKs are divided into two subfamilies, with the group I containing PAK1 to 3 and the group II containing PAK4 to 6 [START_REF] Brown | The actin cytoskeleton in myelinating cells[END_REF]. Interestingly, PAKs are able to directly impact the cytoskeleton assembly but also regulate signalling pathways involved in OL differentiation and maturation. The double suppression of PAK1 and PAK3 in mice, two PAKs highly expressed in OPCs, impair brain development but only during postnatal stages. These mice also present a clear hypomyelination and cognitive deficits [START_REF] Huang | p21-Activated Kinases 1 and 3 Control Brain Size through Coordinating Neuronal Complexity and Synaptic Properties[END_REF]. Interestingly, PAK3 KO induces alone a reduction of cultured OPCs differentiation without affecting their morphology or proliferation and PAK3 KO mice present a hypomyelination at P14 compensated in adult mice (Maglorius [START_REF] Renkilaraj | The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation[END_REF].

Glial support of myelinated axons

The wrapping of multiple layers of myelin membrane sheaths around an axon is critical for the function of the nervous system. Its first role is of course to protect axons from the extracellular medium. If this protection is destroyed, like in demyelinating disease such as MS, axons enter wallerian degenerescence, ending by the total destruction of the axon (Nave KA, nov 2010).

Historically, myelin was first considered as an axonal insulator allowing faster conduction of axon potentials by saltatory conduction compared to unmyelinated segments [START_REF] Wang | Aberrant Methylation and Differential Expression of SLC2A1, TNS4, GAPDH, ATP8A2, and CASZ1 Are Associated with the Prognosis of Lung Adenocarcinoma[END_REF]. This saltatory conduction is the basis for fast processing of information. Myelin sheaths also clustered Na 2+ channels into non-myelinated areas dispatched regularly along the axon called the nodes of Ranvier. Initially, these nodes were thought to result from damage or disruption of myelin. We know now that they concentrate a high density of voltage dependent sodium channels. This way, action potentials are able to jump from node to node without losing any strength between each internode. Interestingly, these cytoplasmic channels, found in the nascent membrane, are present during the development but their number reduces quickly from P10 to P14 in mice.

Oligodendrocytes also provide metabolic support to the axons (Nave KA, april 2010).

To maintain the BBB, neurons, which mostly use lactate instead of the glucose as an energy source, never interact directly with the blood vessels and fully depend on glial cells for their metabolic needs. Oligodendrocytes import glucose from the blood vessels, metabolize it into lactate and deliver it to myelinated neurons, through the myelin sheath, with the help of MCT proteins [START_REF] Rinholm | Regulation of Oligodendrocyte Development and Myelination by Glucose and Lactate[END_REF]Lee et al., 2012)). Interestingly, oligodendrocytes also use this lactate as a source of energy and a precursor for lipid synthesis (Sánchez-Abarca et al, 2001). Astrocytes are also able to capture the glucose from the brain capillary, metabolize it into lactate, carrying it to synapses or neuronal cell bodies, and could even transfert it to oligodendrocytes by coupling with them (Fig. 011). Therefore oligodendrocytes and astrocytes each contribute to the whole axonal trophic support, [START_REF] Amaral | Metabolic Aspects of Neuron-Oligodendrocyte-Astrocyte Interactions[END_REF]. It is even considered that changes in myelination could represent a new kind of CNS plasticity (reviewed by [START_REF] Fields | Change in the Brain's White Matter[END_REF][START_REF] Bercury | Dynamics and Mechanisms of CNS Myelination[END_REF] Figure 011 Glucose metabolization into lactate by the astrocytes and oligodendrocytes to provide trophic support to neurons. Astrocytes and oligodendrocytes are able to capture the glucose from blood vessels and to transform it into lactate, an energy source predominantly used by neurons.

Oligodendrocytes provide lactate to myelinated segments where astrocytes support neurons at the level of their soma and synapses. Astrocytes are also able to directly export the lactate to oligodendrocyte to indirectly support neurons' myelinated segments. From Saab, Tzvetanova and Nave, 2013 PNS Schwann cells, which myelinate a unique axon, also provide metabolic support through localized myelin uncompaction named the Schmidt-Lanterman incisures. This structure is formed by the engulfment of the outer cytoplasmic layer to create a network of interconnected cytoplasmic pockets (1.9 pockets per 10 mm sheath length [START_REF] Velumian | Visualization of cytoplasmic diffusion within living myelin sheaths of CNS white matter axons using microinjection of the fluorescent dye Lucifer Yellow[END_REF]. It allows the Schwann cells to distribute nutrients and molecules to the neurons through the internode, all along the axon.

Finally, glial cells are also involved in angiogenesis. Stable HIF1/2α expression in OPCs, a critical angiogenic factor, induces inhibition of OPCs maturation through the Wnt canonical pathway but also an increased postnatal angiogenesis. On the contrary, OPC-specific HIF1/2α deletion strongly impairs the formation of blood vessels in the corpus callosum and leads to a critical neuronal loss. In Vitro studies suggest that OPCs are able to stimulate endothelial cell proliferation through paracrine effect [START_REF] Yuen | Oligodendrocyte-encoded HIF function couples postnatal myelination and white matter angiogenesis[END_REF]. During the retina development, astrocytes expressed high levels of VEGF, which are hypothesized to attract endothelial cells and promote angiogenesis.

VEGF/HIF suppression specifically in astrocytes provides contradictory data with 3 teams over 4 supporting the pro-angiogenic effect of retinal astrocytes [START_REF] Rattner | Roles of HIFs and VEGF in angiogenesis in the retina and brain[END_REF]. Finally, CNS vascular defects could be observed in MS lesions [START_REF] Buch | Revealing vascular abnormalities and measuring small vessel density in multiple sclerosis lesions using USPIO[END_REF], contributing significantly to disease pathogenesis and progression and highlighting the contribution of neuron-glia interactions to a proper CNS vascularisation and its importance for brain health.

Oligodendroglial pathologies and therapeutics

Oligodendrocytes in Neurodegenerative disorders

Most human developmental disorders and neurodegenerative diseases involve subtle or pronounced white matter deficiencies. Alterations of glial structure, function or interaction with neurons are seen in human psychiatric disorders [START_REF] Haroutunian | Myelination, oligodendrocytes, and serious mental illness[END_REF] and developmental disorders, including autism spectrum disorder (ASD), sensory processing delay disorder [START_REF] Owen | Test-Retest Reliability of Computational Network Measurements Derived from the Structural Connectome of the Human Brain[END_REF], attention deficit hyperactivity disorder [START_REF] Li | Increased fractional anisotropy in white matter of the right frontal region in children with attention-deficit/hyperactivity disorder: a diffusion tensor imaging study[END_REF]Wu et al., 2014), and Rett syndrome [START_REF] Mahmood | White Matter Impairment in Rett Syndrome: Diffusion Tensor Imaging Study with Clinical Correlations[END_REF].

Myelin malfunctions are also seen in other adult neurodegenerative disorders.

Alzheimer's disease (AD) patients present a reduction of their cortex thickness [START_REF] Cho | Longitudinal changes of cortical thickness in early-versus late-onset Alzheimer's disease[END_REF] and lesions in their white matter favor the transition from mild cognitive impairment to established AD [START_REF] Defrancesco | Impact of White Matter Lesions and Cognitive Deficits on Conversion from Mild Cognitive Impairment to Alzheimer's Disease[END_REF][START_REF] Defrancesco | Changes in White Matter Integrity before Conversion from Mild Cognitive Impairment to Alzheimer's Disease[END_REF]. The white matter from the frontal and temporal parts of the brain from Amyotrophic Lateral Sclerosis (ALS) patients present atrophy and their corticospinal tract also tend to degenerate [START_REF] Lillo | Grey and White Matter Changes across the Amyotrophic Lateral Sclerosis-Frontotemporal Dementia Continuum[END_REF][START_REF] Pettit | Executive deficits, not processing speed relates to abnormalities in distinct prefrontal tracts in amyotrophic lateral sclerosis[END_REF]. An increased proliferation of NG2+ cells have been observed in ALS mouse models and Oligodendrocyte genes are impaired prior to the typically observed motoneurons degeneration [START_REF] Kang | Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis[END_REF]). Parkinson's disease patients also present reduced white matter thickness [START_REF] Kim | Alterations of mean diffusivity in brain white matter and deep gray matter in Parkinson's disease[END_REF]. In Huntington's disease, finally, an early demyelination of the corpus callosum have been described even before the onset the onset of the disease in the patients, suggesting that oligodendroglial dysregulation is one of the first step of the disease (Philips et al., 2013).

Oligodendroglia could also become malignant cancerous cells. Glioma, the most common intracranial tumor, represents 81% of malignant brain tumors and could originate from astrocytes (astrocytomas), oligodendroglia (oligodendroglioma) or both of them (oligoastrocytoma). Gliomas are graded in 4 classes by the World Health Organisation (WHO) where glioblastoma (WHO type IV gliomas) are the most malignant and the deadliest, with only 0,05% to 4,7% of patients surviving the 5 years after their diagnosis [START_REF] Ostrom | The epidemiology of glioma in adults: a "state of the science" review[END_REF]. In general, gliomas with an oligodendroglial component have increased survival, as opposed to those with an astrocytic component [START_REF] Sant | Survival of European patients with central nervous system tumors[END_REF]. Due to a quicker diagnosis and new treatments, there has been an increasing trend in survival from oligodendroglioma in the population [START_REF] Nielsen | Incidence of and survival from oligodendroglioma in Denmark, 1943-2002[END_REF]. By example, WHO grade III oligodendrogliomas, which are not significantly receptive for radiotherapy alone, have been shown to be uniquely sensitive to chemotherapy. A study on a European cohort has reported a progression free survival of 2.0 years after radiotherapy plus administration of procarbazine, lomustine and vincristin chemotherapy compared with 1.1 years after radiotherapy alone (Lecavalier- [START_REF] Lathia | Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K[END_REF].

Multiple Sclerosis

MS is an autoimmune neurodegenerative disease affecting 2.5 million people worldwide, with 75% of the case being women. MS also has the highest prevalence both in developed and developing countries (Dobson and Giovannoni, 2018).

Even if the precise causes of MS remain unknown, several risk factors have been clearly identified. Smoking, for example, increases by 50% the risk of MS. There is also a clear corelation between the evolution of smoking habits of women and the MS sex ratio during over than one century, starting from 1900 [START_REF] Palacios | Smoking and Increased Risk of Multiple Sclerosis: Parallel Trends in the Sex Ratio Reinforce the Evidence[END_REF]. A symptomatic infection by the Epstein-Barr virus also doubles the chance to develop MS later on [START_REF] Handel | An Updated Meta-Analysis of Risk of Multiple Sclerosis following Infectious Mononucleosis[END_REF]. Several other environmental factors could interact with the individual genetic background and induce MS, like Vitamin D [START_REF] Manousaki | Low-Frequency Synonymous Coding Variation in CYP2R1 Has Large Effects on Vitamin D Levels and Risk of Multiple Sclerosis[END_REF] MS is an auto-immune disease where the myelin sheath of patients is targeted by their own immune system. It is composed of 3 phases: the asymptomatic, the prodromal and the symptomatic phase. During the early phases of the disease, the patient's CNS undergoes multiple localized demyelinating assaults by the immune system. It is estimated that for each clinical attack, approximately 10 'asymptomatic' lesions could be detected using magnetic resonance imaging (MRI). These lesions stimulate the differentiation of the residents OPCs and are quickly repaired but the new internodes are thinner and shorter [START_REF] Blakemore | Quantitative examination of internodal length of remyelinated nerve fibres in the central nervous system[END_REF][START_REF] Ginhoux | Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment[END_REF].

After 6 months of recovery, the newly remyelinated fibres are similar in length and thickness to developmentally myelinated axons [START_REF] Powers | Remyelination reporter reveals prolonged refinement of spontaneously regenerated myelin[END_REF].

Recent work uncovered genetic differences between patients, categorizing MS in two types [START_REF] Jia | Tensin2 is a novel mediator in thrombopoietin (TPO)-induced cellular proliferation by promoting Akt signaling[END_REF]. The relapsing-remitting MS (RRMS) is characterized by an alternance of phases where the immune system attacks, which could induce clinical symptoms (the relapsing phases), and of remyelination phases where the patient recovers from the attacks (the remitting phases). The primary progressive MS (PPMS), on the contrary, is based on an accumulation of neurological disabilities from the disease's onset. It was previously undetected probably due to the underrepresentation of PPMS in genome-wide association studies cohorts [START_REF] Jia | Tensin2 is a novel mediator in thrombopoietin (TPO)-induced cellular proliferation by promoting Akt signaling[END_REF].

In the two forms, neuroinflammation increases constantly as the disease progresses until patients reach the final phases where no repairs of the demyelinated lesions is possible.

As the disease progresses, OPCs fail to differentiate into OL and repair the myelin defects. Unprotected axons undergo neuronal death through Wallerian degenerescence, and symptoms, based on the lesion localisation, appear. MS differential diagnosis is notably based on monosymptomatic acute optic neuritis, often associated with severe visual loss), brainstem defects (strokes, space occupying lesions, brainstem encephalitis, …) or spinal cord syndromes (Oedema, Spinal cord compression, artery occlusion, …). The broad range of symptoms and their transiency in RRMS make MRI a powerful tool to directly detect MS lesions in patients after the appearance of several symptoms (Dobson et al., 2018).

CHAPTER IV : Tensins proteins

State of the art

General overview

The Tensin (Tns) is a family of focal adhesion proteins composed of 4 member in mammals: Tensin1 (Tns1), Tensin2 (Tns2 or TENC1), Tensin3 (Tns3) and Tensin4 (Tns4 or cten). Tensin 1, 2 and 3 proteins interacts respectively with the actin cytoskeleton and integrins through an actin binding domain (ABD) at their C Terminal part, and SH2-PTB (Phospho-tyrosine binding) domain at N terminal part, anchoring actin cytoskeleton to focal adhesion sites. Tns4 is a truncated version of the other Tensins, sharing the SH2-PTB domain with the other without any ABD. The N-Ter and C-ter part of Tensins are highly conserved but the medial part could vary a lot. This could explain the difference in specific functions that have been described over years for each tensins [START_REF] Liao | Tensinsemerging insights into their domain functions, biological roles and disease relevance[END_REF].

Interestingly, a unique homologous protein, blistery (by), has been described in the drosophila for the whole Tensin family, critical for the wings unfolding. In mammals, tensins are broadly expressed and can be found in many various tissues such as intestines, lung, heart, liver and even the brain. The first Tensin, Tns1, has been described by [START_REF] Davis | Presence of an SH2 domain in the actin-binding protein tensin[END_REF] as a 90 KDa protein fragment immunoprecipitated from a chicken smooth muscle actin extract. A decade later, three other members, Tns2 [START_REF] Chen | Tensin1 and a previously undocumented family member, tensin2, positively regulate cell migration[END_REF], Tns3 [START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF] and Tns4 [START_REF] Lo | Cten, a COOH-Terminal Tensin-like Protein with Prostate Restricted Expression, Is Down-Regulated in Prostate Cancer[END_REF] were identified.

Tns1 is a gene coding for 1735 amino acid proteins with an estimated size of 186 KDa, localized on chromosome 2 in humans. Tns1 is the biggest Tns and is characterised by the presence of a secondary Actin binding domain localised in its medial area (between amino acid 783 and 882). It's secondary ABD have been shown to regulate actin barbed end polymerisation (Lo SH et al, 1994). Over 20 splicing variants have been identified for Tns1 but surprisingly the regular full-length isoform, containing the full coding sequence, is predominantly expressed in the brain (Fig. 012). Tns2 is a gene coding for 1409 amino acid proteins with an estimated size of 153 KDa, localized on chromosome 12 in humans. Tns2 is the second Tensin discovered and was initially characterized as a phosphatase homologue to Tensin (1) and containing a C1 domain, which explains its first name: TENC1, for C1-Ten. A lot of Tns2 splicing variants have also been identified but only three of them have been found in humans. They all contain this exclusive C1 domain, absent in other Tns, and are expressed in brain tissues (Fig. 012). In order to understand theTns2 C1 domain function, a C1-GFP fusion protein has been expressed in human prostate adenocarcinoma cells.

Interestingly, C1-GFP has been detected in the nucleus, suggesting that this domain could be a Nuclear localisation signal [START_REF] Hafizi | Individual domains of Tensin2 exhibit distinct subcellular localisations and migratory effects[END_REF]. Furthermore, no full-length Tns2 endogenous expression has yet been characterised in the cell nucleus, so the real function of the Tns2 C1 domain remains to be defined.

Tns3 is a gene coding for a 1445 amino acid protein with an estimated size of 155KDa and an observed size of 180KDa, localized on chromosome 7 in humans. It contains 32 tyrosines, 13 of them could be potentially phosphorylated. Of the 12 Tns3 isoforms identified, two are found in brain tissues. Indeed, a small 60 Kda isoform containing only the Tns3 C Terminal part, that we will call small Tns3, is expressed in addition to the full-length protein. The small Tns3 isoform is very similar in structure to Tns4 and is also the most expressed isoform in total brain tissues (Fig. 012). Tns3 is the only Tensin enhancing GAP activity of DLC1 in osteoclasts [START_REF] Touaitahuata | Tensin 3 is a novel partner of Dock5 that controls osteoclast podosome organization and activity[END_REF], glioblastoma cell lines (Chen HY et al., 2017) and EGF-treated MCF10A nonmalignant mammary cells.

Tns4, formerly c-ten for C-terminus homology Tensin, is a gene coding for 745 amino acid proteins with an estimated size of 77 KDa, localized on chromosome 17 in humans. Tns4 shares only its C-terminus with the other Tensins. It also contains a specific focal adhesion binding (FAB), including a Nuclear Export sequence (NES).

Finally, Tns4 contains a Nuclear localisation signal (NLS) in its SH2-PTB domain and is currently the only Tensin member known to translocate into the nucleus. Only 4 Tns4 isoforms have been identified and none of them is predominantly expressed in brain tissues (Fig. 012). By its tridimensional structure, Tns4 could act as a competitive antagonist for the other Tensin binding to Focal Adhesion.

Tensins proteomic structures:

Tns1, Tns2 and Tns3 shared a similar C2-PTP at their N-terminus, named FAB-N (Nterminus Focal Adhesion Binding domain). These two domains are highly similar to those found in PTEN, a phospholipid binding protein. [START_REF] Zhang | Identification of Novel Families and Classification of the C2 domain Superfamily Elucidate the Origin and Evolution of Membrane Targeting Activities in Eukaryotes[END_REF]. Tns1 C2 domain contains a specific binding site to recruit at focal adhesion sites the protein phosphatase-1α. Tns1 PTP domain lacks an essential cysteine residue and is considered as an inactive PTP [START_REF] Alonso | The extended human PTPome: a growing tyrosine phosphatase family[END_REF]). Tns2's PTP domain is lacking an arginine residue in its signature motif but still has a PTP activity comparable to PTEN in vitro [START_REF] Koh | C1-Ten Is a Protein Tyrosine Phosphatase of Insulin Receptor Substrate 1 (IRS-1), Regulating IRS-1 Stability and Muscle Atrophy[END_REF]. Finally, even if the Tns3 motif contains both the arginine and the cysteine, it has yet never been described to have a PTP activity. The association of these two domains form the Tensins Actin binding domain 1 (ABD I). It is therefore important to precise that F-actin binding activity has only been attested for Tns1 and confirmed on chicken embryos (Lo SH et al, 1994;[START_REF] Chuang | Molecular cloning, expression, and mapping of the high affinity actincapping domain of chicken cardiac tensin[END_REF] but is only assumed by similarity of structure for Tns2 and for Tns3. Tns3 ABD I has also been reported to bind to Dock5 in osteoclast [START_REF] Touaitahuata | Tensin 3 is a novel partner of Dock5 that controls osteoclast podosome organization and activity[END_REF]. As present before, Tns1 possesses a secondary actin binding domain (ABD II). Tns1 fragments containing the ABD I or the ABD II are the only ones to bind to actin filaments. Exposition of ABD II fragment, but not ABD I, to actin barbed end filament show an actin polymerization delay similar to the full-length Tns1 (Lo SH et al, 1994) All Tensins shared their C-terminus domain, composed of a SH2 (Src Homology 2) and a PTB (Phosphotyrosine binding) domain closely spaced, named FAB-C (Cterminus Focal Adhesion Binding domain). This tandem allows Tns to bind to a great variety of proteins at focal adhesion such as integrins. Despite all these common protein domains, their middle region varies a lot across Tensins, granting each of them unique properties poorly studied to this date (Fig. 013).

Tensins known functions

Cell adhesion

Due to their binding to integrins, Tensins could be found under focal adhesion plates.

The highly dynamic Tns2 is enriched in focal adhesions at the leading edge of cells, whereas Tns3 is mainly found in fibrillar adhesions. Interestingly, Tns1 is equally distributed between focal and fibrillar adhesions [START_REF] Clark | Tensin 2 modulates cell contractility in 3D collagen gels through the RhoGAP DLC1[END_REF]. Tns1 could interact with Hic5 in a Src dependent manner, allowing fibrillar focal adhesion plates formation [START_REF] Goreczny | Hic-5 Regulates Fibrillar Adhesion Formation to Control Tumor Extracellular Matrix Remodeling through Interaction with Tensin1[END_REF]. Interestingly, AMPK KO in fibroblasts, both for α1 and α2 subunits, induce an increase of Tns1 and Tns3 expression resulting in more focal adhesion formation. Both Tns1 KO and Tns3 KO cells show a reduced fibrillar adhesion formation and fibronectin fibrillogenesis compared to WT cells levels, indicating that Tns1 and Tns3 plays a crucial role in focal adhesion genesis, negatively controlled by AMPK [START_REF] Georgiadou | AMPK negatively regulates tensin-dependent integrin activity[END_REF]. However, Tns1 KO , Tns2 KO or Tns3 KO cells do not exhibit issues in focal adhesion formation, suggesting that Tensin's function in cell adhesion could be compensated by other proteins [START_REF] Clark | Tensin 2 modulates cell contractility in 3D collagen gels through the RhoGAP DLC1[END_REF][START_REF] Rainero | Ligand-Occupied Integrin Internalization Links Nutrient Signaling to Invasive Migration[END_REF].

A specific Tns3 interaction in cell adhesion has also been reported. Osteoclast's resorbing activities are based on the formation of a podocytes belt named the sealing zone. Proteomic analyses have shown that Tns3, but not the other Tensins, bind with and activate Dock5 in the sealing zone. Interestingly, this interaction has been observed in individual podosomes [START_REF] Touaitahuata | Tensin 3 is a novel partner of Dock5 that controls osteoclast podosome organization and activity[END_REF]. More generally, the sealing zone formation is based on two separate phenomena. From one side the recruitment of Dock5 by Tns3 to activate Rac [START_REF] Nagai | p130Cas, Crk-Associated Substrate, Plays Important Roles in Osteoclastic Bone Resorption[END_REF], from the other side, Tns3 bind through it's SH2 domain to Src phosphorylated p130Cas, a focal adhesion protein, and link the actomyosin network to promote the podosome belt formation [START_REF] Touaitahuata | Tensin 3 is a novel partner of Dock5 that controls osteoclast podosome organization and activity[END_REF].

Finally, TP63 KO in non-malignant prostatic epithelial cells induce a reduction of Tns4 expression and impair cell adhesion. The ectopic re-expression of Tns4 in these cells restores cell adhesion, indicating that Tns4 also plays a crucial role in this phenomenon (Yang et al., 2016). TP63 KO in MCF-10A cells also show cell adhesion loss and a reduction of β1 integrin, β4 integrin and EGFR levels (Carroll DK et al., 2006). The re-expression of one these receptors partially restore cell adhesion, indicating that Tns4 interacts with these 2 integrins and the EGF receptor (Katz et al., 2007;[START_REF] Seo | Integrin-β4-TNS4-Focal Adhesion Kinase Signaling Mediates Keratinocyte Proliferation in Human Skin[END_REF], promoting cell adhesion very likely by stabilizing β1 integrin, β4 integrin, and EGFR.

Cell Proliferation

All Tensins have been extensively demonstrated to promote cell proliferation.

Endothelial cells from Tns1 KO mice or siRNA knock-down present reduced proliferation, migration, and decreased RhoA activity, which is restored after DLC1 silencing. These data suggest that Tns1 promotes endothelial cell proliferation and migration by inhibiting DLC1-GAP activity toward RhoA (Shih et al., 2015). Recent clinical studies have also shown that the downregulation of Tns1 expression by Prazosin in acute myeloid leukaemia cells was inhibiting their proliferation and survival. Indeed, the depletion of Tns1 impairs the PI3K/Akt/mTOR signaling pathway, inhibiting cell viability. On the contrary, the over-expression of Tns1 is sufficient to reverse the effects of prazosin on viability and apoptosis, confirming the positive role of Tns1 in cell proliferation and survival [START_REF] Sun | Prazosin inhibits the proliferation and survival of acute myeloid leukaemia cells through down-regulating TNS1[END_REF].

On the contrary, Tns2 has a general negative effect on cell proliferation. Indeed Tns2 overexpression reduces HEK293 cell proliferation and survival [START_REF] Hafizi | C1-TEN is a negative regulator of the Akt/PKB signal transduction pathway and inhibits cell survival, proliferation, and migration[END_REF] and its silencing promotes cell proliferation, colony formation and xenograft growth of HeLa cervical cancer cells and A549 lung cancer cells. But interestingly, Tns2 seems also to be required to mediate thrombopoietin-induced cell proliferation.

Thrombopoietin binding to its receptor c-Mpl induces Tns2 phosphorylation. Therefore, Tns2 KO in the megakaryocytic UT7-TPO cell line induces a reduction of cell proliferation and Akt signalling activation, which indicates that p-Tns2 have a positive role in cell proliferation by activating PI3K/Akt signaling (Jung AS, 2011).

Tns3 is highly expressed in tonsil derived mesenchymal stem cells. Tns3 KO induces an increased expression of the cyclin-dependent kinase (CDK) inhibitors p16 and p21 (CDKN2A and CDKN1A), and reduces cell proliferation [START_REF] Park | Tensin-3 Regulates Integrin-Mediated Proliferation and Differentiation of Tonsil-Derived Mesenchymal Stem Cells[END_REF]). An accumulation of CDK inhibitors p21 and p27 (CDKN1B) is observed in Tns4 KO nonmalignant prostate epithelial cells, as well as an attenuation of their proliferation [START_REF] Wu | Downregulation of C-Terminal Tensin-Like Protein (CTEN) Suppresses Prostate Cell Proliferation and Contributes to Acinar Morphogenesis[END_REF]. By interacting with E3 ubiquitin ligase c-Cbl, Tns4 also reduces ligand-induced EGFR degradation in cancer cells, which prolongs EGF signaling cascade and therefore promotes cell proliferation [START_REF] Hong | CTEN prolongs signaling by EGFR through reducing its ligand-induced degradation[END_REF]. MET and β1 integrin are also known to form a stable complex with Tns4 to prevent their internalization and degradation [START_REF] Muharram | Tensin-4-Dependent MET Stabilization Is Essential for Survival and Proliferation in Carcinoma Cells[END_REF]. Tns4 overexpression has also been linked to an increased cell proliferation. In human keratinocytes, it binds and activates β4 integrins, which triggers FAK and ERK activation and promotes cell proliferation [START_REF] Seo | Integrin-β4-TNS4-Focal Adhesion Kinase Signaling Mediates Keratinocyte Proliferation in Human Skin[END_REF], and expression of nuclear Tns4 enhance HeLa cells proliferation [START_REF] Hong | Identification of subcellular targeting sequences of Cten reveals its role in cell proliferation[END_REF].

The Tns3/Tns4 cell motility switch EGF signalling directly impacts Tns3 and Tns4 mRNA expressions. Interestingly, low EGF level are associated with high Tns3 and low Tns4 expression. This induces a strengthening of the actin cytoskeleton and therefore inhibits the cell motility. At high EGF levels, Tns4 is upregulated and its protein will compete with Tns3 to bind to the cytoplasmic tail of β1-integrin. As Tns4 does not bind to the actin cytoskeleton, this will induce a disintegration of actin stress fiber and a general relaxation of the actin cytoskeleton, favouring cell motility (Katz et al, 2007). It is interesting to note that OPCs, which are highly motile, are very sensitive to EGF for their survival and lose this dependence to growth factors when they become immature oligodendrocytes, where the Tns3 is highly expressed (Fig. 014). Katz et al, 2007 In addition to these competitive effects that will relax the actin cytoskelteon, EGFR activation also induces the dephosphorylation of FAK and p130Cas and, therefore, the dissociation of the Tns3-FAK-p130Cas complex. In parallel, EGFR activation induces its tyrosine phosphorylation, which will enhance Tns3 binding to this residue through it's SH2 domain [START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF]. So high levels of EGF will not only induce downregulation of Tns3 but also a change of its binding partners. Under EGFR activation, the main protein to which the residual Tns3 could bind is EGFR. Both Tns3 and Tns4 are able bind DLC1 through their SH2-PTB domains but Tns3's ABD, not present in Tns4, binds the SAM domain of DLC1. The DLC1 SAM interaction with Tns3 replaces its auto-inhibiting interaction with its own RhoGAP domain and will activate it. Activated DLC1 favours the RhoA coupling with GDP and therefore induces a cell resting state. As Tns4 doesn't have ABD, its binding to DLC1 does not activate DLC1 RhoGAP activity, which favours opposite cell migration through RhoA coupling to GTP (Fig. 015).

In conclusion, there is a Tns3/Tns4, EGF dependent, motility switch in cells. Low levels of EGF are associated with Tns3 high expression and Tns4 low expression. Tns3 is able in this situation to bind to integrins, stabilising the cytoskeleton, and to activate DLC1, which both induces a cell resting state. After EGFR activation, Tns3 is downregulated and Tns4 will replace it at integrins, relaxing the cytoskeleton, and at DLC1, which in contrast induce cell motility. The remaining Tns3 is captured by EGF Receptors, reducing again its chances to win the competition against Tns4.

External cues and mechanical sensing

β1-integrin (Itgb1), to which Tensins bind, are activated by an increased fibronectin density or Mn 2+ concentration, two external cues (Lin et al, 2013). This activation leads to the generation of cellular traction forces. Integrins have been extensively described as the main mechanotransducers of the cell (Schwarz and Gardel, 2012), especially β1-integrin, which is a major positive mediator of the cell mechanical state [START_REF] Baker | Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer[END_REF].

By following the movement of internalised microspheres inside AMPK KO fibroblasts, it appears that AMPK loss also increases the intracellular rigidity. Interestingly, both Tns1 and Tns3 expression are increased, suggesting they could bind and activate βintegrins without AMPK [START_REF] Georgiadou | AMPK negatively regulates tensin-dependent integrin activity[END_REF]. The activation of β-integrins promotes a wide range of intracellular processes including cell spreading, ECM assembly, mechanotransduction and intracellular stiffness. Therefore, it seems that the Tensin activity is indirectly regulated by external cues through AMPK inhibition.

In stiff substrates, Tns1 turnover slows down significantly, increasing cell spreading and migration [START_REF] Stutchbury | Distinct focal adhesion protein modules control different aspects of mechanotransduction[END_REF]. Integrin stabilization and the formation of fibrillar adhesions are both based on the interaction between Tns1 and Hic-5, which require Src and is sensitive to the substrate's stiffness [START_REF] Goreczny | Hic-5 Regulates Fibrillar Adhesion Formation to Control Tumor Extracellular Matrix Remodeling through Interaction with Tensin1[END_REF].

A recent study also shown a shortening of fibrillar adhesion length in Tns1 KO fibroblasts cultivated on stiffness-gradient gel, demonstrating the Tns1 requirement for stiffnessinduced adhesion elongation [START_REF] Barber-Pérez | Mechanoresponsiveness of fibrillar adhesions on stiffness-gradient gels[END_REF]. Finally, in response to a stretching stimulus, Src phosphorylates a tyrosine of p130Cas. Tns1 binds this p130Cas residue and anchors it to the actin cytoskeleton, thus promoting cell migration [START_REF] Zhao | Association between tensin 1 and p130Cas at focal adhesions links actin inward flux to cell migration[END_REF]. It is interesting to note that Tns3 has also been shown to bind to p130Cas [START_REF] Qian | The tensin-3 protein, including its SH2 domain, is phosphorylated by Src and contributes to tumorigenesis and metastasis[END_REF] but not to Hic-5. Together, these data show the implication of Tensins in sensing external mechanical cues.

Physio-pathology of Tensins dysfunction

Morphological defects of Tensins knockout in different organisms

Contrary to mammals, Drosophila and Caenorhabditis Elegans possess only one Tensin. In Drosophila, Tensin homologous gene is named blistery, highly similar to Tns4 structure and is mainly localized in focal adhesion sites. Blistery overexpression in flies wing disc during development increase JNK activity and induce a massive cell apoptosis [START_REF] Lee | blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development[END_REF] where as blistery KO flies present issues in the wing unfolding during development, due to a destabilisation of integrins adhesive contacts [START_REF] Tong | Genetically encoded calcium indicators and astrocyte calcium microdomains[END_REF]. On the contrary, C. elegans tensin is similar in sequence to Tns1. Interestingly, Tns KO doesn't seem to affect worm development or morphology but reduce pharyngeal pumping and their impair defecation cycles [START_REF] Bruns | Tensin regulates pharyngeal pumping in Caenorhabditis elegans[END_REF].

In Zebrafish, Tns1 ortholog knockout impairs heart mitral valve development [START_REF] Dina | Genetic association analyses highlight biological pathways underlying mitral valve prolapse[END_REF]. Tns1 KO mice present disruption of their cell-matrix junction and tubule cells polarity in the kidney. Even if Tns1 is expressed in a wide variety of tissues, no other organs were affected, implying a probable compensating effect of Tns2 and Tns3 for the loss of Tns1 [START_REF] Lo | Progressive Kidney Degeneration in Mice Lacking Tensin[END_REF]. In ICGN mouse, a mouse model for nephrotic syndrome, it appears that loss of Tns2 is associated with renal defects (Cho AR et al., 2006). Tns3 3'UTR mutation is also associated with higher nephropathy risks in the chinese Han population [START_REF] Feng | 3′UTR variants of TNS3, PHLDB1, NTN4, and GNG2 genes are associated with IgA nephropathy risk in Chinese Han population[END_REF], indicating the involvement of Tns3 in renal development.

Growth retardment in Tns3 KO animals models has been described both in mice (Chiang MK et al., 2005) and Pekin ducks (Deng MT et al., 2019). In mice, Tns3 KO leads to a sublethal phenotype, meaning an increased risk of premature death for homozygous mutated mice (data from the International Mouse Phenotyping Consortium; https://www.mousephenotype.org/) and various impairments during tissue development. Tns3 KO impaired villus formation in small intestine, alveologenesis in lung, with an increased air space in each alveola, and fewer proliferating cells in the resting zone of their tibias (Chiang MK et al., 2005), probably linked to Tns3-Dock5 control on osteoclast podosome organization and activity [START_REF] Touaitahuata | Tensin 3 is a novel partner of Dock5 that controls osteoclast podosome organization and activity[END_REF].

Tensins dysfunctions in cancer

Tensins dysfunctions have been extensively reported in cancer studies. Each of them have been reported to bind to deleted-in-liver-cancer protein 1 (DLC1). DLC1 binding to Tns3 through its actin binding domain induces the RhoGAP activation that suppresses tumor growth. Both Src and Akt are able to phosphorylate DLC1, which leads to destabilisation of the Tns3-DLC1 interaction and favours DLC1 sterile alpha motif and DLC1 Rho-GAP domain autoinhibitory interaction. The decrease in RhoGAP activation reduces DLC1 anti-tumorigenic properties, which explain why Src or Akt inhibitors are able to reduce tumor growth and metastasis formation in a DLC1dependent manner and stabilise DLC1-Tns3 interaction (Cao et al., 2012).

Tns1 binding to DLC1 through its C2, SH2 and PTB domains is also known to enhance RhoA activity and induce tumor suppression (Shih et al., 2015). Tns4 also binds to DLC1 via its SH2 and inhibition of the DLC1-Tns4 interaction greatly impairs DLC1 tumor suppressor activity (Liao et al., 2007). Tns2, which is by far the least studied Tns in oncology, also interacts with DLC1 through its PTB domain. Disruption of this interaction also leads to reduced Rho GTPase activity and less tumor suppression [START_REF] Chen | Solution Structure of the Phosphotyrosine Binding (PTB) Domain of Human Tensin2 Protein in Complex with Deleted in Liver Cancer 1 (DLC1) Peptide Reveals a Novel Peptide Binding Mode[END_REF]. Therefore, it is clear that Tensins impacts tumorigenicity through the regulation of DLC1 All Tns are also dysregulated in gastro-intestinal tract associated cancers (gastric, intestine, liver, bladder and pancreas cancers). This finding is coherent with Tensin's pattern of expression, both at the mRNA and the protein levels, from Allen Brain Atlas (https://mouse.brain-map.org/) and the GTEX database

[CP1]
(https://gtexportal.org/home/). Analysis from Gene Expression Omnibus (GEO) microarray data has designed Tns1 as one of 4 top gene hub genes whose expression was dysregulated in gastric cancers [START_REF] Yang | Identification of Potentially Functional CircRNA-miRNA-mRNA Regulatory Network in Gastric Carcinoma using Bioinformatics Analysis[END_REF]. A recent study of Tensins' expression in gastric cancers showed that Tns1 was more often observed in metastatic tumours compared to the others. Tns2 was also more common in moderately differentiated tumours and those with peritumoral inflammation. Finally, Tns3 was more prevalent in moderately differentiated tumours than in the poorly or non-differentiated ones (Nizioł et al., 2021).

Tns1, Tns3 and Tns4 have been studied in breast cancer, the first worldwide cause of cancer in 2020 [START_REF] Sung | Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[END_REF]. The involvement of Tns1 is yet debated. Tns1 is a downstream target of MaTAR lncRNA, a long non-coding RNA known to play a role in mammary tumorigenicity, and MaTAR KO results in Tns1 reduced expression and a decreased tumorigenicity (Chang KC et al., 2020). A high expression of miR-548j in breast cancer cells, which targets Tns1 mRNA, is also correlated with higher invasiveness and Tns1 shRNA inhibition suppress this pro-tumorigenic effect of miR-548j [START_REF] Zhan | MicroRNA-548j functions as a metastasis promoter in human breast cancer by targeting Tensin1[END_REF]. However, Tns1 expression studies have not yet shown significant changes in breast cancer tumours compared to non-cancerous tissues, both in mRNA [START_REF] Kotepui | Quantitative Real-Time RT-PCR of ITGA7, SVEP1, TNS1, LPHN3, SEMA3G, KLB and MMP13 mRNA Expression 202 in Breast Cancer[END_REF] and in lncRNA levels [START_REF] Behtaji | Identification of oxytocinrelated lncRNAs and assessment of their expression in breast cancer[END_REF].

Therefore, it is more likely that Tns1 has a limited involvement in breast cancer tissues.

Tns3 expression is reduced by the apport of SUV420H2, a H4K20-specific methyltransferase downregulated in various cancers, in MDA-MB human breast cancer cells, reducing cell invasiveness [START_REF] Shinchi | SUV420H2 suppresses breast cancer cell invasion through down regulation of the SH2 domain-containing focal adhesion protein tensin-3[END_REF]. It appears that Tns3

expression is reduced in breast cancer tissues (Veß A et al., 2017) whether Tns4 is increased (Katz M et al., 2007). Therefore, the Tns3/Tns4 switch, previously described, favours cell motility in breast cancerous tissues and is associated with a higher tumour invasiveness and metastasis.

Lung and colorectal cancers are also well represented in Tns oncogenicity studies.

Tns1 expression has been linked, in association with transgrelin (another actin-binding protein) with colorectal cancer [START_REF] Zhou | Elevated transgelin/TNS1 expression is a potential biomarker in human colorectal cancer[END_REF]. Tns1 is upregulated in non-small cell lung cancer and miRNA Tns1 inhibition impairs their proliferation [START_REF] Duan | miR-152/TNS1 axis inhibits non-small cell lung cancer progression through Akt/mTOR/RhoA pathway[END_REF]. Tns2 silencing in A549 human lung cancer cells and HELA cells up-regulates the activities of Akt, Mek, and IRS1, and increases tumorigenicity (Hong SY et al., 2016). Single nucleotide polymorphism in Tns3 3' untranslated region is associated with lung lymphatic metastasis [START_REF] Yan | Single nucleotide polymorphism in the 3′ untranslated region of LPP is a risk factor for lung cancer: a case-control study[END_REF] and cases of EGFR fusion with

Tns3 have already been reported in lung adenocarcinoma [START_REF] Zhang | Decitabine inhibits the proliferation of human T-cell acute lymphoblastic leukemia molt4 cells and promotes apoptosis partly by regulating the PI3K/AKT/mTOR pathway[END_REF].

Finally, Tns4 is upregulated in colorectal animal models [START_REF] Raposo | Investigating TNS4 in the Colorectal Tumor Microenvironment Using 3D Spheroid Models of Invasion[END_REF]. As described before, this upregulation increases cell motility by playing on the Tns3/Tns4 switch but the calpain, a Tns4 inhibitor, seems to act in colorectal cancer without affecting this switch [START_REF] Thorpe | Multiple pathways regulate Cten in colorectal cancer without a Tensin switch[END_REF]. Tns4 is highly expressed in the lung (Wang X et al., 2020) and high levels of Tns4 are again associated with higher tumour cell invasion [START_REF] Bennett | C-terminal tensin-like protein mediates invasion of human lung cancer cells and is regulated by signal transducer and activator of transcription 3[END_REF].

More generally, Tns are often dysregulated during oncogenesis as they play a crucial role on cell motility and migration. Overexpression of Tns4 has been observed in tumours derived from breast, colon, lung, stomach, skin and pancreas. The growth factors and cytokines associated with those cancers are known to increase Tns4 expression, which is correlated with an increased tumorigenicity [START_REF] Lo | Tensin: a potential link between the cytoskeleton and signal transduction[END_REF]). It appears that reduced levels of Tns3 and increased levels of Tns4 in cancer tissues relax the actin cytoskeleton and favour metastasis formation by affecting the Tns3/Tns4 motility switch. Furthermore, Tns1 and Tns4 also play crucial roles in the epithelial-mesenchymal transition, a process where stable epithelial cells convert themselves into invasive mesenchymal cells [START_REF] Asiri | Cten promotes Epithelial-Mesenchymal Transition (EMT) in colorectal cancer through stabilisation of Src[END_REF]. Finally, Tns3 is the only Tensin described to be involved in glioblastoma. This is of particular interest because glioblastoma, or WHO grade IV astrocytoma, are extremely aggressive tumours derived from glial cells. Musashi1, a RNA binding protein broadly expressed in various cancer tissues including glioblastoma, binds to Tns3 mRNA to repress its translation in this context. All of these data indicates that Tensins are good targets to develop new treatments to impair tumour metastasis, especially Tns3 for glioblastomas.

Material and methods

Animals and genotyping

Generation of Tns3 KO mice lines by CRISPR Cas9

Mice were generated at the ICM mice facility in collaboration with Phillipe RAVASSARD. In a mice egg cell were microinjected with the Cas9 protein, a crRNA, a tracrRNA and a targeting vector containing a DNA sequence surrounded by 2 homology arms in order to be inserted by the CRISPR-Cas9 machinery by Homology Directed Repair (HDR). Egg cells were then implanted in receptor BL6;JK mice, to give birth to F0 heterozygous mutated pups. Each pup was genotyped by Sanger sequencing on PCR amplify DNA segment to detect mutation at the mutation site.

We first designed this approach to integrate a tag in Tns3 coding sequence by HDR but unfortunately, no knockin mice were generated in this experiment. Instead, Non-Homologous End Joining (NHEJ) occurred, randomly inserting or suppressing nucleotides and generating different Tns3 KO mutated alleles: 1 nucleotide insertion, 4 nucleotide deletion, 14 nucleotide deletion. Mice were then crossbred in order to generate homozygous mice for each mutation.

Tns3 V5 ; Rosa STOP-Cas9-GFP mice Tns3 V5 mice were generated at Currie Institute Animal Facility with a similar approach.

Two different sites for the V5-tag insertion were tested, one at the 3' part of the protein and the other at 5' terminus. 100 mice embryos were microinjected (50 with the 5' plasmid and 50 with the 3'). 55 of them succeed to birth, with only 8 injected with the 5' plasmid and 47 with the 3', and only 27 survived the first postnatals weeks, with only 3' mutated mice. Tns3 flox/WT heterozygous mice were bred together in order to obtain homozygous Tns3 flox/flox mice then with a PdgfRa-CreER T ; Rosa26 YFP mice.

Oligodendroglia is known to be altered by the Cre recombinase expression in PdgfRa-CreER T homozygous mice, so Tns3 WT ; PdgfRa-CreER T/WT ; Rosa YFP were exclusively used as controls. This mice line allows us to induce an OPC-specific deletion of Tns3 with a subcutaneous tamoxifen injection. Quantifications were performed in doubleblind and only on sagittal brain slices presenting a fully formed fimbriae to study similar brain areas.

Flox Tns3 knockout by tamoxifen injection

30μl from a 20mg/ml tamoxifen solution (diluted in Corn Oil) was injected at P7 in Tns3 flox ; PdgfRa-CreER T ; Rosa YFP (experimental) or Tns3 WT ; PdgfRa-CreER T ;

Rosa YFP (Control) pups to induce Tns3 KO. After 14 or 21 days, mice were anesthetized with a lethal dose of pentobarbital, then perfused with 30mL of paraformaldehyde 2% (diluted in fresh PBS). Brains were left overnight in a 10%

sucrose solution (diluted in PBS), then transferred in a 20% sucrose solution for half a day. Brains hemispheres were separated and frozen in OCT using -80°C isopentane.

For the Live imaging experiment, pups were instead injected at P5 with 30μl of a 20mg/ml tamoxifen solution and sacrificed at P7.

Electroporation

The postnatal brain electroporation protocol described in Boutin C et al., 2008 was adapted to target the dorsal SVZ. P2 pups were cryo-anesthetized for 2min on ice and 1,5 μl of plasmid were injected into the left ventricle using a glass capillary. Plasmids were injected at the concentration of 5ug/μl. Electrodes (Nepagene CUY650P10) were applied in the dorso-ventral axis with the positive pole dorsal. Five electric pulses of 100V, 50ms pulse on, 850ms pulse off were applied using a Nepagene CUY21-SC electroporator. Pups were immediately warmed up in a heating chamber and brought

to their cages at the end of the experiment. Tns3 V5 ; Rosa STOP-Cas9-GFP mice were injected only with a Tol2-pCAG-gRNA plasmid containing a Tol2 sequence, to allow CRISPR tools genomic insertion and gRNA sequences. We used a alternatively a gRNA targeting a region downstream the start codon of the Tns3 gene (exon6 gRNA)

or two gRNA targeting upstream and downstream regions of either Tns3 exon9 (exon9 gRNA) or the whole Tns3 locus . A plasmid without gRNA (empty condition) was used as a Control for half of the electroporated littermates.

MACS

Dissociation of cortex and corpus callosums from V5 mice was done using neural tissue dissociation kit (P) (Miltenyi Biotec;. Briefly, cortices from P7, P14 or P21 mices were dissected then dissociated using Miltenyi MACS dissociator (Miltenyi Biotec;. Dissociated cortices were then filtered using Smartstainer 70um (Miltenyi Biotec;. For P14 and P21 samples, an DMEM/F12 (Gibco) up to 50ml, 250µl HEPES buffer 1M (Gibco), 666µl glucose 45% (Sigma), 0.5ml penicillin/streptomycin (Sigma), 0.5ml N2 supplement (Gibco), 1ml B27

(Gibco), EGF to a final concentration 20ng/ml (Peprotech), FGF to a final concentration 10ng/ml (Peprotech) and Insulin to a final concentration 20µg/ml (Sigma),and mechanically dissociated using a pipette. Approximately 1×10^6 cells/mL were cultured in 5 ml NSC fresh medium in a 25 ml flask, and maintained at 37 °C in a humidified atmosphere of 5% CO2. Under these proliferating conditions, the cells grow as free-floating neurospheres. After 5 days of proliferation, Neurospheres were dissociated by accutase treatment followed by a mechanical dissociation with a pipet (Passage 1). Neurospheres were used directly or freezed in DMSO for a later use.

OPCs differentiation

Legend 8: Protocol for OPC differentiation in vivo culture

Before the culture, neurospheres were pre-incubated in flask for 3 days with OPCs proliferation medium (NSC fresh medium supplemented with PDGFa at the final concentration of 10ng/ml). This condition will favor NCS specification toward the oligodendroglial lineage. After dissociation, NSCs were plated at 30000 to 40000 cells per mm 2 in glass coverslip previously coated with poly-L-Ornithine (Sigma Aldrich, P4957-50ML). NSCs were left 3 to 4 days in OPCs proliferation medium to proliferate.

Differentiation was induced by switching the culture medium for growth factors free differentiating medium. Cells were fixed with 4% paraformaldehyde diluted in PBS for 10 minutes and processed for immunostaining characterization.

Tns3 KO with viral vectors

AAV Tns3KO in NSCs from Tns3 flox ; Pdgfra-CreER T ; Rosa YFP mice

We design twoo AAV9 vectors to target Tns3 flox ; Pdgfra-CreER T ; Rosa YFP neurospheres. AAV were produced at the ICM vectorology facility, with one containing an empty gRNA ("AAV Empty") and the other, the two 5' and 3' exon9 gRNA ("AAV exon9'") in addition to the typical AAV sequence. AAV were used at MOI of 500 000, determined empirically. AAV concentrations were determined by AAV ITR sequence qPCR, following the protocol described in Aurnhammer et al., 2012 (PMID: 22428977).

specification medium (NSC fresh medium + Pdgfa) to favorise the NSCs specification toward the oligodendrocyte lineage. Neurosphere have been dissociated mechanically with a pipette and chemically with an acute treatment and plate and previously P-L-O coated coverslips at the concentration of 40 000 cells/mm 2 . We wait 1 hour, in order to let the NSCs adherate to the coverslip, then we discard the medium and add 300uL of a fresh specification medium with viral vectors (AAV vectors for Tns3 V5 ;Rosa STOP- Cas9-GFP NSCs and Adeno-Cre or Adeno-GFP vectors for Tns3 flox ;PdgfRa-CreER T ;Rosa YFP NSCs). We wait overnight and 700uL of new specification medium.

Cells were left to proliferate for 2 to 3 days, then medium was discarded and replaced by differentiation medium (NSC fresh medium w/o growth factors). Cells were left to differentiate for 1 to 5 days and then have fixed with an incubation with a 4% PFA (diluted in PBS) solution during 10 minutes. Cells were then stained and analysed as described before.

Live cell Imaging

Legend 11: In vitro timelapse Live Imaging protocol for MACSorted Tns3 flox OPCs

Same mediums have been used to specify the neurosphere toward the oligodendroglial lineage and make the OPCs proliferate then differentiate into OL, but in order to avoid cytotoxicity, all of them have been prepared without phenol Red.

Tns3 flox ;PdgfRa-CreER T ;Rosa YFP pups have been injected at P5 and OPCs from their

Cortex have been MACSed at P7 using anti-Pdgfra coupled magnetics beads. This

OPCs have been plated at 40 000 cells/mm2 in PLO coated IBIDI glass bottom box.

OPCs were left in the specification medium for 2 days to amplify and 2 days in the differentiation medium to start their differentiation.

At 2 days of differentiation, we discard the old meidum to get rid of cell debris then we add 250uL of fresh differentiation medium. Cells were imaged using Axio-Observer 7

provided by David AKBAR from CELIS ICM cell culture facility during 2x24h, with one picture taken every 10 minutes in phase contrast and in the GFP channel. After the timelapse, cells were fixed in 4% PFA, stained and imaged as described before.

Demyelination Lesions

Before surgery, adult (2-3months) WT mice were weighted and anesthetized with an intraperitoneal injection of a ketamine (0.1 mg/g) and xylazine (0.01 mg/g) mix. An analgesic (buprenorphine, 30 mg/g) was administered intraperitoneally to prevent postsurgical pain. Focal demyelinating lesions were induced by stereotaxic injection of 1μl of lysolecithin solution (LPC, Sigma, 1% in 0.9%NaCl) into the corpus callosum (CC; at coordinates: 1 mm lateral, 1.3 mm rostral to bregma, 1.7 mm deep to brain surface) using a glass-capillary connected to a 10μl Hamilton syringe. Animals were left to recover in a warm chamber before being returned into their housing cages.

Objectives and Hypothesis

Our lab aimed to understand how oligodendrogenesis is regulated in order to provide insights for new therapeutic approaches in the neurodegenerative context. In MS patients, OPCs differentiate into OLs to repair the demyelinated lesions and a better understanding of the natural oligodendrogenesis mechanisms could shed light on new ways to cope and treat these diseases.

In a previous study, the team screened for the targets of key oligodendroglial factors, both in OPCs or OLs, using genome-wide transcriptome analysis and chromatin immuno-precipitation. We identified several gene candidates, like Chd7 and Chd8 that we and collaborators characterized to be key chromatin remodelers during oligodendrogenesis (He et al, 2016;Marie C et al, 2018). Among them, I focussed on Tns3 for my work, an adaptor protein between the integrins and the cytoskeleton, and which is strongly but transiently expressed during iOLs maturation.

Any induction of a stable mutation in the Tns3 gene is surprisingly challenging. In humans, human genome studies show a small prevalence of Tns3 mutation in the human population [START_REF] Karczewski | The mutational constraint spectrum quantified from variation in 141,456 humans[END_REF][START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF]. Tns3 SNP has been linked to lung carcinoma [START_REF] Yan | Single nucleotide polymorphism in the 3′ untranslated region of LPP is a risk factor for lung cancer: a case-control study[END_REF] or nephropathy [START_REF] Feng | 3′UTR variants of TNS3, PHLDB1, NTN4, and GNG2 genes are associated with IgA nephropathy risk in Chinese Han population[END_REF] in the Chinese Han population. Tns3 KO animal models have all shown strong phenotypic impairment with reduced growth (Chiang et al, 2005) and body mass [START_REF] Deng | Genome-wide association study reveals novel loci associated with body size and carcass yields in Pekin ducks[END_REF]. Finally, Hatem Hmidan, who started this project, demonstrated during his PhD that the Tns3 bgeo mice model was still expressing Tns3 protein in brain cells. These preliminary data indicates a crucial role of Tns3, but its role in CNS, particularly oligodendrocytes, is however completely undocumented.

In this project, I aimed 1) to fully characterized the expression of Tns3 all along the oligodendrogenesis in (re)myelination context, 2) to decipher whether Tns3 is required for the production of oligodendrocytes in the brain and 3) to provide firsts insights to explain the function of Tns3 protein in OLs, in order to provide knowledges on the natural generation of OLs and help to design new efficient remyelinating therapies for neurodegenerative diseases. [START_REF] Funfschilling | Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity[END_REF]Lee et al., 2012). Furthermore, oligodendroglia and myelin pathologies have been recently link, not only to the development of glioma [START_REF] Liu | Mosaic Analysis with Double Markers Reveals Tumor Cell of Origin in Glioma[END_REF], but to neurodevelopmental [START_REF] Castelijns | Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients[END_REF][START_REF] Phan | A myelin-related transcriptomic profile is shared by Pitt-Hopkins syndrome models and human autism spectrum disorder[END_REF], neurodegenerative [START_REF] Bryois | Genetic identification of cell types underlying brain complex traits yields insights into the etiology of Parkinson's disease[END_REF][START_REF] Grubman | A single-cell atlas of entorhinal cortex from individuals with Alzheimer's disease reveals cell-type-specific gene expression regulation[END_REF], and psychiatric [START_REF] Nott | Brain cell type-specific enhancer-promoter interactome maps and diseaserisk association[END_REF] diseases.

INTRODUCTION

OPCs originate from focal regions of germinal zones in the brain and spinal cord [START_REF] Spassky | Multiple restricted origin of oligodendrocytes[END_REF][START_REF] Tekki-Kessaris | Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon[END_REF], and through proliferation and migration occupy the whole CNS before starting to differentiate into myelinating OLs [START_REF] Rowitch | Glial specification in the vertebrate neural tube[END_REF]. Unlike most precursor cells, OPCs remain abundant in the adult CNS, being one of its neural cell subtypes [START_REF] Ffrench-Constant | The oligodendrocyte-type-2 astrocyte cell lineage is specialized for myelination[END_REF][START_REF] Suzuki | Multiple cell populations in the early postnatal subventricular zone take distinct migratory pathways: a dynamic study of glial and neuronal progenitor migration[END_REF]. OPCs need to keep a tight balance between proliferation, survival, and differentiation. This balance is crucial to maintain the OPC pool while contributing to myelin plasticity in adult life, and to remyelination in diseases such as multiple sclerosis (MS). Being an inflammatory degenerative disease of the CNS, MS therapies are mainly based on immuno-suppressant, immuno-modulatory and anti-inflammatory drugs [START_REF] Compston | Multiple sclerosis[END_REF][START_REF] Kieseier | Treatment and treatment trials in multiple sclerosis[END_REF] but no treatment are yet available to directly promote remyelination. Demyelinated plaques can be normally repaired by endogenous OPCs especially in early stages of the disease but with time this repairing process become more and more inefficient with aging and OPC differentiation seems to be partially impaired [START_REF] Chang | Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis[END_REF][START_REF] Compston | Multiple sclerosis[END_REF]Neumann et al., 2019b). Therefore, understanding the mechanisms inducing OPC differentiation is a critical event for successful remyelination and myelin pathologies.

A large diversity of extrinsic signals [START_REF] Bergles | Oligodendrocyte Development and Plasticity[END_REF] as well as many intrinsic factors, including transcription factors (TFs, reviewed in [START_REF] Emery | Transcriptional and Epigenetic Regulation of Oligodendrocyte Development and Myelination in the Central Nervous System[END_REF]), and chromatin remodelers (reviewed in (Parras et al., 2020)), has been involved in OPC proliferation, survival, and differentiation. However, the mechanism for how these signals are integrated in the nucleus to balance OPC behavior is largely unknown. OPC differentiation requires profound changes in chromatin and gene expression [START_REF] Emery | Transcriptional and Epigenetic Regulation of Oligodendrocyte Development and Myelination in the Central Nervous System[END_REF][START_REF] Küspert | SomethiNG 2 talk about-Transcriptional regulation in embryonic and adult oligodendrocyte precursors[END_REF][START_REF] Wheeler | Extracellular cues influencing oligodendrocyte differentiation and (re)myelination[END_REF]. TFs, such as Olig2, Sox10, Nkx2.2 or Ascl1, are key regulators of OL differentiation by directly controlling the transcription of genes implicated in this process (Nakatani et al., 2013;Qi et al., 2001;Stolt et al., 2002;Yu et al., 2013) but being already expressed in OPCs, it is still unclear how these TFs control the induction of differentiation. A growing body of evidence suggests that some of these TFs work together with chromatin remodeling factors during transcriptional initiation/elongation to drive robust transcription [START_REF] Zaret | Pioneer transcription factors, chromatin dynamics, and cell fate control[END_REF]. Accordingly, Olig2, Sox10 TFs have been shown to cooperate with chromatin remodelers such as Brg1 (Yu et al., 2013), Chd7 (He et al., 2016;Marie et al., 2018), Chd8 (Marie et al., 2018;[START_REF] Zhao | Dual Requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Repair[END_REF], and EP400 [START_REF] Elsesser | Chromatin remodeler Ep400 ensures oligodendrocyte survival and is required for myelination in the vertebrate central nervous system[END_REF], to promote the expression of OL

RESULTS

Tns3 is a direct target gene from key regulators of oligodendrocyte differentiation

There is a major need to develop treatments enhancing the process of myelin repair that requires a better understanding of the mechanisms promoting effective oligodendrocyte differentiation and remyelination (Neumann et al., 2019b). To find new factors involved in oligodendrocyte differentiation, we screened for gene targets of key regulators of oligodendrogenesis, Olig2 transcription factor, and Chd7 and Chd8 chromatin remodelers (He et al., 2016;[START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF]Lu et al., 2000;Marie et al., 2018;Parras et al., 2020;Yu et al., 2013;[START_REF] Zhao | Dual Requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Repair[END_REF]. To this aim, we generated and compared the genome-wide binding profiles for these key regulators in oligodendroglial cells. After purifing O4 + cells by magnetic cell sorting (MACS) from postnatal mouse brain cortices, composed of both PDGFRa + OPCs and Nkx2.2 + /CC1 + immature oligodendrocytes (iOLs), we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) for Olig2 and key regulatory histone marks (H3K4me3, H3K4me1, H3K27me3, and H3K27ac; Fig. S1A), and combined them with our previously generated datasets for Chd7 and Chd8 (Marie et al., 2018). Olig2 was found bound in 16,578 peaks corresponding to 8,672 genes (Fig. S1B), including key regulators such as Ascl1, Sox10, Myrf, Chd8, and Smarca4/Brg1. Histone marks activity code (Fig. S1C) showed that Olig2 bound to regulatory elements (60% being promoters and 40% enhancers) both in active and more poised/repressive state (Fig. S1D). We obtained 1764 peaks commonly bound by Olig2, Chd7 and Chd8, from which 47% (832 peaks) were found at active promoters (H3K4me3/H3K27ac marks) corresponding to 654 protein-coding genes (Fig. S1E). Among these genes, Tensin 3 (Tns3), coding for a cell adhesion protein deregulated in certain cancers [START_REF] Martuszewska | Tensin3 Is a Negative Regulator of Cell Migration and All Four Tensin Family Members Are Downregulated in Human Kidney Cancer[END_REF], was the third with the highest expression in iOLs relative to other brain cell-types [START_REF] Zhang | An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex[END_REF]; Fig. 1D). Olig2, Chd7 & Chd8 binding together in Tns3 alternative promoters (for the 2 isoforms), together with active marks (Fig. 1A) To assess whether Tns3 is a direct target of these key regulators, we interrogated the transcriptomes of postnatal OPCs/iOLs (P7) purified from brain cortices of Chd7iKO (Pdgfra-CreER T ; Chd7 flox/flox ) and Chd8cKO (Olig1 Cre ; Chd8 flox/flox ) and their respective controls (Marie et al., 2018;[START_REF] Zhao | Dual Requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Repair[END_REF]. Indeed, Tns3 transcripts were largely downregulated upon acute deletion of each of these factors in postnatal OPCs (Fig. 1B,C), indicating that Tns3 expression in OPCs/iOLs is directly controlled by Chd7 and Chd8, key regulators of oligodendrocyte differentiation.

We then fetch to characterize Tns3 expression pattern in the brain. We first verified that the high expression levels of Tns3 transcripts found in iOLs compared to its low expression in other oligodendroglial stages or brain cells in the postnatal mouse brain (Fig. 1D; [START_REF] Zhang | An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex[END_REF], was paralleled by the sparse labelling detected by in situ hybridization with Tns3 probes, enriched in white matter regions of the postnatal and adult brain (Fig. 1E; Allen Brain atlas, https://portal.brain-map.org/). To better characterize Tns3 expression pattern in oligodendroglia, we took advantaged of recent resources profiling the transcriptome of single cells (scRNA-seq) including oligodendrocyte lineage cells at embryonic, postnatal, and adult stages (Marques et al., 2016[START_REF] Marques | Transcriptional Convergence of Oligodendrocyte Lineage Progenitors during Development[END_REF]. We selected the neural stem/progenitor cells and oligodendroglial cells of these datasets and integrated them following Seurat3 strategy (Stuart et al., 2019). Unsupervised clustering and visualization of cells in two dimensions (with uniform manifold approximation and projection, UMAP), identified nine different clusters following a differentiation trajectory from neural stem cells (NSCs) to mature oligodendrocytes (MOLs, Fig. 1F). Based on cell subtype specific markers, we could identify these clusters as (Fig. 1F,H Interestingly, Tns3 transcripts were strongly expressed in both iOL1 and iOL2 clusters, similar to the proposed iOL markers Itpr2 and Enpp6 (Fig. 1G,H), indicating that Tns3 expression is strongly upregulated during oligodendrocyte differentiation and downregulated in mature oligodendrocytes.

Tns3 protein is enriched in the cytoplasm and processes of immature oligodendrocytes

Given the high expression level of Tns3 transcripts in immature oligodendrocytes, we then fetch to characterize Tns3 protein expression pattern in the postnatal brain using commercial and homemade Tns3-recognizing antibodies. Optimization of immunofluorescence protocols demonstrated signal in CC1 + oligodendrocytes in the postnatal brain with four different antibodies (P24, Fig. S1). To our surprise, while all antibodies show signal localized to the main iOL processes (Fig. S1A-E), one Tns3 antibody (Millipore) also presented a strong nuclear signal never reported for Tns3 localization in other tissues (lung, liver, intestine, mammary cells, etc.) [START_REF] Cao | A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration[END_REF]Katz et al., 2007;[START_REF] Nishino | Distinct Distribution of the Tensin Family in the Mouse Kidney and Small Intestine[END_REF]. To better characterize Tns3 protein expression pattern and its subcellular localization, we generated a knock-in mice tagging Tns3 C-terminal side with V5-tag (Tns3 Tns3-V5 mice) by microinjecting mouse zygotes with a single strand oligodeoxinucleotide (ssODN) containing V5 sequence together with Cas9 protein and a gRNA targeting Tns3 stop codon region (see methods and Fig. S2A-C). We first, validated by immunofluorescence that Tns3-V5 protein in Tns3 Tns3-V5 mice presented the expression pattern reported for Tns3 in other tissues such as the lung and the kidney (Fig. S2D,E). We then characterized Tns3 protein expression in oligodendroglia using V5 antibodies, and found that Tns3 protein can be detected at high levels in the cytoplasm and main processes of CC1 + iOLs but not in their nucleus (Fig. 2A). We then used an antibody recognizing Itpr2, suggested iOLs marker (Marques et al., 2016), and showed that Tns3 largely overlapped with Itpr2, despite the lower quality of immunofluorescence obtained with Itpr2 antibody (Fig. 2B). Using Nkx2.2 and Olig1 cytoplamic expression distinguishing iOL1 and iOL2 respectively, we could find that high levels of Tns3 are detected in iOL1s (Nkx2.2 + /Olig1 -cells) and a fraction of iOL2s (Nkx2.2 -/Olig1 cytoplamic cells; Fig. 2C), suggesting that Tns3 protein expression peaks in early iOLs. Moreover, comparison with Opalin protein localized in the cell body, processes, and myelin segments of oligodendrocytes, showed that Tns3 levels decreased with increasing levels of Opalin, and that myelinating oligodendrocytes, that is Opalin + /CC1 + cells presenting myelinated segments (internodes), had downregulated Tns3-V5 expression (Fig. 2D, arrowheads). Therefore, the characterization of Tns3 Tns3-V5 mice suggests that Tns3 protein present at strong levels in early immature oligodendrocytes. As Tns3 isoforms can be detected at the transcript level in the human brain (Fig. S2F, GTEX project, gtexportal.org/home/gene/TNS3), we performed Western blot analysis using V5

antibody in purified O4+ cells from P7, P14, and P21 Tns3 Tns3-V5 brains to assess its specificity. Indeed, we could detect both the full-length and the Tns3 short 3-term isoforms at P7 and P14 containing many iOLs, but not at P21 having mainly mOLs, validating the specificity of V5 antibodies to recognize Tns3-V5 protein. Eventually, we found a Tns3 antibody recognizing the c-Term of Tns3 protein (Sigma Ct) giving optimal immunofluorescence labeling, and confirming Tns3 expression pattern obtained with V5 antibodies. Combined with Nkx2.2 and Olig1 immunofluorescence allowed us to distinguishing with more precision different stages of immature oligodendrocytes. Indeed, we could find that Tns3 is strongly detected in the cytoplasm and main cellular processes of all iOL1s, defined as Nkx2.2 high /Olig1 -cells having a round nucleus and small cytoplasm (Fig. 2E, white arrows), and segmenting iOL2s, defined as Nkx2.2 -/CC1 high cells, into three stages: (1) Tns3 high /Nkx2.2 -/Olig1 -cells (Fig. 2E, arrowheads), (2) Tns3 high /Nkx2.2 -/Olig1 high-cytoplamic (Fig. 2E, grey arrows), and

(3) Tns3 -/Nkx2.2 -/ Olig1 high-cytoplamic (Fig. 2E). Therefore, Tns3 protein is strongly expressed in early iOLs (iOL1s and early iOL2s) and then downregulated upon oligodendrocyte maturation (late iOL2s and mOLs). Similar expression pattern and Tns3 localization was found in neonatal neural progenitors differentiating cultures, where Tns3 immunofluorescence was detected in the cytoplasm and cell processes expressing CNP myelin protein, and excluded from the nucleus (Fig. 2F,G).

Altogether, these results indicate that Tns3 protein is expressed at high levels in early immature oligodendrocytes, and localized to the cytoplasm and cell processes (Fig. 2H,I).

Finally, we investigated whether other Tensin family members (Tns1, Tns2/Tenc1, and Tns4/Cten) were expressed in oligodendroglia and found that Tns1 and Tns2 could be also detected at very low levels in iOLs by immunofluorescence (Fig. S3A,B), in accordance with their low transcript expression (Fig. S3C; brainrnaseq.org). Therefore, Tns3 is the main Tensin expressed during oligodendrocyte differentiation, suggesting that Tns3 function in immature oligodendrocytes is likely to be evolutionarily selected, and of biological importance for CNS myelination.

Tns3 expression is found in immature oligodendrocytes during remyelination

Given the strong Tns3 expression in iOLs during postnatal myelination, we hypothesized that Tns3 expression could be enriched during remyelination in differentiating oligodendrocytes contributing to remyelination. To address this hypothesis, we preformed LPC focal demyelinating lesions in the corpus callosum of adult (P90) Tns3 Tns3-V5 and wild-type mice, and assessed for Tns3 expression at the peak of oligodendrocyte differentiation (8 days post-lesion) in this remyelinating model [START_REF] Nait-Oumesmar | Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination[END_REF]. We found that while non-lesioned adult brain regions contained only sparse iOLs (CC1 high /Olig1 cyto-high cells) also expressing Tns3, remarkably many Tns3 + iOLs were found in the remyelinating area both with V5 (Fig. S5A,C, arrows) and Tns3 antibodies (Fig. S5B, arrows). Quantification of Tns3 + cells showed a 3-fold increase in Tns3 + iOLs around the lesion borders compared to the corpus callosum far from the lesion area (Fig. S5D), strongly suggesting that Tns3 expression could be used as a hallmark of on-going remyelination and lesion repair.

Of note, we could also detect Tns3 expression in some microglia/macrophages cells in the lesion area using a combination of F4/80 antibodies (Fig. S5C, arrowheads).

Altogether, all this data indicates that Tns3 expression peaks at the onset of oligodendrocyte differentiation, labeling immature oligodendrocytes during both myelination and remyelination, which prompt us to study its function this process.

Tns3 knockout mice present normal numbers of oligodendroglia in the postnatal brain and still express Tns3 full length transcripts

To explore the role of Tns3 in oligodendrocyte differentiation, we first analyzed a Tns3 gene trap mouse line (Tns3 bgeo ) already characterized outside the CNS (Chiang et al., 2005), where the bgeo cassette is inserted after Tns3 exon 4 (Fig. S6A) driving LacZ transcription, and predicted to be a Tns3 loss-of-function mutation by inserting a stop poly-A sequence. Despite the original report of postnatal growth retardation in Tns3 bgeo/bgeo mice, these mice were kept in homozygosity for several generations in a different genetic background (XX, Shu-Hao Lo). We thus analyzed the impact in oligodendrogenesis in the postnatal brain of Tns3 bgeo animals at P21. We first found bgal immunofluorescence in Olig2 + /CC1 + OLs of Tns3 bgeo postnatal brain (Fig. S6B,C), paralleling our characterization of Tns3 expression with V5 and Tns3 antibodies. We then quantified the number of OPCs and differentiating oligodendrocytes present in two main white matter areas (corpus callosum and fimbria) of Tns3 bgeo/bgeo and Tns3 bgeo/+ littermates, at P21 when many oligodendrocytes are differentiating. The density of PDGFRa + OPCs or CC1 + oligodendrocytes was the same in both genotypes (Fig. S6D,D',E). Moreover, quantification of three different stages of oligodendrocyte differentiation (iOL1, iOL2, and mOL) using Olig2/CC1/Olig1 immunofluorescence did not reveal changes in the rate of oligodendrocyte differentiation (proportion of each stage) in Tns3 bgeo/bgeo mice compared to control littermates (Fig. S6F,F',G). We therefore verified whether the Tns3 bgeo allele was present in homozygosity in Tns3 locus of Tns3 bgeo/bgeo compared to wild type mice. Indeed, we could amplify the expected PCR products from genomic DNA of P21 brains using primers recognizing Exon-2 and bgeo only in Tns3 bgeo/bgeo mice but not using Exon-2 and Exon-14, that only amplified in wild type mice; Fig. S6H). We then checked for Tns3 full-length transcripts using cDNA generated from P21 brains, and to our surprise, primers flanking Exons 17 and 31 were similarly amplified from cDNA of Tns3 bgeo/ bgeo and wild type brains (Fig. S6I), suggesting that the Tns3 bgeo allele still produces Tns3 full-length transcript, and thus Tns3 protein, in the brain of Tns3 bgeo/ bgeo mice. Altogether, these results indicate that Tns3 bgeo allele do not lead to Tns3 loss-of-function in the brain, different from what previously reported in other tissues (Chiang et al., 2005), and thus it is not suitable for assessing Tns3 function in the CNS.

Therefore, we decided to generate a new Tns3 knockout mice using CRISPR/Cas9 technology. In order to generate Tns3 loss-of-function mutations (indels), we generated integrative plasmids (Fig. S7A) driving Cas9 expression and gRNAs targeting the start of Tns3 coding sequence (Exon-6), using as control, plasmids without the Tns3-targeting sequence of the gRNA. We validated the cutting efficiency of two Tns3-targeting gRNAs by lipofection of neural progenitors (Fig. S7B-F). We then, used these optimized tools to induce CRISPR-mediated mutations in mouse zygotes (methods), generating and characterizing two mouse lines having small deletions after the coding ATG of Tns3 in exon 6 (4-deletion and 14-deletion; Fig. S8A) and presumably leading to frame shifts and Tns3 loss-of-function.

Remarkably, homozygous animals were found in reduced numbers compared to mendelian ratios (Fig. 8B), with many of them dying during embryonic development (Fig. S8C) and most homozygous animals presenting major growth retardation in the second and third postnatal week compared with their littermates (Fig. S8E,F), similar to the original report of Tns3 bgeo mice (Chiang et al., 2005). Furthermore, we could still detect Tns3 protein in CC1+ immature oligodendrocytes of these homozygous mice by immunofluorescence with at least two different Tns3 antibodies (Fig. S8G) and

Tns3 transcripts the expression of exons corresponding to Tns3 full-length transcript by qPCR (Fig. S8H). Altogether, these results suggest that mice carrying constitutive Tns3 loss-of-function mutations seems to escape the full Tns3 deletion, at least in the brain, and thus we considered these animals not suitable to study Tns3 function in oligodendrogenesis.

Finally, to assess whether TNS3 is potentially required during human fetal development, we queried genetic loss-of-function data obtained from the human population by the GNOMAD project [START_REF] Karczewski | The mutational constraint spectrum quantified from variation in 141,456 humans[END_REF][START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF]. These Tns3 near the first coding ATG (exon 6) and expressing the GFP reporter (Fig. 3A), to transfect neonatal NSCs by postnatal electroporation (Fig. 3B). We targeted the dorsal subventricular zone (SVZ) NSCs, which generate large number of oligodendroglial cells during the first postnatal weeks (Kessaris et al., 2006;Nakatani et al., 2013), and quantified the GFP + cells, progeny of targeted NSCs, three weeks later (P22, Fig. 3B,C). We focused our study in glial cells by quantifying the GFP + cells outside the SVZ and located in dorsal telencephalon (i.e. corpus callosum and cortex). We identified the fate of GFP + cells by immunofluorescence for GFP and glial subtype markers, using high levels of CC1 for oligodendrocytes, PDGFRα for OPCs, and recognized astrocytes by their unique branchy morphology and low levels of CC1.

Remarkably, brains electroporated with two different CRISPR plasmids targeting

Tns3, had a 2-fold reduction in GFP + oligodendrocytes compared to the brains electroporated with control plasmids, while GFP + OPCs were found in similar proportions, and even the proportion of GFP + astrocytes was increased by 1.5-fold as a result of the large reduction in oligodendrocytes, the number of GFP + astrocytes was not changed (61.3 ± 10.9 in experimental versus 57.2 ± 11.8 in controls; Fig. 3D,D',E).

To assess whether the reduction in oligodendrocytes from Tns3-deleted NSCs was the consequence of a reduction in OPCs generated, we assessed for possible changes in numbers, proliferation, and survival of OPC at P11 (Fig. 3F), when most cortical OPCs have not yet started differentiation. Noteworthy, we found no differences in either proportion of GFP cells being OPCs (Fig. 3G), in the proliferative status of GFP + OPCs (MCM2 + /PDGFRa + cells; Fig. 3H,H',J), nor GFP + OPCs showing traits of cell death (Fig. 3I,I'), between experimental and control brains, while the reduction of oligodendrocytes was already marked (Fig. 3G).

Given the presence of two Tns3 isoforms expressed in the brain, we asked whether a deletion of both isoforms would have a greater impact in the reduction of oligodendrocytes. To assess this, we took advantage of our two gRNA efficiently targeting and cutting the beginning and the end of Tns3 coding sequence to delete the whole Tns3 locus (Fig. S10A-C). Remarkably, the loss of the two Tns3 isoforms had a similar effect in the reduction of oligodendrocytes to the mutations affecting only Tns3 full-length (Fig. S10D,D',E), suggesting that the small Tns3 isoform is not functionally redundant with Tns3 full-length for oligodendrocyte formation. Altogether, these results indicate that Tns3 loss-of-function mutations in neonatal SVZ-NSCs impairs normal OPC differentiation without affecting OPC generation, proliferation, or survival, and thus suggesting that Tns3 is largely required for OPC differentiation into mature oligodendrocytes in the postnatal brain (Fig. 3K). Altogether, these results indicates that acute deletion of Tns3 in OPCs reduces by 2fold oligodendrocyte differentiation in the postnatal brain, without major changes in OPC proliferation or survival (Fig. 4G).

OPC-specific

Tns3-iKO immature oligodendrocytes present an increased apoptosis in primary cultures

To get some insight into the cellular defects produced by Tns3-deletion in oligodendroglia, we investigated their cellular morphology and behavior during their differentiation by video microscopy. To this goal, we MACS-purified OPCs from Tns3-iKO and control mice at P7, two days after administration of tamoxifen, plated them in proliferating medium for three days, and then recorded their behavior during three days in the presence of differentiation medium. Having the expression of the YFP as a readout of Cre-mediated recombination, we could compared the behavior of YFP + cells (Tns3-iKO) with neighboring YFP -cells (internal control) in the same cultures. In parallel, we used MACSorted cells from control mice as external control.

Quantification of the proportion of YFP+ cells over time, showed a neat reduction (from 80% to 60%) of YFP+ cells during the 3 days in proliferation medium followed by a reduction to 50% by day 3 in differentiation medium (Fig. 5B), suggesting a competitive disadvantage of Tns3-mutant cells. Live imaging monitoring of cell behavior showed that once YFP+ cells had developed multiple branched morphology, characteristic of differentiating oligodendrocytes in culture, they showed 4-fold increase their probability to die compared to YFP-cells of the same culture (Fig. 5C-E, yellow and white arrows, respectively) or to cells from control cultures (Fig. 5D). This increase in cell death was more pronounce in the third day of culture (Fig. 5E). Together, this result point to cellular defects of Tns3-iKO differentiating oligodendrocytes ending up in an increased apoptosis.

DISCUSSION

The tight balance of oligodendrocyte precursor cells (OPCs) between proliferation and differentiation ensures the capacity to respond to myelination needs of the CNS by generating new oligodendrocytes on demand, and avoid generation brain gliomas uncontrolled OPC proliferation. Furthermore, the observation that OPCs are present within demyelinating MS lesions, but fail to differentiate into myelinating cells with disease progression [START_REF] Chang | Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis[END_REF], suggests that efforts to foster OPC differentiation are a critical event for successful remyelination in MS patients. In this study, we combined the genome wide binding profile of key regulators of oligodendrocyte differentiation, Olig2, Chd7, and Chd8 (He et al., 2016;[START_REF] Küspert | SomethiNG 2 talk about-Transcriptional regulation in embryonic and adult oligodendrocyte precursors[END_REF][START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF]Lu et al., 2000;Marie et al., 2018;[START_REF] Zhao | Dual Requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Repair[END_REF]Zhou and Anderson, 2002;Zhou et al., 2000), to identify their common gene targets, and focused our analysis in Tensin3 (Tns3) whose expression matches oligodendrocyte differentiation. To study Tns3 expression and function, we generate several genetic tools, including CRISPR/Cas9 vectors to induce in vivo and in vitro Tns3 mutations, a Tns3 Tns3-V5 knock-in mice, two lines Tns3 knockout mice, and a Tns3 Flox mice. Using these tools, we provide several lines of evidence showing that Tns3 is expressed in immature oligodendrocytes (iOLs) and is required for normal oligodendrocyte differentiation. First, Tns3 expression is strongly induced at the onset of oligodendrocyte differentiation, localized to the cytoplasm and main cell processes, and downregulated in mature oligodendrocytes both at the transcript and protein levels, thus constituting a novel marker for iOLs, for whom we provide an optimal immunofluorescence protocol with a commercial antibody (Sigma, Ct). Second, we show that during remyelination, Tns3 is also expressed in newly formed

Tns3 is a novel marker for immature oligodendrocytes

Recent studies has started to uncover genes enriched in iOLs, such as Itpr2 (Marques et al., 2016;Zeisel et al., 2015), Enpp6 (Xiao et al., 2016), and Bcas1 [START_REF] Fard | BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions[END_REF], that could be used as markers for these transient cell population particularly interesting to label areas of active myelination and remyelination in the context of oligodendrocyte and myelin pathology, such as preterm brain injury and multiple sclerosis. Here, we report for the first time that Tns3 protein is a hallmark of iOLs (figure 2). Tns3 is expressed at high levels in iOLs and downregulated in mature oligodendrocytes, showing a complete overlap with Itpr2. We found that Tns3 antibody from Millipore also recognizes a nuclear epitope different from Tns3 that remarkably, like Tns3 in the cytoplasm, labels at high levels iOLs, similarly to what reported for CC1 antibody recognizing both APC and Quaking-7 proteins [START_REF] Bin | The oligodendrocyte-specific antibody 'CC1' binds Quaking 7[END_REF][START_REF] Lang | Adenomatous Polyposis Coli Regulates Oligodendroglial Development[END_REF]. Remarkably, upon trying several antibodies, we found one (Sigma Cterminus) working optimal in immunofluorescence on brain sections and oligodendroglial cultures, while Itpr2 commercial antibody did not match this high quality of immunofluorescence. The use of BCAS1 antibodies to label iOLs, despite is peak expression in iOLs, has the caveat of being also expressed in mature oligodendrocytes and myelin [START_REF] Zhang | An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex[END_REF]. Finally, Enpp6 has a very specific expression in iOLs at the transcript level (Xiao et al., 2016) but to our knowledge Enpp6 recognizing antibodies producing good quality immunofluorescence are yet not available. Therefore, Tns3 protein expression in the CNS is a hallmark of iOLs and Tns3 Sigma Cterminus antibody constitute an optimal reagent to label iOLs during both myelination and remyelination.

Tns3 is required for oligodendrocyte differentiation

Oligodendrocyte differentiation implicates a large generation of membrane and cell processes composing the 40-60 myelin segments formed by mature oligodendrocytes [START_REF] Hughes | Myelin remodeling through experience-dependent oligodendrogenesis in the adult somatosensory cortex[END_REF]. Actin cytoskeleton remodeling is an important driver of the OL morphological changes undergone during their differentiation [START_REF] Nawaz | Actin Filament Turnover Drives Leading Edge Growth during Myelin Sheath Formation in the Central Nervous System[END_REF]Zuchero et al., 2015). Tensin proteins, linking the extracellular signals received by integrins with the actin cytoskeleton, are well placed to play a relevant role in these morphological changes. Indeed, high levels of Tns3 expression coincides which the phase when iOLs display a large cytoplasm and produce large number of processes, suggesting an active role in this cellular remodeling. The guiding of the OL cell processes' growth cone is based on the sequential activation of Fyn, FAK and RhoGAP (Thomason et al., 2020). On the other hand, PDGF survival signaling in OPCs and myelin formation are dependent of α6β1-integrin binding to Fyn (Colognato et al, 2004). Interestingly, Tns3 can bind β1-integrin, through its PTB domain [START_REF] Georgiadou | AMPK negatively regulates tensin-dependent integrin activity[END_REF], but also binds FAK by its SH2 domain [START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF]) (Liao et al., 2007), and is thus well placed to mediate integrin signaling to the actin cytoskeleton. Moreover, β1-integrin, FAK, Fyn, p130Cas, and Tns3 are all highly expressed in iOLs (https://www.brainrnaseq.org/). Here, using three independent approaches, we show that loss of Tns3 in iOLs reduces by half the numbers of oligodendrocytes in the postnatal brain, and increases their apoptosis in oligodendrocyte differentiating cultures. It is therefore very likely that Tns3 act as a mediator of α6β1-integrin signaling to promote OL survival and differentiation by helping their actin cytoskeletal remodeling. If so, an exogenous activation of α6β1integrin in cultured OPCs with Mn2+ (Colognato et al., 2004) should not increase oligodendrogenesis in Tns3-iKO oligodendroglia. Finally, by its binding to EGFR [START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF], whose activation is another driver of oligodendroglial differentiation, Tns3 could also be required to mediate growth factors receptors activation in early iOLs, explaining the increased death in OLs lacking Tns3.

Tns3 and cancer

Although deregulation of Tensins' expression is frequently associated with cancer [START_REF] Lo | Cten, a COOH-Terminal Tensin-like Protein with Prostate Restricted Expression, Is Down-Regulated in Prostate Cancer[END_REF]Sasaki et al., 2003a;Sasaki et al., 2003b), the roles of the different Tensins in tumorigenesis and metastasis remain largely undefined (Cao et al., 2012).

All four family members are downregulated in human kidney cancers [START_REF] Martuszewska | Tensin3 Is a Negative Regulator of Cell Migration and All Four Tensin Family Members Are Downregulated in Human Kidney Cancer[END_REF], and reduced levels of Tns3 are detected in breast tumors (Veß et al., 2017), glioblastoma (Chen et al., 2017) or thyroid carcinoma (Maeda et al., 2009).

EGF is an important regulator of dynamic Tensin expression given that EGF treatment of epithelial cells decrease Tns3 expression (Katz et al., 2007). Mechanistically, it has been shown in mammary cells in culture that Tns3 can bind DLC1 (Deleted in Liver Cancer 1) by its actin binding domain, releasing an auto-inhibitory interaction in DLC1, and thereby leading to inactivation of RhoA and thus decreasing cell migration (Cao et al., 2012). Furthermore, EGF-driven cell migration and cellular transformation is dependent on the inhibition of DLC1 and it implies Tns3 downregulation likely mediated by p-ERK (Cao et al., 2012). Therefore, all these data suggest a role of Tns3 in maintaining focal cell adhesion and thus preventing migration and metastasis in tumor cells. Interestingly, EGFR is involved in OPC proliferation and survival [START_REF] Aguirre | A functional role for EGFR signaling in myelination and remyelination[END_REF] and it is downregulated in iOLs in parallel with the increase expression of Tns3.

Intolerance to Tns3 loss-of-function in mice and humans

Tns3 bgeo gene trap mice has been reported as a null-allele in the lung and intestine, resulting in growth retardation and in some cases death within 3 weeks after birth (Chiang et al., 2005). Histological analysis of this mutant showed smaller digestive tracts with defects in both villi and enterocyte differentiation as well as a failure in secondary septation of lung alveoli, with BrdU birth dating experiments indicating a migration defect of Tns3-mutant epithelial cells of the small intestine (Chiang et al., 2005). Given that interactions between epithelia and surrounding mesenchyme are critical for the development of both organs [START_REF] Minoo | Epithelial-mesenchymal interactions in lung development[END_REF][START_REF] Roberts | Epithelial-mesenchymal signaling during the regionalization of the chick gut[END_REF][START_REF] Garaud | Calmodulin in epithelial intestinal cells during rat development[END_REF] and EGF induced migration of epithelial cells requires modulation of cell-cell and cell-matrix interaction [START_REF] Pignatelli | Modulation of cell adhesion during epithelial restitution in the gastrointestinal tract[END_REF], it is possible that lacking Tns3 impairs the ability of the epithelia to fully process environmental cues, such as EGF, which normally induces tyrosine phosphorylation of Tns3 (Chiang et al., 2005;[START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF]. In this study, we have described specific expression of bgalactosidase in differentiating oligodendrocytes that corresponds to immunofluorescence with Tns3 specific antibodies. Analysis of different oligodendroglial stages in the postnatal brain of Tns3 bgeo/bgeo mice did not show any changes compared to control littermates. Unexpectedly, we found that Tns3 immunofluorescence signal is still present in Tns3 bgeo/bgeo brains and detected Tns3 transcripts containing the main exons are present in Tns3 full-length isoform, indicating that Tns3 bgeo allele is not a Tns3-null allele in the CNS. We obtained similar results using two different CRISPR/Cas9-generated alleles (Tns3 4del and Tns3 14del ) predicted to be loss-of-function by inducing a frame shift at the beginning of Tns3 coding sequence, suggesting that animals escaping embryonic or postnatal death have prevented the loss Tns3 at least in certain cells or tissues. This result parallels, the intolerance found in the human population for Tns3 loss-of-function predicted variants, where less than 20% of the expected alleles could be found in 60,000 human genomes involved in the GNOMAD project (https://gnomad.broadinstitute.org).

MATERIAL & METHODS

Animals and genotyping

All animal procedures were performed according to the guidelines and regulations of the Inserm ethical committees (authorization #A75- [START_REF] Aurnhammer | Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences[END_REF][START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF][START_REF] Bacon | N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination[END_REF][START_REF] Baker | Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer[END_REF][START_REF] Barber-Pérez | Mechanoresponsiveness of fibrillar adhesions on stiffness-gradient gels[END_REF][START_REF] Barrera | Organization of Myelin in the Mouse Somatosensory Barrel Cortex and the Effects of Sensory Deprivation[END_REF][START_REF] Barres | The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease[END_REF] and animal experimentation license A75-17-72 (C.P.). Both males and females were included in the study. Mice were maintained in standard conditions with food and water ad-libitum in the ICM animal facilities. For staging of embryos, midday of the vaginal plug was calculated as E0.5. Polymerase chain reaction was performed with wild type and knock-out primers to genotype the embryos. Wild type, heterozygous and homozygous mutant embryos were obtained from intercrosses of heterozygous mice. Genotyping of the different Tns3 alleles was performed by PCR using the following primers and conditions:

Tensin3 gene trap mice line (Tns3 bgeo ) details are from Su Hao Lu lab (UC Davis, USA).

Mice used for ChIP-seq analysis are Wild-type Swiss obtained from Janvier Labs.

Tns3 flox were crossed with PdgfRa-CreER T ; Rosa26 stop-YFP mice to generates Tns3 flox ;

Pdgfra-CreER T ; Rosa26 stop-YFP mice line. Due to the reported subtle effects of Cre protein in the biology of cells expressing it, all mice used as control for in Tns3 flox study are Tns3 WT ; Pdgfra-CreER T ; Rosa26 stop-YFP mice.

Generation of Tns3 4del and Tns3 14del knockout mice

Tns3 KO mice were generated at the ICM mice. Briefly, the Cas9 protein, the crRNA, the tracrRNA and a targeting vector for the Tns3 gene have been microinjected in a mice egg cell transplanted in a receptor C57BL/6J. Pups with NHEJ mutations inducing a gene frameshift were selected after genotyping and Sanger sequencing verification. Finally, only two lines contained indels of 4 and 14 nucleotide deletions were maintained and studied.

Generation of Tns3 Tns3-V5 knockin mice

Tns3 V5 mice were generated at the Curie Institute mice facility. Briefly, the Cas9 protein, the crRNA, the tracrRNA and a ssODN targeting vector for the Tns3 gene have been microinjected in a mice egg cell transplanted in a receptor C57BL/6J-BALB/cJ female. Pups pups presenting HDR insertion of the V5 tag were selected after genotyping. Non-bound beads-coupled antibodies have been washed by centrifugation then bound cells have been sorted using MultiMACS Cell24 Separator Plus (Miltenyi Biotec;. Sorted cells were either plated in culture plates for in vitro cell study or centrifuged at 1200 rpm and use for Western blot analysis.

Generation of Tns3

O4 + MACSorted cells from P7 wild-type mouse cortices (80% PDGFRα + OPCs and 20% Nkx2.2 + -Cnp + iOLs) were used for Olig2 ChIP-seq. O4 + MACSorted cells from P12 wild-type mouse cortices (30-50% PDGFRa + OPCs and 50-70% Nkx2.2 + -Cnp + iOLs) were used for histone marks ChIP-seq.

Chromatin immunoprecipitation (ChIP)

ChIP-seq assays were performed as described previously (Marie et al;2018) The ChIP-seq libraries were prepared using ILLUMINA Truseq ChIP preparation kit and sequenced with ILLUMINA Nextseq 500 platform.

ChIP-seq analysis

All ChIP-seq analysis were done using the Galaxy Project (https://usegalaxy.org/).

Reads were trimmed using Cutadapt (--max-n 4) and Trimmomatic (TRAILING 1; SLIDINGWINDOW 4 and cutoff 20;LEADING 20;MINLEN 50), and mapped using Bowtie2 onto mm10 mouse reference genome (-X 600; -k 2; --sensitive). PCR-derived duplicates were removed using PICARD MarkDuplicates. Bigwig files were generated with bamCoverage (binsize=1). Peak calling was performed using MACS2 callpeak with Input as control and with options: --qvalue 0.05; --nomodel; --keep-dup 1; --broad (only for histone marks). Blacklisted regions were then removed using bedtools Intersect intervals.

Visualization of coverage and peaks was done using IGV ( [START_REF] Robinson | Integrative genomics viewer[END_REF]; http://software.broadinstitute.org/software/igv/home). Intersection and analysis of bound genes were done using Genomatix (https://www.genomatix.de/).

Two replicates were done for Olig2, with one of them of better quality [START_REF] Chiang | Inactivation of tensin3 in mice results in growth retardation and postnatal lethality[END_REF]960 peaks for replicate 1 and 14,242 peaks for replicate 2). The peaks found in both replicates [START_REF] Allen | Gliamore than just brain glue[END_REF]781) and/or peaks from replicate 1, which were found in regulatory elements (13948) were considered (16578 in total). Three replicates were done for H3K4me3, two replicates were done for H3K27me3 and one replicate was done for H3K27Ac and H3K4me1. Intersection of these datasets was done using bedtools Intersect intervals.

Peaks overlapping with regions between 1000bp upstream of transcription start site (TSS) and 10bp downstream of TSS were identified as "promoters" (Genomatix).

"Active promoters" contain peaks for H3K4me3 and H3K27Ac. "Repressed promoters" contain peaks for H3K27me3 and no active marks. "Poised promoters" contain H3K4me1 and no active or repressed mark. Regions outside promoters containing histone marks were considered as "enhancers". "Active enhancers" contain peaks for H3K27Ac. "Repressed enhancers" contain peaks for H3K27me3 and no active marks.

"Poised enhancers" contain H3K4me1 and no active or repressed mark. Genes are considered associated if the peaks is present in promoter or a range of 100kb from the middle of the promoter and the gene expression is medium to high ("active"), low ("poised") or not ("repressed") expressed (based on control RNA-seq dataset in (Marie et al;2018))

Analysis of scRNAseq

Counts per gene were downloaded from GEO datasets GSE75330 and GSE95194, and processed in R (4.0) using the following packages: Seurat (3.0) for data processing, sctransform for normalization, and ggplot2 for graphical plots. Seurat objects were first generated for each dataset independently using CreateSeuratObject function (min.cells = 5, min.features = 100). Cell neighbors and clusters were found using FindNeighbors (dims = 1:30) and FindClusters (resolution = 0.4) functions.

Clusters were manually annotated based on the top 50 markers obtained by the

Postnatal electroporation

Postnatal brain electroporation (Boutin et al., 2008) was adapted to target the dorsal SVZ. Briefly, postnatal day 2 (P2) pups were cryoanesthetized for 2 min on ice and 1.5 ml of plasmid were injected into their left ventricle using a glass capillary. Plasmids were injected at a concentration of 2-2.5ug/ul. Electrodes (Nepagene CUY650P10) coated with highly conductive gel (Signagel, signa250) were positioned in the dorsoventral axis with the positive pole dorsal. Five electric pulses of 100V, 50ms pulse ON, 850ms pulse OFF were applied using a Nepagene CUY21-SC electroporator. Pups were immediately warmed up in a heating chamber and brought to their cages at the end of the experiment.

Immunofluorescence

Embryonic mice were taken after anesthetizing the mother by 100 μL phenobarbital (euthasol)(Virbac USA) at the time point we wanted and their brains were dissected out and fixed for 30 minutes in 2% paraformaldehyde (PFA) freshly prepared from 32%

PFA solution (Electron Microscopy Sciences, 50-980-495). Postnatal mice brain till 15 days post-natal (P15) were perfused with 15ml of 2% PFA, and the older stages were perfused with 25 ml of 2% PFA, brains from post-natal were dissected out, cryoprotected in PBS with 20% sucrose overnight, and included in OCT (BDH) before freezing and sectioning (16µm thickness) in a sagittal plane with a cryostat microtome (Leica). Sections were then either processed for immunohistochemistry after drying In vitro, the same blocking solution and the same antibodies were used than in vivo. Fixed coverslips were blocked for 30 minutes at RT, incubated in the primary antibodies for 45 minutes at RT and washed 3 times in 1x PBS. Secondary antibodies were apply for 45 minutes at RT and washed 3 times in 1X PBS. Coverslips were then incubate with DAPI solution for 5 minutes at RT. A final washing was done before mounting the coverslips on slides to be visualized under the microscope.

Immunofluorescence was visualized with Zeiss® Axio Imager.M2 microscope with Zeiss® Apotome system. Pictures were taken as stacks of 5-10µm with 0.5µm between sections. Image acquisition and processing are achieved by ZEN Microscopy and imaging Software, Z-projections and orthogonal projections were done in ImageJ and processed with Adobe Photoshop. Figures were made using Adobe Illustrator.

Western blot

Proteins from MACsorted cells were extracted 30 minutes at 4°C in RIPA buffer from

Demyelinating lesions

Before surgery, adult (2-3months) WT mice were weighted and anesthetized by intraperitoneal injection of mixture of ketamine (0.1 mg/g) and xylacine (0.01 mg/g).

An analgesic (buprenorphine, 30 mg/g) was administered intraperitoneally to prevent postsurgical pain. Focal demyelinating lesions were induced by stereotaxic injection of 1µl of lysolecithin solution (LPC,Sigma, 1% in 0.9%NaCl) into the corpus callosum (CC; at coordinates: 1 mm lateral, 1.3 mm rostral to bregma, 1.7 mm deep to brain surface) using a glass-capillary connected to a 10µl Hamilton syringe. Animals were left to recover in a warm chamber before being returned into their housing cages.

Development of CRISPR/Cas9 tools

Design of gRNA

In order to produce indel mutations by Non Homologous End Joint (NHEJ) repair mechanisms after Cas9/gRNA targeting of the coding region we decided to find efficient sgRNA in the region downstream of the coding ATG. To this purpose, we used CRISPOR software (http://crispor.tefor.net/) to choose gRNA with predicted cutting efficiency and minimal off-target and for PCR amplification primer design.

Validation of gRNA cutting efficiency

The validation of tns3-targeting CRISPR/Cas9 system was performed in 3T3 cell lines by transfection with lipofectamine 3000 of PX459 plasmids containing 4 different sgRNA sequences. After 2-days incubation, puromycin was added to medium for 4 days allowing survival of cells containing the PX459 plasmid. Three days after proliferation in fresh medium without puromycin, DNA was extracted using DNeasy blood & tissue kits (Qiagen), and target DNA for 5' Tns3 region was amplified by PCR using primer sequences: Forward: 5'-AGG TGG CCT TCA GCT CAGT -3', Reverse: [START_REF] Zhang | An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex[END_REF]brainrnaseq.org). (E) In situ hybridization in sagittal sections of the adult (P56) mouse brain at the level of the lateral ventricles showing Tns3 transcript expression in sparse white matter cells (Allen brain atlas, portal.brain-map.org). (F) UMAP representation of neural progenitors and oligodendroglial cells extracted and integrated using Seurat from scRNA-seq datasets (Marques et al., 2016;2018) Finally, to better characterize Tns3 expression pattern in oligodendroglia, we took advantaged of recent resources profiling the transcriptome of single cells (scRNA-seq)

including oligodendrocyte lineage cells at embryonic, postnatal, and adult stages (Marques et al., 2016[START_REF] Marques | Transcriptional Convergence of Oligodendrocyte Lineage Progenitors during Development[END_REF]. We selected the neural stem/progenitor cells and oligodendroglial cells of these datasets and integrated them following Seurat3 strategy (Stuart et al., 2019). Notably, we found that Tns3 was mostly expressed in iOL1/iOL2 clusters (Figure 1F-G), with Tns3 expression correlating well with iOLs markers such as Itpr2, Nkx2.2 or Enpp6 (Marques et al., 2016;Zeisel et al., 2015;Xiao et al., 2016).

On the contrary, Tns2 or Tns1 were lowly expressed in the whole dataset, mostly in NPC and NSC clusters. Finally, Tns4 mRNA was not detected in any cluster of this dataset (Figure 1H). Again, these data confirm that Tns3, and not the other Tensins, is strongly regulated by key oligodendroglial factors and suggest an involvement of Tns3 in oligodendrogenesis. As expected, Tns3 expression shows an almost complete overlap with immature markers, with only a small population of Itpr2+ and almost no Nkx2.2+ cells being Tns3-(Figure 3C). I summarize the Tns3 expression pattern in a graphical scheme (Figure 3D). Tns3 peculiar pattern of expression is of particular interest for oligodendroglia analysis as it represents a specific marker of a precise step of oligodendrocytes differentiation. [START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF][START_REF] Georgiadou | AMPK negatively regulates tensin-dependent integrin activity[END_REF]Liao and[START_REF] Liao | Tensinsemerging insights into their domain functions, biological roles and disease relevance[END_REF].

I confirmed these findings using in vitro primary NSCs cultures. Tns3 is found in almost all CNP+ and Nkx2.2+ cells after 5 days (Figure 4A). In these conditions, it is possible to study more in detail the subcellular localization of Tns3 expression. Indeed, Tns3 is not restricted to OLs soma and can also be found in sporadic dots all along OLs processes (Figure 4B). These Tns3 dots colocalized with β1-integrins expression (Figure 4C), suggesting these areas could be focal adhesion plates but an extensive study remains to be done. Indeed, Tns3 is known to interact with β1-integrins and key cytoskeletal proteins in fibroblasts at focal adhesion (Figure 4D), helping the cell anchoring to its environment [START_REF] Georgiadou | AMPK negatively regulates tensin-dependent integrin activity[END_REF].

From all these data, I proposed a model describing the expression of Tns3 all along the OL differentiation. Tns3 is not expressed in OPCs but shows a clear peak of expression in iOLs which decrease quickly in the late stages of OL maturation. From all of these, it appears clear that Tns3 expression is strictly regulated during all the oligodendrogenesis, therefore representing a novel marker of iOLs stages. To assess whether Tns3 was also involved in oligodendrogenesis during remyelination, I induced an LPC demyelinating lesion in the corpus callosum of 3month-old Tns3 Tns3-V5 mice and look for remyelination after 7 days of recovery. I found a Tns3 expression in immature OLs surrounding the lesion core (Figure 5A), indicating that Tns3 is not only expressed in oligodendroglia during developmental myelination, but also during remyelination. Interestingly, Tns3 was also expressed in Cd48-F4/80+ microglial cells (Figure 5B). The precise nature of these cells and the involvement of Tns3 in microglia during demyelination remain to be characterized more precisely.

In order to see whether Tns3 expression was really correlated with remyelination, I quantify the Tns3+ oligodendrocytes density every 50m from the lesion core (without quantifying the lesion core itself, due to the background signal induce by the myelin debris and the high density of microglial cells). I found an accumulation of Tns3+ oligodendrocytes in a 100m area surrounding the lesion core (Figure 5C). Altogether, this data suggest that Tns3 is not only expressed during myelination, but also in remyelination context to generate new OLs at the lesion site.

Tns3 constitutive KO induce a sublethal phenotype

In our first attempt to generate the V5-tagged mice line, we created three Tns3 KO mouse lines by CRISPR-induced Non-Homologous End Joining. Each presented their own nonsense mutation changing the Tns3 reading frame: 1 nucleotide insertion, 4

nucleotides deletion and 14 nucleotides deletion. No clear deleterious phenotype was observed in heterozygous Tns3 KO mice but Tns3 4del and Tns3 14del homozygous mice presented decreased survival rates (Figure 6A). We asked whether this reduced number of Tns3 KO homozygous mice were present before birth and genotype E14.5

embryo. Even at E14.5, a significantly lower number of Tns3 KO homozygous was found (Figure 6B), suggesting an involvement of Tns3 during foetal development. Tns3 KO homozygous mice presented also a growth defect (Figure 6B, arrows) at P8 (Left) and P14 (Right), coherent with the literature's data (Chiang et al, 2005) In parallel with these studies, Tns3 KO mice were also generated by the International Mouse Phenotype consortium (IMPC, https://www.mousephenotype.org/). Several test to attest the severity of this mutation are currently ongoing but they already found a so-called "sublethal" phenotype, similar to ours (Figure 6D) and a female specific increase in bone mineral density in heterozygous Tns3 KO mice (Figure 6E). The reduction in body weight and the increased death for homozygous Tns3 KO mice is not a surprise. Indeed, previous attempts to generate Tns3 knockout models or the study of natural Tns3 mutants revealed that the loss of this protein is associated with growth defects and reduction of the carcass size (Chiang et al, 2005;[START_REF] Deng | Genome-wide association study reveals novel loci associated with body size and carcass yields in Pekin ducks[END_REF].

A consequent part of our Tns3 KO homozygous mice later died after birth at P14 due to unknown reasons. We sadly lost these three mice lines during the COVID-19 lockdown, blocking us to study more in detail this phenotype. Nevertheless, we were able to detect Tns3 protein by immunofluorescence (Figure 6E) and Tns3 transcripts by RT qPCR from Tns3 KO mice brain, indicating that the surviving mutant mice still expressed Tns3 in the brain. To tackle this problem, we changed our strategy for a co-electroporation of three plasmids according to the efficient transfection obtained in the lab of our collaborators, the team of Lucas Tiberi (Trento, Italy), implicating the PiggyBAC transposition system having better efficiency that the Tol2 system we previously used. In this new strategy, we first generated a plasmid containing the gRNA and the Cre-recombinase sequence un a backbone plasmid with pCAG promoter and PB integrative sequences, a PBase transposase expressing plasmid and a PB integrative plasmid containing the pCAG-VENUS reporter gene. The strong VENUS fluorescence and expression after host cell genome integration decreased the number of false negative cells and helped us to phenotype cells by using their morphology in addition to marker expression. Finally, in a tour de force, we used our Tns3 Tns3-V5 ;Rosa STOP-Cas9-GFP mice line, expressing the Cas9 after Cre recombination under the control of an ubiquitous locus, together with and two plasmids pB-pCAG::Cre-pU6::gRNA plasmid and pBase, in order to quicken Cas9 cutting after the gRNA is produced. With the high VENUS signal, I also decided to use an automatic scanner, which quickened the analysis of the brain slices. With these images, it is also easier to show the overall efficiency of our electroporation strategy (Figure 8A).

With this new protocol, over five times more NSCs were targeted and five times more glial were quantified (Figure 8B). I quantified the targeted glial cells, using again CC1

and PDGFRa staining, and also found a decrease by 40% of the number of OL generated by mutated NSCs at P21 (Figure 8C). Interestingly, it seems that the phenotype is weaker in this new setup. This could be explained by the fact that our new co-eletroporation system despite targeting more cells, also seem to induce more false positive results. Indeed, as a mutagenesis is based on the integration of 3 plasmids, if the VENUS plasmid is integrated without the CRISPR plasmid, some nonmutated cells could exhibit GFP expression. Still, the strong decrease of the proportion OLs reinforce our previous results indicating a clear involvement of Tns3 in oligodendrogenesis.

Tns3 proteomic expression in oligodendroglia

Transcriptomic resources from human (gtexportal.org/home/gene/TNS3) and mouse tissues already suggested that two Tns3 transcripts were expressed in several brain regions and that the smaller isoform was expressed at higher levels (Figure 9A). But our electroporation CRISPR-Cas9 system was designed in order to get rid of the expression of only the full length Tns3. OPCs and iOLs, both Tns3 isoforms can be found, with the small one predominantly expressed (Left).

At P14, the isoforms are expressed at similar levels in iOLs (Middle). And at P21, only the small tns3 isoform is expressed and at low levels (Right).

My Western blot from Tns3 V5 ;Rosa STOP-Cas9-GFP P35 mice brain confirms literature data with a high expression of Tns3 small splicing variant(60KDa) and a weak full length tns3 expression (Figure 9B Left).

I asked whether these data from total brain extracts were representative of the oligodendroglial population, so I decided to investigate for the presence of Tns3 isoforms in cultured NSCs after 3 days of proliferation and 4 days of differentiation.

Surprisingly, these cells show a clear expression of both isoforms and at similar levels (Figure 9B). To confirm this in vitro test, I MACSorted OPCs/iOLs from P7 cortex, iOLs at P14 and more mature OLs at P21 and look at the relative expression of each isoform by Western Blot using a specific anti-V5 antibody. Interestingly, at P7, the two isoforms are highly expressed, but the small isoform is more than 5 five time more expressed than the full length (Figure 9C Left). At P14, iOLs still expressed the two isoforms at lower but similar levels (Figure 9C Middle). Finally, P21 mOLs expressed general low levels of Tns3 and only the small isoform remains detectable (Figure 9C Right).

Interestingly, it seems that full length Tns3 is specific to early iOLs in the brain, and it is downregulated during OL maturation only levels of Tns3 can be detected, and mainly of the small isoform.

Deletion of the whole Tns3 locus by CRIPR-Cas9

Our first CRISPR-Cas9 strategy induces a Tns3 frameshift at the level of the exon 6, which is the first translated exon of full length Tns3 isoform. On the contrary, this Knockout is supposed to not affect the small Tns3 60 KDa isoform, by far the most expressed in the brain and oligodendrocytes. I decided to check whether small Tns3 isoforms could be also involved in oligodendrogenesis by comparing the effect of Tns3 frameshift, affecting only Tns3 full length expression, and the deletion of the whole locus, affecting the expression of both isoforms.

Instead of inducing an exon6 frameshift by NHEJ, I used either a combination of two gRNA to get rid of the 9th exon, which deletion induces a frame shift after 100 aa in the coding region, or two gRNAs to delete the whole Tns3 locus. I also kept a control condition in which a simple exon6 frameshift was induced (Figure 10A). As before, mice were electroporated at P1 and perfused at P21 (Figure 10B) to follow the progeny of the (GFP+) electroporated NSCs' progeny (Figure 10C).

Immunostaining against PDGFRa, to look at the OPC population, and CC1, characterizing our OLs, show a good number of GFP+ cells both in control (Figure 10D) or KO conditions (Figure 10D'). The quantification of the NSCs progeny after the deletion of the whole Tns3 locus show surprisingly a similar reduction of the generation of OLs (Figure 10E), compared to controls and Exon6 frameshift (Figure 10F-G). To verify the efficiency of CRISPR Cas9 cutting in these two models and their effect on Tns3 expression in iOLs cells, I quantified the GFP+/Nkx2.2+/Tns3+ and the GFP+/Nkx2.2+/Tns3-cells proportion in mutant mice using immunostaining (Figure 11A). Interestingly, I found a similar drop of the Tns3 expressing GFP+/Nkx2.2+ cells (Figure 11B) both with Exon6 frameshift and whole locus depletion (Exon6 deletion: -64%, p value: 0,02; whole locus deletion: -59%, p value: 0,03), without any effect on the Tns3-population, suggesting that Tns3 does not plays crucial roles in previous oligodendroglial stages

As a complementary strategy we design tools to delete Tns3 in NSC cultures by generating AAVs expressing Tns3-targeting CRISPR tools. In order to do so, I produced Tns3 deletion by transfecting gRNA and Cre expressing AAV9 virus in primary NSCs culture from Tns3 Tns3-V5 ; Rosa26 stop-Cas9-GFP mice. I stained these cultures to look Tns3 expression in OLs (Figure 12A) quantifying the number of CNP+ OLs after 2 and 3 days of differentiation. Interestingly, I found no differences in OL proportions after 2days (Figure 12B) but the NSCs transfected with the Exon6frameshit-AAV tended to produce less CNP+ OLs after 3 days of differentiation, at the onset of Tns3 expression in vitro (Figure 12C). Finally, we combined Tns3 flox animals with Pdgfra-CreER T mice, to induce Cremediated Tns3 deletion specifically in OPCs, in presence of a Cre reporter background (Rosa26 stop-YFP ) in order to trace Tns3-deleted cells (Fig. 14A). I induced Tns3 deletion in pups by administering tamoxifen at P7 and checked for mutated OPCs progeny first at P14 (Figure 14A) in three different brain regions: the corpus callosum, the cortex, the fimbria (Figure 14B). A clear decrease of the CC1+

OLs density compared to the controls could be observed in the cortex (-65%, p value: 0,003) but not in the fimbria nor the corpus callosum (Figure 14C). Interestingly, OPC density was not changed in any of the quantified areas, reinforcing our hypothesis that Tns3 impairs OL generation without affecting OPCs population.

I then looked at the effect of Tns3 flox allele recombination at P21 (Figure 15A), using our CC1/GFP/PDGFRa staining (Figure 15B-B' and C-C'). I quantified CC1+ OLs and PDGFRa+ OPCs density in four different brain area: the corpus callosum, the cortex, the fimbria and striatum, this later one seeming also affected at P14 (Figure 14B, not quantified). I found a clear decrease of CC1+ OLs in recombined mice (Figure 15D), with again no significant effect on the OPCs density.

In order to characterized more precisely the oligodendroglial stage at which Tns3 act, I then stained these brains with an CC1/Olig1 staining (Figure 15E-E') to quantify oligodendroglial cells of different stages: OPCs (Olig1+/CC1-cells), iOL1s (CC1+/Olig1-cells) and iOL2s/mOLs (CC1+/Olig1+ cells). I found a significant decrease of iOL2s/mOLs in the four areas (cortex: -39%, p value 0,003; fimbria: -57%, p value 0,0007; corpus callosum: -37%, p value 0,005; striatum: -38%, p value 0,019).

Interestingly, a significant decrease of iOL1s (CC1+/Olig1-cells) is also observed in the fimbria, the cortex, and the striatum (cortex: -39%, p value 0,005; fimbria: -34%, p value 0,03; striatum: -49%, p value 0,002) but not in the corpus callosum. Again, no significant effect was observed in the OPC density (Figure 15F). prone to die, differentiate slower or proliferate less than non-recombined OPCs from the same animals. Another way to represent this data is to look at the percentage of loss cells from the onset of the differentiation (Figure 19C). During the first 24h, no clear difference is observed between GFP+ and GFP-cells. From 24h to 48h, the percentage of GFP+ cells loss tends to be higher than GFP-cells, until reaching the significancy at 72h. PdgfRaWT littermates similar to the rate of GFP-cells from Tns3flox; PdgfRa-CreERT/WT mice. (E)

Representative section of the liveimaged cultures.

After 72h, GFP+ positive cells lost 5 times more cells/hour compared to GFP-cells.

Interestingly, OPCs MACSorted from Tns3 flox ; PdgfRa WT ; Rosa YFP mice show a profile similar to GFP-OPCs from Tns3 flox ; PdgfRa-CreER T ; Rosa YFP mice (Figure 19D), suggesting that the absence of these cells is not solely due to a paracrine effect between GFP+ and GFP-OPCs in each culture. In these preliminary data, I did not identify yet the cause of this reduced number of GFP+ OPCs in culture. It is very likely to be due to a mix of a slower differentiation, a decrease cell mobility, and an increased cell death, by the loss of cell-ECM adhesion (Figure 19E). We still need to finish the analysis of these data, and to produce the control experiment with MACS OPCs from

Tns3 WT ; PdgfRa-CreER T ; Rosa YFP mice, which will be more pertinent as controls.

Discussion

Tns3 is a crucial protein involved in numerous biological functions. Tns3 mutations have been associated mainly with lung dysfunctions or intestinal tract defects. This is very likely linked to their role in cell-cell GAP junction stabilisation, crucial for epithelial cells adhesion with each other and lumen stabilisation. Tensins are also heavily dysregulated in cancer in order to favour tumour metastasis. But each Tensins also has its own specific function, mostly related to their highly variable medial part. Tns3

seems to be especially involved in the CNS as it is the main expressed Tensin in oligodendroglia and the only Tensin known to be impacted in Glioblastoma (Chen et al, 2017). Therefore, this protein represents an interesting target to provide better insights on brain oligodendrocyte basic biology, both in normal and neurodegenerative context, and to fight brain tumour metastasis.

By Genome wide profiling of chromatin binding of key regulators of oligodendrogenesis, we show that Tns3 is directly targeted by key oligodendroglial transcription factors, such as Olig2, Chd7, and Chd8. Its expression is tightly controlled in order to be quickly but transiently increased during early oligodendrogenesis in myelinating and remyelinating context. We also show that Tns3 was found in the OL soma but was also concentrated in small dots distributed all along the oligodendroglial processes. The nature of these dots remains to be defined.

Tns3 expression is not exclusive to oligodendroglia in the CNS given that endothelial cells also present high levels of Tns3 transcripts, present a sustained Tns3 expression, which could be due to their high level of connectivity. Indeed, Tensins are known to play a role cell adhesion to each other and the ECM, as described for or smooth muscle endothelial cells (Charles et al, 2007) and Human aortic abdominal endothelial cells (Hassanisaber et al, 2018). OLs present instead this peculiar transient peak of expression, suggesting a critical role of Tns3 only at a precise step of oligodendroglial differentiation. Surprisingly, we also found a microglial population expressing Tns3 in demyelinating lesions but the precise nature of these cells remain to be defined. It is likely that Tns3 could be required for microglial motility to help microglial pseudopodia formation and their migration to the lesion site.

Tns3 deletion. These findings corroborate our hypothesis that Tns3 play a role only at a precise step of oligodendrogenesis and is not required for NSCs specification toward the OL lineage or OPC survival, even if we see a tendency of decreased proliferation in Tns3KO OPCs.

In vitro confirmation of these experiments also show a Tns3 requirement only at a precise step. Tns3 CRISPR/Cas9-induced mutation through AAV transfection of NSC cultures does not show any impact on OLs population before the third day of differentiation, which coincides with the start of Tns3 peak in these cultures.

Transfection with Cre-expressing adenovirus of Tns3 flox NSCs also show reduced number of oligodendrocytes only after 4 days of differentiation. Finally, the live imaging experiments clearly show not effects of Tns3-deletion on cell survival between recombined and non-recombined OPCs during the first 24h. However, a clear decrease of the proportion of recombined (GFP+) cells is observed during the 3 days of OPCs proliferation. An explanation of this could be that some of the OPCs start to differentiate during the proliferation step. Indeed, MACS is a stressful protocol, which takes about 5 hours, and the growth factor deprivation is known to induce OPCs differentiation.

In any case, we show that Tns3 plays a crucial role at a precise step of oligodendrogenesis. The involvement of actin remodelling protein in oligodendrogenesis is not new. Before oligodendrogenesis, OPCs migration from their birthplace to reach their differentiation area strongly required actin cytoskeleton. Then, at the onset of OLs differentiation, the formation of actin growth cones at the tip of iOLs processes, to find axons to myelinate, is considered as a major driver for the latter OLs morphological modifications (Thomason et al, 2020). The progression of the OL's edges around the axon during myelination is also extensively based on actin cytoskeletal reorganisation. But Tns3, which could act at all of these three steps, seems to be predominantly required for iOL differentiation as it is almost not expressed in OPCs and myelinating OLs, suggesting a specific role for Tns3 in OPCs processes formation.

[CP1] The precise mechanisms of Tns3 function in differentiating oligodendrocytes remains to be defined.

Surprisingly, none of our efforts to delete Tns3 have succeeded to delete more that 50% of Tns3 expressing cells, even in Tns3-iKO mice where we achieved more than 80% recombination of OPCs. This suggests the presence of unknown compensatory mechanisms, as the Tns3 constitutive KO mice mostly failed to survive after P14.

Tns3 KO have been extensively reported to present growth issues and impairment of gastro-intestinal tract, so this increase in death in Tns3 KO mice could also be due to growth defects or even difficulties for these mice to feed and assimilate the nutrients.

We have not identified a clear neurological phenotype in Tns3-iKO mice at P21, even if they miss half of their oligodendrocytes, but these mice present a non-significant reduction of their body mass compared to the controls. We planned to look at later time points, such as two and three month-old mice to assess for long lasting phenotypic effects of Tns3 deletion. It is likely that these mice end up presenting neurological defects, due to their lack of OLs, but another possibility is that these mice have indeed managed to overcome Tns3-deletion in order to keep the minimal number of OLs required for a functional myelination. In this later scenario, it will be interesting to challenge the brain by inducing partial demyelination (using the LPC or cuprizone demyelinating models) and assess the capacity of Tns3-iKO brains to recover from the induced demyelination. In parallel, it will be important to induce Tns3-deletion using Tns3-iKO mice in the context of adult brain de/remyelination. We are therefore growing our Tns3-iKO colony to be able to perform these experiments in the near future using the LPC-induced demyelination model of the corpus callosum. Tns3 binds to the same protein domains, therefore competing with full-length Tns3 binding, without binding actin. We could therefore ask whether small Tns3 acts as a competitive antagonist to full-length Tns3 to relax the cytoskeleton. Indeed, the small Tns3 isoform is very similar in its structure to Tns4, a protein which acts as a Tns3 competitive antagonist under EGFR activation. As Tns4 is mostly undetected in the whole oligodendroglial lineage and, more generally, the brain cells, I proposed a competitive antagonism model similar to the Tns3/Tns4 switch previously described, with the small Tns3 isoform to play the role of full-length Tns3 regulator.

To challenge or confirm this model, a precise proteomic study of Tns3 isoform remains to be done. We planned to check at each Tns3 isoform expression level and to investigate for changes in their binding affinity to their typical binding partners, like β1-Integrin or Fyn, at the different steps of oligodendrogenesis. A previous study from the ffrench-Constant laboratory (Colognato H et al, 2004) already showed the sequential activation of a gene cascade after β1-Integrin activation during OL differentiation in vitro. Among these genes can be found FAK, coding for a protein bound by Tns3 and involved in various cell signaling pathways, notably in actin cytoskeleton reorganisation. Therefore, Tns3 is perfectly placed to mediate β1-Integrin signaling as it is able to bind with both β1-Integrin, FAK and directly with the actin cytoskeleton. To test this hypothesis, a complex experiment could be to exogenously express the fulllength Tns3, but not of the small Tns3 splicing variant, in β1-Integrin deficient mice

OPCs to see a potential rescue effect. Considering the difficulty to specifically enhance the full-length Tns3 expression specifically in OPCs, another simpler way could be to activate the β1-Integrin pathway, either with genetic mice models or manganese treatment in culture (Colognato H et al, 2004), in Tns3-iKO mice or cells. If Tns3 is really a mediator of the β1-Integrin signalling in oligodendroglial cells, then no difference should be observed after activation.

Résumé en français

La Sclérose en plaques (SEP) est une maladie neurodégénérative causée par la perte des oligodendrocytes (OLs), les cellules myélinisantes du système nerveux central.

Figure 01

 01 Figure 01 Timing of neural tube closures in the embryo. Defect in closure 1 lead to craniorachischisis, closure 2 failing lead to anencephaly and closure 3 defects induces Lumbosacral spina bifida and spina bifida occulta.From[START_REF] Copp | The genetic basis of mammalian neurulation[END_REF]

Figure 02

 02 Figure 02 Neural plate folding mecanisms at different levels of the spinal cord. The neural plate initially undergoes a first general bending (a). Then, the upper spine bending is based on a Median Hinge Point (MHP) (b), the intermediate spine bending is based both on two paired Dorsolateral Hinge Point (DLHP)

Figure 03

 03 Figure 03 Finalisation of neural tube closing. The cells of the two folds first contact each other via their lamellipodia (a). Then the folds adhere to each other (b) and finally, apoptosis and remodelling occurs

Figure 05

 05 Figure 05 Corticogenesis and extrinsic factors. Cortex is organised in an outside-in perspective. At E10, BMPs Wnts, FGF and Shh induces the production of Radial Glial Cells (RGCs) from Neural Stem

Figure 07

 07 Figure 07 Oligodendroglial factors expression during cortical gliogenesis. RGCs first differentiate into an intermediate state, the pre-OPC that will then maturate into early and then late OPC. From Huang et al, 2020

Figure 08

 08 Figure 08 Structure of the myelin sheath. Myelin is composed of an alternance of Dense and non dense layers visibles with electron microscopy. They are formed by superposition of the cell membranes from the concentric wrap. Myelin sheaths contain less compacted lines in their structure called longitudinal incisures. From Simons and Nave et al, 2016

Figure 09

 09 Figure 09 Dynamic of the myelin sheath wrapping. After finding an axon to myelinate, the myelin sheath first extends laterally (A), before wrapping around it (B). In detail, oligodendrocyte growth cones first contact the axon (C-a) and form a triangle-shaped structure (C-b). This structure will progress around

  or UVB exposition. Migration studies support this statement as adult migrants from MS low risk countries to Europe keep a low risk of developing the disease where their children born in Europe present a high MS risk similar to the rest of the population (Kurtzke JF, 2013).

Figure 012

 012 Figure 012 Tensins isoforms expression in the brain. Up: Each line represents a Tensin splicing variant expression in several brain areas (column). Brightness is correlated with the isoform expression, dark blue being a strong expression. Down: Representation of each Tensin isoform detected in the brain to this date. Each square represents an exon. Isoforms from the same Tensin are aligned from their starting exon. No Tns4 isoform is expressed at detectable levels to this date. Data from GTEX database (https://gtexportal.org/home/)

Figure 013

 013 Figure 013 Protein domains of the Tensins family members (Tns). Tns1, 2 and 3 shared a C-Terminal SH2-PTB domain and N-terminal Actin Binding domain (ABD) composed of a PTP and a C2 domain, and SH2. Tns4 only contains the SH2-PTB domain. Tns2 possesses an additional C1 domain at its Cterminus and Tns1, a second ABD in its medial part. From[START_REF] Liao | Tensinsemerging insights into their domain functions, biological roles and disease relevance[END_REF]

Figure 014

 014 Figure 014 EGF driven Tns3-Tns4 (cten) reciprocal motility switch. In resting cells, Tns3 is bound to F-Actin and integrin, stabilising the cytoskeleton. EGF expostion induces the repression of Tns3 and the expression of Tns4. At the intergin level, Tns4 bound to integrin but not to actin, destabilising the cytoskeleton and therefore promoting cell motility. FromKatz et al, 2007

Figure 015

 015 Figure 015 DLC1 regulation by the EGF driven Tns3/Tns4 motility switch. In resting cells, Tns3 activates DLC1 by binding to its autoinhibitory SAM domain. Activated DLC1 favors RhoA coupling to GDP, leading to cell resting state. EGFR activation leads to Tns3 repression and Tns4 enhanced expression. Tns4 does not suppress the autoinhibition of DLC1 by its SAM domain, leading to RhoA coupling to GTP and increased cell migration. From Cao et al, 2012

Legend 1 :

 1 5' (Up) and 3' (Down) Tns3 sequences targeted by our Knock In strategy. Highlighted in red and in blue, the genomic sequence corresponds respectively to the 5' and the 3' homology arms of the targeting vector. In red, the sequence of the V5 tag.The 3' or 5' ends were amplified by PCR and genotype for each mouse in order to characterize their potential mutation. Most of the mice presented random nucleotide insertions or deletions at the mutation locus (indels), like the previous attempt. The eights 5' mutated pups born were both presenting a WT allele and a Tns3 KO mutant allele (mostly 4 nucleotide deletions and 1 nucleotide insertion). However, 2 of the 3' mutated mice presented an insertion of the V5 tag sequence (mice n°531 and 532, legend 2). One of them (mice n°531) also presented a 1 nucleotide deletion before the v5 tag sequence that changed his reading frame.We try to reproduce this experiment by microinjecting 50 new mice egg cells with the 3' end targeting vector. 45 survived the parturition but unfortunately none of them presented an insertion of the V5 tag. Mice n° 532 was bred with Rosa STOP-Cas9-GFP mice in order to produce Tns3 V5 ; Rosa STOP-Cas9-GFP mice both tagged for the Tns3 and expressing Cas9 under Cre recombination.Tns3 flox mice were generated at Copenhagen University animal facility with the help of Javier Martin. We designed a 1.5Kb plasmid construct containing the exon9 gene sequence flanked by 2 loxP sites. This plasmid has been electroporated in Embryonic Stem cells (ESCs) and clones with the correct mutation were selected in vitro. ESCs colonies with a correct floxed Tns3 allele were micro-injected into receiver blastocyst, creating a series of chimeric mice whose progeny are Tns3 flox heterozygous. Genotyping of each mouse has been performed at Copenhagen University Animal facility, for both the LoxP upstream and downstream Tns3 exon 9.

FloxedLegend 4 :

 4 Tns3 exonic sequence at the level of exon9. Highlighted in blue, the loxp sequences.Highlighted in green, the exon9. In blue and in red the sequences corresponding respectively to the left and right homology arms. Note that Tns3 exon9 is composed of 65 nucleotides, which deletions induces Tns3 reading frame shift.

Legend 5 :

 5 additional step using the debris removal kit(Miltenyi Biotec; to eliminate the myelin residues. Cells were saturated in a 0,5% NGS solution then incubated with anti-PdgfRa or anti-O4 coupled-beads(Miltenyi Biotec;. Non-bound beads-coupled antibodies have been washed away by centrifugation then bound cells were sorted using MultiMACS Cell24 Separator Plus(Miltenyi Biotec;. Sorted cells were either plated in culture plates for In Vitro cell study or centrifuged at 1200 rpm and use for Western blot analysisWestern BlotProteins from MACSorted cells were extracted using RIPA buffer from ThermoFisher (ref: 89901), supplemented with Halt™ Protease Inhibitor Cocktail (100X) from ThermoFisher (ref: 87786) at the final concentration of 50µL RIPA buffer + protease inhibitors for 1 million cells. The mix has been shaken at 4°C for 30 minutes before being centrifuged at 12000 rpm for 30 minutes. Protein concentration was estimated using the Pierce Detergent Compatible Bradford Assay Kit from Thermofisher (ref: 23246). For each Western Blot column, 50ug of proteins have been incubated for 10 minutes at 95°C with βmercaptoethanol (24X) and BoltTM LDS Sample Buffer (4X) from ThermoFisher (ref: B0007) to denature proteins. Western Blot have been performed using Mini Gel Tank and Blot Module Set from ThermoFisher (ref: NW2000). 12µl of denatured proteins were first load on Bolt™ 4 to 12%, Bis-Tris, 1.0 mm Mini Protein Gel from ThermoFisher (ref: NW04122BOX) and immersed in 4°C Bolt™ MOPS SDS Running Buffer (20X) from ThermoFisher (ref: B0001). Precision Plus Protein™ All Blue Standard from BioRad (ref: 1610373EDU) has been loaded as a migration control. Proteins were left migrating for 90 minutes at 90V. Then gels have been transferred on Amersham Protran 0,2 µm Nitrocellulose membranes from Dutscher (ref: 10600001), immersed in 4°C NuPAGE Transfer Buffer (20X) from ThermoFisher (ref: NP0006-1), for 90 minutes at 60V. Membranes were incubated 1h in 10% dry milk to saturate non-specific antibodies binding sites. Primary antibodies, diluted in TBS-T, were incubated with the membrane overnight at 4°C with shaking. Then membranes were washed 15 minutes with roomtemperature TBS-T 3 times and incubated with HRP-conjugated secondary antibodies, diluted in TBS-T, for 1h at 4°C with shaking. After being washed 3 times 15 minutes in room temperature TBS-T, proteins in the membranes were revealed using Pierce™ ECL Western Blotting Substrate from ThermoFisher (ref: 32109). Membranes were imaged with the ChemiDoc™ Touch Imaging System from BioRad (ref: 1708370) provided by CELIS ICM facility. GAPDH and Actin were used as loading controls to estimate variations of protein levels in samples.ImmunofluorescenceMice were anesthetized using 100 μL phenobarbital (euthasol / Virbac USA) at the time point we wanted and their brains were dissected out. Buprenorphine (30 mg/g) has been administered to the mice at least 30 minutes before the perfusion in order to prevent the pain caused by the phenobarbital injection. Postnatal mice brain till 15 days post-natal (P15) were perfused with 15ml of 2%PFA (Sigma), and the older stages were perfused with 25 ml of 2%PFA, brains from post-natal were dissected out, cryoprotected in PBS with 10% sucrose overnight, then transferred in PBS with 20% sucrose for 2h and included in OCT (BDH) before freezing and sectioning (14μm thickness) in a sagittal plane with a cryostat microtome (Leica). Sections were then either processed for immunohistochemistry after drying them well for one hour at room temperature (RT) or stored at -80°C for stock. Later on those frozen sections were dry for 1 hour at room temperature before adding the blocking solution (Phosphate Buffered Saline (PBS) with 10% normal goat serum (NGS), 0.1% triton X-100) for one hour at RT.The following antibodies, previously diluted at 50% in glycerol, were diluted in the same blocking solution and used at the concentrations indicated in table 1. Secondary Antibodies (AlexaFluor 488, 594 and 647) were all diluted at 50% in glycerol and used at a final concentration of 1:2000. Immunofluorescence was visualized with Zeiss® Axio Imager.M2 microscope with Zeiss® Apotome system. Pictures were taken as stacks of 5-10μm with 0.5μm between sections. Image acquisition , Z-projections and orthogonal projections and processing are achieved by ZEN Microscopy, image quantification was performed automatically on ImageJ, using a macro provided by ICM Quant Facility or performed manually on ZEN. Figures were made using AdobeIllustrator. CRISPR Cas9 strategy to induce Tns3 exon6 frameshiftWe design a plasmid construct ("Ex6 frameshift" plasmid) containing an Ampicilin resistance gene and its promoter (AmpR), a Cas9 and a GFP reporter under the control of a pCMV promoter and a pCAG promoter followed by an empty sequence containing a restriction site for the HindIII (the only one in the plasmid). Plasmid without gRNA insert has been used as an "Empty" control condition for the electroporation.Using HindIII digestion, I have inserted a linear sequence containing the gRNA in the empty sequence to create our experimental plasmid.Creation and optimization of the gRNA and of the backbone plasmid used in this study has been previously performed by Dr Hatem HMIDAN during his pHD (more details in its thesis manuscript). We use a 2A sequence between the Cas9 and the GFP to produce an equal amount of GFP and Cas9 proteins. All the active parts of the plasmid have been flanked with Tol2 sequence, in order to facilitate the insertion of the plasmid in the genome by a transferase expressed by a co-electroporated plasmid.Triple plasmids co electroporation for Ex6 frameshift (and Empty Ctrl)Co-electroporation of 3 plasmids have been used in WT and Tns3 V5 mice in order to increase the number of targeted cells and the intensity of the GFP signal in late differentiated oligodendroglial cells. We used a combination of a VENUS reporter plasmid, of our previously generated "Ex6 frameshift" plasmid and a pCMV HAhyPBase, encoding for the hyperactive form of the transposase (described in[START_REF] Yusa | A hyperactive piggyBac transposase for mammalian applications[END_REF]. Plasmids have been injected with a ratio of 1/7 of HAhyPBase, 2/7 of "Ex6 frameshift" or "Empty" plasmid and 4/7 of VENUS plasmid Legend 6: VENUS plasmid gRNA plasmids for Tns3V5;Rosa-STOP-Cas9-GFP mice electroporations New lighter plasmids were generated after the creation of the Tns3 V5 ; Rosa STOP-Cas9- GFP mice line. Given the fact these mice already have a mutation reporter gene and express the Cas9 ubiquitously , we discard the GFP and the Cas9 sequences from the electroporated plasmid. We also used two newt gRNA targeting either two sites flanking the exon 9 ("exon9" plasmid) or the whole Tns3 locus ("Whole Tns3" plasmid), in addition to the previous Exon6 gRNA. Pups were euthanized by freezing them in ice for 2 minutes then were rapidly decapitated. Brains were collected in PBS 1X (Invitrogen) containing 1% of penicillin/streptomycin before dissecting the SVZ. First, Olfactory bulbs and cerebellum were cutted out. Then, SVZ were dissected (see figure), washed three times in PBS and transferred in neural stem cell fresh medium prepared as following:

Legend 7 :

 7 Dissected brains area for Neurosphere culture

  differentiation genes. Here, to improve our understanding of the mechanisms of OL differentiation, we searched for novel common targets of these TFs and remodelers, by generating and combining the binding profiles of Olig2, Chd7, and Chd8, together with histone regulatory marks in differentiating oligodendroglia. By this strategy, among the genes commonly regulated by these key regulators, we identified Tensin3 (Tns3) focal adhesion protein. We show that Tns3 is expressed in immature OLs during myelination and remyelination, and thus constituting a hallmark for this oligodendroglial stage. Furthermore, using different genetic strategies to induce Tns3 loss-of-function mutations in vivo, we describe for the first time a function for a Tensin protein in the CNS, demonstrating that Tns3 is required in immature OLs for normal oligodendrocyte differentiation in the postnatal mouse brain.

  ):[START_REF] Aggarwal | A Size Barrier Limits Protein Diffusion at the Cell Surface to Generate Lipid-Rich Myelin-Membrane Sheets[END_REF] two types of neural stem/progenitor cells, that we named NSCs and NPCs according to their expression of stem cell (Vim, Hes1, Id1) and neural progenitor (Sox11, Sox4, Dcx) markers; (2) OPCs expressing their known markers (Pdgfra, Cspg4, Ascl1) and cycling OPCs also enriched in cell cycle markers (Mki67, Pcna, Top2); (3) two stages of iOLs, expressing the recently proposed iOLs markers Itpr2 and Enpp6(Marques et al., 2016; Xiao et al., 2016), which can be distinguished by the expression of Nkx2-2 (iOL1 being Nkx2-2 + and iOL2 being Nkx2-2 -), in agreement with our previous characterization by immunofluorescence(Nakatani et al., 2013; Marie et al., 2018); (4) myelin forming oligodendrocytes (MFOLs), enriched in markers such as Slc9a3r2, Igsf8; and (5) two clusters of myelinating OLs, that we named MOL1 and MOL2, expressing transcripts of myelin proteins(Cnp, Mag, Mbp, Plp1, Mog) and some specific markers of each cluster, including Mgst3, Pmp22 for MOL1, and Neat1, Grm3, Il33 for MOL2.

  analyses reveal that TNS3 has a loss of function observed/expected (LOEUF) score of 0.19 (Fig.S9A; https://gnomad.broadinstitute.org), meaning that complete loss of TNS3 causes ~80% developmental mortality, a rate similar to key neurodevelopmental genes such as SOX10 (LOEUF=0.21; Fig.S9B), CHD7 (LOEUF=0.08; Fig.S9C), and CHD8 (LOEUF=0.08; Fig.S9D), contrary to less broadly required factors such as NKX2-2 (LOEUF=0.67; Fig.S9E) and OLIG1 (LOEUF=1.08; Fig.S9F). Therefore, TNS3 loss-of-function variants are badly tolerated in both mice and humans.In vivo CRISPR-mediated Tns3 loss-of-function in neonatal neural stem cells impairs oligodendrocyte differentiationGiven the abovementioned tendency of cells to escape to the full Tns3 deletion in the whole organism, we then fetch to assess for Tns3 requirement during postnatal oligodendrogenesis in vivo by inducing acute Tns3-deletion only in few neural stem cells (NSCs) of the neonatal brain, combining the postnatal electroporation technique with CRISPR/Cas9 technology. We first used integrative CRISPR plasmids, targeting

  Tns3 deletion impairs oligodendrocyte differentiation in the postnatal brain Given the limitation to assess the in vivo penetrance of Tns3 loss-of-function induced by CRISPR/Cas9-mediated mutations, and in order to address in depth the consequences of Tns3 loss-of-function, we designed a Tns3 conditional knockout allele, by flanking with LoxP sites the essential exon 9 (Fig. S11A,D). In this Tns3floxed allele (Tns3 flox ), Cre-mediated recombination induces a transcription frame shift introducing an early stop codon, leading to a small peptide (109 aa) instead of the full length Tns3 protein (1442 aa; Fig. S11B,C). Mouse embryonic stem cells (ESCs) were transfected with a plasmid expressing Cas9, GFP, gRNAs flanking Tns3 exon 9, and Tns3-floxed targeting vector, to induce CRISPR/Cas9-mediated homologous recombination. After verifying the presence of Tns3-floxed allele in Tns3 locus by Sanger sequencing, positive ESC clones were injected into blastocysts to generate Tns3-floxed (Tns3 flox ) mice. Finally, we combined Tns3 flox animals with Pdgfra-CreER T mice, to induce Cre-mediated Tns3 deletion specifically in OPCs, in presence of a Cre reporter background (Rosa26 stop-YFP ) in order to trace Tns3-deleted cells (Fig. 4A).In order to specifically delete Tns3 in postnatal OPCs, we administered tamoxifen at P7 to Pdgfra-CreER T ; Tns3 flox/flox ; Rosa26 stop-YFP (hereafter called Tns3-iKO mice) and control pups (Tns3 flox/flox ; Rosa26 stop-YFP littermates), and analyze its effects in oligodendrogenesis at P14 and P21 (Fig.4A) both in white matter (corpus callosum and fimbria) and grey matter regions (cortex and striatum). We first assessed for the efficiency of Tns3 deletion in Nkx2.2 + /GFP + iOLs from different regions by immunofluorescence using a Tns3 antibody (Sigma Cterminus), finding an almost complete elimination of Tns3 in Nkx2.2 + /GFP + iOLs of Tns3-iKO compared control (Fig.S12B,B',C, arrows and arrowheads). We then assessed for changes in oligodendrogenesis. Remarkably, the number of oligodendrocytes (CC1 + /GFP + cells) was reduced by half in all quantified regions (reduction of 38.95% in the CC, 48.60% in cortex, 50.88% in the fimbria, 38% in the striatum; Fig.4B-D, Fig.S12E-F', Fig.S13B,B') in Tns3-iKO compared to control, while OPC (PDGFRa + /GFP + cells) density was unchanged (Fig.4B-D). Using markers labeling different stages of oligodendrocyte differentiation (iOL1 and iOL2/mOL), we found that the density of early iOL1s (Nkx2.2 high cells) which express the highest levels of Tns3 protein were not changed (Fig.S12B,B',D), that the density of iOL1s (CC1 + /Olig1 -cells) were reduced by 30% in Tns3-iKO compared to controls, while later oligodendrocyte stages (iOL2/mOLs, CC1 + /Olig1 + cells) were reduced by 50%, suggesting that Tns3 is required for normal oligodendrocyte differentiation (Fig.4E-F). Finally, we assessed for possible changes in OPC proliferation and cell death, by immunofluorescence with Mcm2 and activated-Caspase3 recognizing antibodies respectively, finding only reduction tendency in the proliferation of Tns3-iKO OPCs compared to controls (Fig.S13C,C',D), and no evidence of changes in dying OPCs between genotypes.

  oligodendrocytes and can be used as a hallmark for on-going remyelination. Third, analyzing both Tns3 bgeo gene trap mice and two Tns3 KO mice, we show that constitutive Tns3 deletion is detrimental for normal development and that the predicted loss of Tns3 full-length transcript and protein is bypassed in oligodendroglia from surviving homozygous animals, paralleling the intolerance for TNS3 loss-of-function variants in the human population. Fourth, in vivo CRISPR-mediated Tns3-deletion in neonatal stem cells leads to a 2-fold reduction of oligodendrocytes without changes in OPC generation, proliferation, and survival. Fifth, in vivo Tns3 induced knockout (Tns3-iKO) in postnatal OPCs leads to a 2-fold reduction of differentiating oligodendrocytes, without reducing the OPC population, both in grey and white matter brain regions. Finally, video microscopy of primary OPC differentiation cultures indicate that Tns3-iKO differentiating oligodendrocytes present an increased apoptosis compared to control oligodendrocytes from the same cultures, suggesting that Tns3 function is required for normal oligodendrocyte differentiation.

  Flox mice The Tns3 conditional knockout mouse (LoxP-Exon9-LoxP) was generated at the Transgenic Core Facility of the University of Copenhagen. The repair template contained homology arms of 771bp and 759bp length and LoxP sequencing flanking the Exon-9. It was synthetized by Invitrogen and verified by Sanger sequencing. Two gRNAs were designed at the Transgenic Core Facility that target a DNA sequence in the proximity of each LoxP site. The gRNAs were designed in a fashion where the insertion of the LoxP disrupts the targeting site, thus preventing retargeting of the repaired DNA. We have used a mES (mouse embryonic stem cell) method for the generation of this mouse model by transfection (or electroporating) ES cells with the repair construct (dsDNA) together with two plasmidseach containing each gRNA. Identification of the positive mES clones was done via a combination of a PCR genotyping and Sanger sequencing confirmation. Detailed protocols can be provided upon request.MACSDissociation of cortex and Corpus Callosums from V5 mice was done using neural tissue dissociation kit (P)(Miltenyi Biotec;. Briefly, we dissect cortices from P7, P14 or P21 mice. Cortices have dissociated using Miltenyi MACS dissociator(Miltenyi Biotec;. Dissociated cortices were then filtered using Smartstainer 70um(Miltenyi Biotec;. Myelin residues were eliminated from P14 and P21 mice 'cortices during an additional step using the debris removal kit(Miltenyi Biotec;. Cells were saturated in a 0,5% NGS solution then incubated with anti-PDGFRα or anti-O4 coupled-beads(Miltenyi Biotec;.

  , using iDeal ChIP-seq kit for Transcription Factors (Diagenode, C01010055). Briefly, O4 + MACSorted cells were fixed in 1% formaldehyde (EMS, 15714) for 10 min at room temperature and reaction was quenched with 125 mM glycine for 5 min at room temperature. Lysates were sonicated with a Bioruptor Pico sonicator (Diagenode, total time 8 min) and 4μg of antibodies were added to sheared chromatin (from 4 million cells for Olig2 and from 1 million cells for histone marks) and incubated at 4°C overnight. Antibodies used were: mouse anti-Olig2 antibody (Millipore, MABN50), rabbit anti-H3K4me3 antibody (Active motif, 39060), rabbit anti-H3K27Ac antibody (Active motif, 39034), rabbit anti-H3K4me1 antibody (Ozyme, 5326T), mouse anti-H3K27me3 antibody (Abcam, ab6002). Mock (Rabbit IgG) was used as negative control. Chromatin-protein complexes were immunoprecipitated with protein A/G magnetic beads and washed sequentially. DNA fragments were then purified. Input (non-immunoprecipitated chromatin) was used as control in each individual experiments.

FindAllMarkers

  function, adopting mainly the nomenclature from Marques 2016. Using the subset function, we selected only the clusters containing neural progenitors and oligodendroglia cells. Using the merge function, we combined both oligodendroglial datasets into a single Seurat object (OLgliaDP) containing 5516 cells. The new object was subjected to NormalizeData, FindVariableFeatures, ScaleData, RunPCA, and RunUMAP functions with default parameters. Different OPC clusters were fused into a single one keeping apart the cycling OPC cluster. For DimPlots and Dotplots, clusters were ordered by stages of oligodendrogenesis from neural stem cells (NSCs) to myelinating OLs.

  them well for one hour at room temperature (RT) or stored at -80°C for a later use, and to dry those frozen sections for 20 minutes at room temperature also for 20 minutes before adding the blocking solution (Phosphate Buffered Saline (PBS) with 10% normal goat serum (NGS, Eurobio, CAECHVOO-OU), 0.1% triton X-100) for one hour at RT. The following primary antibodies were diluted in the same blocking solution and used at the concentration indicated in Table1. Fluorescent secondary antibodies included the following: AlexaFluor-488, AlexaFluor-594. Finally, cell nuclei are labeled with DAPI (1/10000, Sigma-Aldrich®, D9542-10MG), and slices are mounted in Fluoromount-G® (SouthernBiotech, Inc. 15586276).

ThermoFisher ( 50

 50 µL per million cells; ref: 89901), supplemented with Halt™ Protease Inhibitor Cocktail (100X) from ThermoFisher (ref: 87786). Protein concentration in the supernatant was estimated using the Pierce Detergent Compatible Bradford Assay Kit from Thermofisher (ref: 23246). For each Western Blot, we used 50ug of proteins denaturated for 10 minutes at 95°C with b-mercaptoethanol (24X) and BoltTM LDS Sample Buffer (4X) from ThermoFisher (ref: B0007). Western Blot have been performed on Bolt™ 4 to 12%, Bis-Tris, 1.0 mm Mini Protein Gel from ThermoFisher (ref: NW04122BOX), immersed in 4°C Bolt™ MOPS SDS Running Buffer (20X) from ThermoFisher (ref: B0001) using Mini Gel Tank and Blot Module Set from ThermoFisher (ref: NW2000). Precision Plus Protein™ All Blue Standard from BioRad (ref: 1610373EDU) has been loaded as a migration control. Proteins have migrated for 90 minutes at 90V. Then gels have been transferred on Amersham Protran 0,2 µm Nitrocellulose membrane from Dutscher (ref: 10600001) immersed in 4°C NuPAGE Transfer Buffer (20X) from ThermoFisher (ref: NP0006-1) for 90 minutes at 60V Membranes were incubated 1h in TBS-T, 10% dry milk to be saturated from nonspecific antibodies reaction. Primary antibodies, diluted in TBS-T, were incubated with the membrane overnight at 4°C with shaking. After three TBS-T wash, membranes were incubated with HRP-conjugated secondary antibodies, diluted in TBS-T, for 1h at 4°C with shaking, then revealed using Pierce™ ECL Western Blotting Substrate from ThermoFisher (ref: 32109) and imaged with the ChemiDoc™ Touch Imaging System from BioRad (ref: 1708370) provided by CELIS ICM facility. Detection of actin has been used as loading control.

5 '

 5 -GCT ATC ATC CCC ACT CAC CA-3'; annealing temperature of 64°c, the PCR result of this primer pairs is 326bp. DNA from 3' Tns3 target region was amplify using primer sequences: Forward: 5'-CCAGTCAGTGGTGACATTGTTT -3', Reverse: 5'-ACTGTTCCCAGGTTGCTATCAT -3'), the annealing temperature 58°c, giving a band of 419bp, Common PCR settings being: Initial denaturation 95°c for 5 minutes (1 cycle), Denaturation 95°c for 30 seconds, Annealing 64/58°c for 30 seconds, Elongation 72°c for3 minutes (35cycles), and final elongation 72°c for 7 minutes. For the PCR reaction mix (20µl final volume): 4µl of 5x FIREPol master mix (Solis BioDyne), 1µl each forward and reverse primer (10pmol/µl), 2µl from the DNA and d H2O to 20µl. Cutting effecieny of sgRNA was verified by T7 endonuclease I, following beta protocol of IDTE synthetic biology for amplification of genomic DNA and detect mutations and using PAGE (Polyacrylamide gel electrophoresis).

Figure legends Figure 1 :

 legends1 Figure legends

Figure

  Figure S1: Tns3 is a gene target of Olig2, Chd7, and Chd8 key oligodendrogenic regulators

Figure 2 :

 2 Figure 2: Tns3 protein is present at high levels in the cytoplasm and main cell processes of immature oligodendrocytes in the postnatal brain

Figure S4 :

 S4 Figure S4: Tns1 and Tns2 proteins are detected at low levels in immature oligodendrocytes

Figure

  Figure S5: Tns3 is expressed in newly formed oligodendrocytes during brain remyelination

Figure S6 :

 S6 Figure S6: Oligodendrocyte differentiation is normal in Tns3 bgeo/bgeo mice, which express full-length Tns3 transcripts in the brain

Figure S10 :

 S10 Figure S10: CRISPR-mediated Tns3 coding sequence deletion in NSCs reduces oligodendrocyte differentiation in the postnatal brain

Figure S11 :

 S11 Figure S11: Generation of Tns3 conditional allele

Figure 4 :

 4 Figure 4: OPC-specific Tns3 deletion reduces oligodendrocyte differentiation in the postnatal brain

Figure

  Figure S12: Efficient OPC-specific Tns3 deletion in the postnatal brain and reduced oligodendrocyte differentiation

Figure

  Figure S13: OPC-specific Tns3 deletion reduces oligodendrocyte differentiation in the postnatal brain

Figure 3 :

 3 Figure 3: Tns3 expression pattern during oligodendrogenesis. (A) Tns3 is expressed in the less mature Opalin+ cells (arrow) but not in the most mature oligodendrocytes (arrowhead). (B) Tns3 starts to be expressed in Nkx2.2 positive cells (1) stay expressed after Nkx2.2 extinction (2) and even in Olig1+ cells (3) but decrease as they mature (4). (C) Tns3 expression correlates well with Itpr2 and Nkx2.2, two iOLs markers. Schematic of the Tns3 pattern of expression during oligodendrogenesis.

Figure 4 :

 4 Figure 4: Tns3 In Vitro expression in oligodendroglia. (A) At 5 days of differentiation, Tns3 is expressed in almost all CNP+ cells. (B) Tns3 is expressed not only in oligodendrocyte soma but also in their processes in small dots. (C) These Tns3 dots overlap with β1 integrin expression in CNP cells. (D) Schematic of Tns3 binding in cells based on the literature (based on data found in[START_REF] Cui | Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3[END_REF]

Figure 5 :

 5 Figure 5: Tns3 is expressed in newly formed oligodendrocytes during brain remyelination (A,B) Tns3expressing cells in the CC of adult (P90) mice, 7 days after demyelination showing strong Tns3 expression in CC1 + /Olig1 -iOL1s (arrowheads) and CC1 + /Olig1 citoplasmic iOL2s (arrows) around the lesion using either anti-V5 antibody in Tns3 Tns3-V5 mice (A-A1) or anti-Tns3 antibody in wild-type mice (B-B1). Note the absence of CC1+/Olig1+ OLs in the lesion. (C-C1) Tns3 expression in CC1 high iOLs (arrows) around the lesion and in some F4/80+ microglia (arrowheads) in the lesion area. A1, B1 and C1 are higher magnification of corresponding insets (squares). (D) Quantification of the Tns3+ OLs density every 50mm away from the lesion. Scale bar, 20 m.

Figure 6 :

 6 Figure 6: Homozygous Tns3 KO induces a sublethal phenotype. (A) Genotypes of the newborn mice in double Tns3 KO/WT breedings. (B) Genotypes of E14.5 embryos from double Tns3 KO/WT breedings. (C) Growth defects in homozygous Tns3 KO pups (blue arrows) at P8 (left) and P14 (right). (D) Sublethal

Figure 7 :

 7 Figure 7: CRISPR-mediated Tns3 mutation in NSCs reduces oligodendrocyte differentiation in the postnatal brain. (A) Schematics of the Tol2-PX458 CRISPR/Cas9 expression vector allowing Tol2-DNA integration driving Cas9 and GFP expression (from polycistronic 2A-mediated cleaved) from CAG promoter and gRNA expression from U6 promoter. (B) Schematics of the dorsal SVZ electroporation of Tol2-P458 plasmid at postnatal day 1 (P1) and immunofluorescence analysis carried out in sagittal brain sections at P22. (C) Schematics of the Tns3 CRISPR-Cas9 mutation in NSCs and of their differentiation. (D) Immunofluorescence of representative P22 sagittal sections of the dorsal telencephalon showing GFP+ cells being either PDGFR+ OPCs (arrowheads), CC1high OLs (arrows) or CC1low astrocytes (asterisks) progeny of P1 NSCs electroporated either with Ctrl plasmid (D) or Tns3-gRNA#1 plasmid (D'). Scale bar, 20 m. (E) Histogram showing the percentage of GFP+ glial cell-types found in Ctrl, gRNA#1 or gRNA#2 electroporated brains being PDGFR+-OPCs, CC1high-OLs and CC1low-astrocytes. Note the 2-fold reduction of CC1+ OLs in Tns3-gRNA transfected brains,

Figure 8 :

 8 Figure 8: Optimisation of the CRISPR Cas9 strategy. (A) Immunostaining of the oligodendroglia in P21 mice telencephalon electroporated at P1 with a Exon6 frameshift gRNA. (B) Number of cells quantified per brain. In light grey, the total number of cells targeted with the old protocol. In dark grey, the mean number of cells quantified with the new system. (C) Histogram showing the percentage of GFP+ glial cell-types found in Ctrl or gRNA#2 electroporated brains being PDGFR+-OPCs, CC1high-OLs and CC1low-astrocytes. Note the 40% reduction of CC1+ OLs in Tns3-gRNA transfected brains.

Figure 9 :

 9 Figure 9: Tns3 isoforms expression in oligodendroglia. (A) Schematic of the two main TNS3 isoforms exon expressed in the human brain (gtexportal.org/home/gene/TNS3). Dark blue is associated with higher expression. (B) V5 Western Blot from P35 Tns3 V5 mice and oligodendrocytes (OLs) after 4 days of differentiation in vitro. Note the absence of the Tns3 full length isoform in the total brain extract but not in cultured OLs. (C) V5 Western Blot from MACSorted oligodendroglial cells. At P7, MACSorted

Figure 10 :

 10 Figure 10: CRISPR-mediated Tns3 mutation in NSCs reduces oligodendrocyte differentiation in the postnatal brain. (A) Schematic of the integrative CRISPR/Cas9 expression vector allowing the expression in transfected cells of 2 gRNA targeting the Tns3 3' and 5' and the expression of the Cas9 protein. (B) Schematic of the dorsal SVZ electroporation of Tol2-P458 plasmid at postnatal day 1 (P1) and immunofluorescence analysis carried out in sagittal brain sections at P22. (C) Schematics of the Tns3 CRISPR-Cas9 whole deletion in NSCs and of their differentiation. (D) Representative P22 sagittal sections of the dorsal telencephalon showing GFP+ cells being either PDGFR+ OPCs (arrowheads), CC1high OLs (arrows) or CC1low astrocytes (asterisks) progeny of P1 NSCs electroporated either with Ctrl plasmid (D) or Tns3-5'-3' plasmid (D'). Scale bar, 20 m. (E) Histogram showing the percentage of GFP+ glial cell-types found in Ctrl or 3'-5' gRNA electroporated brains being NSCs in the SVZ or

Figure 11 :

 11 Figure 11: Tns3 expression is suppressed in similar proportions with Exon6 frameshift and whole locus deletions. (A) Representative P22 sagittal sections of the dorsal telencephalon electroporated with the Empty (Right) or the Exon6 plasmid (Left). (G) Histogram showing the percentage of GFP+ Nkx2.2+ cells found in Ctrl or Exon6 gRNA electroporated brains. In dark grey, the GFP+ Nkx2.2+ cells expressing Tns3. In light grey, the GFP+ Nkx2.2+ cells with no Tns3 expression. Note the 2-fold reduction of GFP+ Nkx2.2+ Tns3+ in both conditions and the absence of effect on the GFP+ Nkx2.2+ Tns3-population.

Figure 12 :

 12 Figure 12: Tns3 slightly impair OLs differentiation in vitro at 3 days of differentiation. (A) Representative staining of primary Tns3 V5 NSCs cultures transfected with the Empty (Up) or the Exon6 AAV (Down). (B) Quantification of the OLs percentage among the transfected cells at 2 days (2dd) and at 3 days of

Figure 13 :

 13 Figure 13: Generation of Tns3 conditional allele. (A) Schematic of Tns3 locus, Tns3 Flox allele and targeting vector with LoxP sites flanking Exon9, used to induce homologous recombination in mouse ESCs. (B) Full-length Tns3 amino-acid sequence indicating its protein domains. Squared in red highlights the 109 amino-acid sequence of the Tns3 peptide produced upon Cre-mediated recombination in Tns3 flox expressing cells. (C) Peptide sequence of predicted for Tns3-iKO expressing cells.

Figure 14 :

 14 Figure 14: Tns3 iKO impairs OLs generation at P14 in the Cortex but not in the fimbria and the corpus callosum. (A) Scheme of Tns3 recombination in P7 mice before being analysed at P14. (B) Representative sagittal sections of Tns3 flox or Tns3 WT brains. (B) Histograms showing the percentage of GFP+ glial cell-types found in Tns3 flox or Tns3 WT brains being NSCs in the SVZ or PDGFR+-OPCs, CC1high-OLs and CC1low-astrocytes in the CC, the Ctx and the fimbria. Note the 2-fold reduction of CC1+ OLs only in the Cortex of Tns3 flox brains, as shown in B.

Figure 15 :

 15 Figure 15: OPC-specific Tns3 deletion reduces oligodendrocyte differentiation in the postnatal brain: (A) Scheme of tamoxifen administration to Tns3-iKO and control (Cre + ; Tns3 +/+ ) mice, Cre-mediated genetic changes, and timing of experimental analysis. (B-B', C-C') Immunofluorescence in P21 sagittal brain sections for CC1, GFP and PDGFRa illustrating similar density of OPCs and 2-fold reduction in OL density in Tns3-iKO (B', C') compared to control (B, C) in the fimbria (B) and the cortex (C). (D) Histograms showing OPC and OL density in P21 Tns3-iKO and control (Ctrl) mice, in the corpus callosum, fimbria, cortex, and striatum. Note the systematic OLs decrease of 40-50% in each region. (E-E1') Immunofluorescence in P21 sagittal brain sections for Olig1, GFP and CC1 to distinguish three stages of oligodendrogenesis: OPCs (Olig1 + /CC1 -), iOL1s (CC1 + /Olig1 -) and iOL2s/mOLs (CC1 + /Olig1 + ) in Ctrl (E) or Tns3-iKO mice (E'). E1 and E1' are higher magnification of the squared area in E and E'. (F) Histograms showing the OPCs, the iOL1s and the iOL2s/mOLs density in P21 Tns3-iKO and control mice, in the corpus callosum, fimbria, cortex, and striatum. Note the decrease of iOL1s and iOL2s over 40% in each area quantified (except for iOL1 density in the corpus callosum). (G) Schematic representing defects in oligodendrogenesis found in Tns3-iKO compared to control.

Figure 16 :

 16 Figure 16: Efficient OPC-specific Tns3 deletion in the postnatal brain and reduced oligodendrocyte differentiation: (A) Scheme of tamoxifen administration to Tns3-iKO and control (Cre + ; Tns3 +/+ ) mice, Cre-mediated genetic changes, and timing of experimental analysis. (B-B') Immunofluorescence for Tns3, GFP and Nkx2.2 in P21 mice sagittal brain sections at the level of the corpus callosum, illustrating the loss of Tns3 signal in Nkx2.2 high iOL1s (arrows) but not in vessels (asterisk) of Tns3-iKO (B') compared to control (B). Note that Nkx2.2 high iOL1s do not change in density in Tns3-iKO compared to control. (C) Histograms showing the Tns3+ cells density in the corpus callosum, fimbria, cortex, and striatum, in Tns3-iKO and control. (D) Histograms showing the Nkx2.2 high iOL1s density in the corpus callosum, fimbria, cortex, and striatum, in Tns3-iKO and control.

Figure 17 :

 17 Figure 17: (C-C'). Immunofluorescence for MCM2 proliferative marker, GFP, and PDGFRa illustrating similar proportion of proliferative OPCs in control (C) and Tns3-iKO mice (C'). (D) Histograms quantifying the proliferative fraction of GFP + OPCs in the fimbria (Up) and corpus callosum (Down). Note the slight reduction (non-significative) in proliferation in Tns3-iKO OPCs compared to control OPCs.

Figure 19 :

 19 Figure 19: Cellular defects of Tns3iKO iOL by videomicroscopy of OPC differentiating cultures. (A) Schematics of the videomicroscopy protocol. Tns3flox mice were injected with tamoxifen at P5 and their OPCs were MACS at P7, cultivated on P-L-O coated culture plates and left proliferating for 3 days. Cells were imaged every 10 minutes during 72h in differentiating medium. (B) Histogram showing the reduction of the GFP+ cells proportion from the plating (-72h) to the end of the experiment (72h after differentiation onset). (C) Time curve showing the increase of mean loss percentage for GFP+ compared to GFP-cells during the 72h of differentiation. (D) Histogram showing the mean number of loss cells per hour during the 72h differentiation, with a 5 time increase for GFP+ cells from Tns3flox mice compared to the GFP-non-recombined cells. Note the mean death per hour in Tns3flox;

  The precise involvement of the different Tns3 isoforms in oligodendrogenesis is still an open question. Indeed, Tns3 mRNA RT-qPCR and Western Blot analyses both indicate that the small 60kDa Tns3 isoform is the most expressed one in all oligodendroglial lineages, except in the most matures stages where almost no Tns3 is detected. But this isoform only contains the C-terminus which contains the SH2-PTB domain, allowing it to bind only to the C-term interacting proteins of full-lengh Tns3, especially β1-Integrin, but not to actin. It means that, contrary to full-length Tns3 whose function is mainly to stabilise the actin cytoskeleton at focal adhesion plates, small

  Même si l'attaque auto-immune contre les oligodendrocytes peut désormais être réduite grâce à l'administration d'immunosuppresseurs, aucun traitement permettant la remyélinisation des lésions n'existe à ce jour. Chez le cerveau des patients atteints de SEP, une remyélinisation par les précurseurs d'OLs (OPCs) a lieu mais est inefficace et diminue avec l'âge. La présence d'OPCs dans ces lésions échouant à se différencier en cellules myélinisantes suggère que l'induction de la différenciation des OPCs est une étape indispensable à une remyélinisation efficace. Nous avons découvert que Tensin3 (Tns3) était une cible directe pour des facteurs oligodendrogeniques tel que Chd7, Chd8 ou Olig2. Tns3 est fortement induit au début de la différentiation oligodendrogliale mais est réprimé dans les OLs matures, ce qui en fait un bon marqueur des OLs immatures. J'ai caractérisé le schéma d'expression de Tns3 dans durant l'oligodendrogenèse en montrant que la protéine Tns3 était 1) spécifiquement trouvée durant les stades immatures des OLs, 2) localisée dans le cytoplasme et les extensions cytoplasmiques oligodendrogliales, et 3) exprimée en même temps que d'autres marqueurs connus des oligodendrocytes immatures tel que Itpr2 ou Nkx2.2. Tns3 est aussi exprimée dans les nouveaux OLs formés en réponse à une démyélinisation induite par injection de LPC, ce qui en fait un nouveau marqueur des OLs immatures. La perte à la naissance de Tns3 par CRISPR-Cas9 dans les cellules souches neurales de la zone sous-ventriculaire bloque leur différenciation en oligodendrocytes sans affecter la prolifération ou la survie des OPCs. Il semble que la délétion totale du locus produise les mêmes effets qu'une suppression de l'isoforme complète de Tns3, par décalage du cadre de lecture. De plus, une suppression de Tns3 spécifique aux OPCs, dans un modèle de souris floxée pour Tns3, réduit également la production d'OLs sans affecter les OPCs et les OLs immatures positifs pour Nkx2.2. Enfin, des données préliminaires suggèrent que la délétion de Tns3 dans des OPCs triés par MACS retarderait leur différenciation et réduirait leur survie durant les 72 première heures. Tout cela démontre que Tns3 est impliquée dans la formation d'oligodendrocytes matures.

Table 1

 1

	Reagent or Resource		Source	Identifier	Concentration
					1023627600
	DAPI			Sigma-Aldrich	1	1:10000
	Fluoromount-G®		SouthernBiotech, Inc.	0100-01
	Antibodies
	Rabbit anti Tensin1		Su Hao Lu, UC davis	None	1:100
	Rabbit anti Tensin2		Su Hao Lu, UC davis	None	1:100
				Sassan HAFIZI, University of
	Rabbit anti Tensin3		Portsmouth	None	1:1000
	Rabbit anti Tensin3		Millipore	AB229	1:500
	Rabbit anti Tensin3		Thermofisher	PA5-116022 1:1000
	Mouse	monoclonal	anti
	Tensin3			Santa Cruz Biotech	sc-376367	1:500
					SAB420020
	Rabbit anti Tns3 Cterminus	Sigma-Aldricht	5	1:200
	Rabbit anti V5 tag		Millipore	AB3792	1:2000
	Mouse monoclonal anti V5 tag Invitrogen	R960-25	1:1000
	Rat anti PDGFRα		BD Biosciences	558774	1:250
	mouse monoclonal anti Olig1 NeuroMab	75-180	1:1000
	Mouse monoclonal anit Olig2 Millipore	MABN50	1:500
	Rabbit anti Sox10		Ozyme	89356	1:500

Acknowledgements

ACKNOWLEDGMENTS We thank D. Bergles for the PDGFRa::CreER T mice. All animal work was conducted at the ICM PHENOPARC Core Facility. Data generated relied on ICM Core Facilities: bioinformatics (ICONICS), sequencing, CELIS, histology, and ICM Quant, and we thank all personnel involved for their contribution and help. The Core Facilities were supported by the "Investissements d'avenir" (ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS) and the "Fondation pour la Recherche Médicale". This work was supported by funding by grants from the National Multiple Sclerosis Society (NMSS RG-1501-02851), and the Fondation pour l'Aide à la Recherche sur la Sclérose en

Plaques (ARSEP 2014(ARSEP , 2015(ARSEP , 2018(ARSEP , 2019(ARSEP , 2020)). E.M, H.H, and C.M. were supported by funding from Sorbonne Université. C.M. was also supported by Fondation pour la Recherche Médicale (FRM, FDT20160435662) and ARSEP grant 2018-2020. 139 140

Neurospheres extracted from Tns3 WT ; Pdgfra-CreER T/WT ; Rosa26 YFP mice were used as control. Adeno-Cre was used at a MOI of 5. Frozen Tns3 V5 ;Rosa STOP-Cas9-GFP or Tns3 flox ;Pdgfra-CreER T ;Rosa YFP neurospheres, have been thawed in NSC fresh medium then pre-incubated during 3 days in

Viral transduction in primary Neurospheres cultures

Results

In this manuscript, I will first present the article we wrote and are currently editing before sending it for publication to a peer-reviewed journal and to BioRxiv. In a second time, I will detail the experiments presented in the article with supplementary information to present an exhaustive review of our knowledge on this topic.

ABSTRACT

Oligodendroglia play a key role in brain development and neuronal activity support, and accumulating evidence indicates its fundamental contribution to brain plasticity, memory, and pathology underlying glioma, neurodevelopmental, neurodegenerative, and psychiatric diseases. Oligodendrocyte precursor cells (OPCs) constitute a stable population of the CNS, that generates oligodendrocytes (OLs) on demand both during development and adulthood, by keeping a tight balance between proliferation, survival, and differentiation. Both during aging and pathology OPC differentiation diminishes affecting normal brain homeostasis and myelin repair (Neumann et al., 2019a), such as in multiple sclerosis patients. To better characterize the mechanisms promoting OPC differentiation and myelination, we screened for genes regulated by Olig2, Chd7, & Chd8, key regulators of OL differentiation and identified the focal adhesion protein Tensin3 (Tns3). Developing new genetic tools to characterize Tns3 expression and function in the brain, we provide several lines of evidence showing that Tns3 is expressed in immature oligodendrocytes (iOLs) and is required for normal oligodendrocyte differentiation. Using that both V5-recognizing antibodies in Tns3 Tns3- V5 knock-in mice and Tns3 validated antibodies, we show that Tns3 protein is restricted to iOL stage during myelination and remyelination, thus constituting a novel marker for iOLs. We show that Tns3 loss-of-function mutations are badly tolerated in mice, as well as in the human population, and that constitutive Tns3 mutant mice escape to Tns3 deletion in OLs. We demonstrate that postnatal Tns3-deletion reduces by half the number of OLs generated during the first postnatal month, both by CRISPRmediated Tns3-deletion in neonatal neural stem cells and by Tns3 induced knockout (Tns3-iKO) in postnatal OPCs. Finally, video microscopy of primary oligodendroglial cultures indicates that Tns3-iKO differentiating OLs present an increased apoptosis, suggesting that Tns3 function is required for normal OL differentiation.

Generation of Tol2-integrative plasmids

In order to generate plasmids that will insert CRISPR tools into the genome of the tranfected cells and lead to permanent expression of the targeting tools, we subclone the PX458 (GFP) or PX459 (Puromycin) plasmids into Tol2 containing sequences backbone (obtained from Tol2-mCherry expressing plasmid kindly provided by Lean Livet, Institut de la Vision, Paris).

Data Resources

Raw data files have been deposited in the NCBI Gene Expression Omnibus under accession number GEO: XX

Contact for Reagent and Resource Sharing

Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author Carlos Parras (carlos.parras@upmc.fr).

Detailed results

Tns3 is regulated by keys oligodendroglial factors

Prior to this project, we performed a chromatin immunoprecipitation analysis (ChIP) in order to identify the direct binding targets of Olig2, Chd7 and Chd8, three keys oligodendrogenic regulators, together with key histone marks (H3K4Me1, H3K4Me3, H3K27Ac, H3K27Me3) for activity of regulatory elements, in OPCs and OLs MACsorted from mouse brains (Figure1 A). Interestingly, we found three genomic positions commonly bound by these key regulators, corresponding to Tns3 putative promoters (middle and right square, Figure 1A Down) of the different Tns3 isoforms. Indeed, our profiling of histone marks in oligodendroglia indicated that they correspond to actively expressed promoters in these cells and having histone marks of active promoter elements (H3K4Me3 and H3K27Ac marks, Figure1 A Down), suggesting an involvement of Tns3 during oligodendrogenesis.

We observed a clear reduction Tns3 transcripts both in OPCs in which a deletion of Chd7 (Chd7iKO, Figure 1B) or Chd8 (Chd8iKO, Figure 1C) was acutely induced in the postnatal brain (Marie C et al, 2018), indicating that Tns3 is upregulated by these two key regulators of oligodendrocyte differentiation. Transcriptomes from the purified brain cells (Zhang and Barres Brain RNA-Seq database, www.brainrnaseq.org), show that Tns3 mRNA was detected at low levels in all mice glial cells (astrocytes, microglia and OLs) but presented a 5-fold stronger expression in immature OLs (Figure 1D). This expression pattern was paralleled by the sparse strong labelling detected by in situ hybridization with Tns3 probes from the Allen mouse Brain Atlas (https://mouse.brain-map.org), enriched in white matter regions of the postnatal and adult brain (Figure 1E). To identify the nature of this signal, we hypothesize this could be a non-specific recognition of similar proteins, like Tns1 or Tns2. Quickly, we looked at the Tns1 and Tns2 expression in oligodendroglia and surprisingly found a low cytoplasmic signal excluded from the nucleus (Figure 2F-F'). Therefore, this nonspecific signal is not caused by a cross recognition of Tns1 or Tns2. However, considering the high levels of staining detected in OL nuclei with the non-specific Tns3, the identification of the protein causing this non-specific signal could provide an interesting new marker of the oligodendrocytes in the brain.

Tns3 is highly expressed in an iOL1 subpopulation

In order to confirm that this signal was really non-specific of Tns3, we generated a mice knockin line in which the Tns3 was tagged with a V5 sequence (Tns3 Tns3-V5 mice), not found in the mice genome. Immunostaining revealed a cytoplasmic Tns3-V5 expression (Figure 1G), confirming that the nuclear signal previously observed was non-specific of Tns3 is OLs. For the rest of this study, Tns3-V5 and the Sigma Aldrich

Tns3 expression during remyelination phenotype identified by the IMPC (https://www.mousephenotype.org/). (E) Histogram of the mean bone mineral content in Tns3 KO compared to WT mice. (F)

Immunofluorescence showing that Tns3 is still detected in CC1+ OLs of Tns3 4de/4del P21 mice, with four different Tns3 recognizing antibodies (Millipore, SantaCruz, Sigma, and Sassan Hafizi lab)

. (G) Histogram representing RT qPCR on cDNA from P21

brains showing no differences in the amplification of Tns3 Exon 17 and Exon31 (C1) between Tns3 4de/4del , Tns3 4de/+ or WT mice, indicating the presence of a long Tns3 transcript containing these exons.

Tns3 exon6 frameshift by CRIPSR-Cas9 in NSCs

Given that Tns3 constitutive knockout mice models were not suitable to study the requirement of Tns3 during oligodendrogenesis, we decided to create our own knockout model in brain cells. We designed a CRISPR-Cas9 strategy in order to target the Tns3 expression of only a small OL subpopulation (Figure 7A), to avoid compensatory effects, by electroporating plasmids in telencephalic NSCs between P1 to P2 (Figure 7B). This strategy was based on the integration of a single plasmid expressing both the guidance RNA (gRNA), the Cas9 protein and a GFP reporter. We then quantify the progeny of the mutated NSCs either at P21, after the end of most OPC differentiation in the cerebral cortex (Figure 7C).

The two best gRNA for their cutting efficiency in vitro and for presenting the lowest off target effects were selected and used for electroporation. At P21, we stained brain sections finding a GFP signal in NSCs, astrocytes, OPCs and OLs both in Ctrl (Figure 7D), gRNA#1 and gRNA#2 electroporated mice (Figure 7D'). We found a clear reduction by over 50% of the OLs generation from NSCs with both Tns3 targeting gRNAs compared to control (same CRISPR plasmid without Tns3 targeting sequencing) with no effect on the OPC population (Figure 7E), suggesting that Tns3 is required during oligodendrogenesis. I then performed similar experiment and analysed the brain at P11 when most cortical OPCs have not yet started differentiation. Interestingly, I found no changes in the number of OPCs between experimental and control conditions, and detected a tendency to decrease in the number of OLs in mutated mice (Figure 7G), suggesting that Tns3 deletion in NSCs do not impairs OPC generation. No clear differences between mutant and control OPC proliferation (Figure 7J), and no clear evidence of increased cell death could be observed in mutant OPCs using a Caspase3 staining (not shown).

as illustrated in D' compared to D. (F) Schematics of experimental design of data provided in G-J panels. (G) Histograms representing the percentage of GFP+ differentiated cells at P11. Note the lack of changes in OPCs, and the incipient reduction in OLs. (H-I') Immunofluorescence for PDGFR from P11 mice electroporated with Ctrl plasmid (H, I) or Tns3-gRNA#1 plasmid (H', I') together with MCM2 (H, H') and activated Caspase3 (I-I'), illustrating similar MCM2 labelling and the absence of

All together, these data suggest that Tns3 is required for OL differentiation without affecting OPC proliferation or OPC survival (Figure 7K). However, the small proportion of targeted cells make it hard to make a clear conclusion from P14 caspase3 and MCM2 staining. We needed a way to affect a greater number of cells to study Tns3+ cells in detail, as they represent a small transient fraction of the whole oligodendroglial cell population.

Optimisation of the Tns3 CRISPR-Cas9 knockout system

During my first PhD year, I optimized the CRISPR-Cas9 strategy in order to target a greater number of cells. I switched the former pCMV promoter controlling the gRNA expression to a pCAG, known to induce a greater gene expression than the short cytomegalovirus reporter. In our first strategy, the part containing the gRNA, the Cas9 and the GFP sequences was flanked by two Tol2 sequences, in order to allow their stable integration in the transfected cell genome by a transposase, encoded a coelectroporated plasmid. However, it appears this integration was not occurring as the GFP signal was faint in the most differentiated oligodendrocytes. It is likely that the GFP produced was indeed diluted among NSCs' daughter cells at each division, making it harder and harder to detect recombined oligodendrocytes as they differentiate.

differentiation (3dd). Note the absence of difference at 2dd and the 30% decrease at 3dd. (C) Histogram

showing the percentage of GFP+ Tns3+ cells found in cultures after 3dd.

This tendency is associated with a non-significant reduction of Tns3+ expression in CNP+ OLs in cultures transfected with Tns3-targeting gRNA AAVs. These preliminary results suggest that the effects of the Tns3 KO are also present in culture, starting at 3 days of differentiation. Nevertheless, none of these data reached statistical significance, very likely due to the fact we only looked at the onset of Tns3 in vitro expression, without doing later time point. We are thus currently planning to reproduce these experiments looking at the effect of Tns3 deletion after 4-5 days differentiation.

In vivo Tns3 induced knockout of the postnatal OPCs population

Even after optimization, one major drawback of the CRISPR-induced Tns3-mutations by electroporation is the limited number of mutated cells. Given this limitation and the difficulty to assess the in vivo penetrance of Tns3 loss-of-function induced by CRISPR/Cas9-mediated mutations, and in order to address in depth the consequences of Tns3 loss-of-function, we designed a Tns3 conditional knockout allele, by flanking with LoxP sites the essential exon 9 (Fig. 13A,D). In this Tns3-floxed allele (Tns3 flox ), Cre-mediated recombination induces a transcription frame shift introducing an early stop codon, leading to a small peptide (109 aa) instead of the full length Tns3 protein (1442 aa; Fig. 13B,C Considering these findings, I first tried to record a timelapse Live Imaging of the OPCs differentiation of these cultures (not shown). Unfortunately, the wide diversity of generated cells makes it hard to record one precise OLs. To tackle this issue, I decided to reproduce this timelapse Live Imaging but using MACSorted OPCs from Tns3 flox ; Pdgfra-CreER T ; Rosa YFP P7 pups previously recombined by a Tamoxifen injection at P5. OPCs were left 3 days to proliferate and imaged every 10 minutes during 3 other days both for YFP fluorescence and phase contrast (Figure 19A).

A first quantification after the plating of these OPCs in culture shows a mean recombination rate of 80% in OPCs (72h before the medium change to induce their differentiation). After 3 days of differentiation, GFP+ positive cells represented less than 50% of the cells (Figure 19B), indicating either that recombined OPCs are more Tns3 mutations are broadly associated with lethal or strongly deleterious phenotypes.

Human genomic studies have identified few genomic Tns3 mutations, being linked with severe disease such as lung [START_REF] Yan | Single nucleotide polymorphism in the 3′ untranslated region of LPP is a risk factor for lung cancer: a case-control study[END_REF] or thyroid cancer (Maeda et al, 2009) and even nephropathy [START_REF] Feng | 3′UTR variants of TNS3, PHLDB1, NTN4, and GNG2 genes are associated with IgA nephropathy risk in Chinese Han population[END_REF]. Tns3 knockout models present strong lethality and we have a strong difficulty to obtain viable Tns3 mutant animals. We have also previously demonstrated the non-utility of the Tns3 bgeo gene-trap mice to study its function in oligodendrogenesis due to apparent compensatory effects to re-establish Tns3 expression in the CNS.

All of these indicate that Tns3 is involved in various biological processes and that is critical during the development, which makes it hard to study solely its role in brain development. For this reason, we first focused on the induction of CRISPR/Cas9mediated point mutation only in a subpopulation NSCs by the technique of postnatal electroporation of the lateral ventricle of the telencephalon, in order to induce viable Tns3-deleted brains. We found that the knockout of Tns3 impair NSCs' ability to generate OLs during the third postnatal week. Interestingly, we didn't see any effects on OPC population, suggesting that Tns3 play doesn't play a crucial role for NSCs specification into the oligodendroglia or for OPCs proliferation.

Knowing the reported presence of a 60KDa Tns3 isoform being predominantly expressed in the brain (GTEX resource, gtexportal.org/home/gene/TNS3), by Western blot analysing of MACSorted iOLs, we showed that purified iOLs had significantly higher levels of Tns3 full-length protein, despite that the small Tns3 isoform remains highly expressed. Moreover, the CRISPR/Cas9-mediated deletion in NSCs of the whole Tns3 coding sequence locus induced similar effects to the frameshifting at the beginning of the full-length isoform, suggesting that the Tns3 full-length isoform is the Tns3 variant mainly required for OL differentiation.

We also showed, using a Tns3 flox mice model we generated, that Tns3 OPC-specific induced knockout at P7 induces a decrease by 40% to 50% of the mature OL density two week later. Interestingly, the cortex shows already an effect of the Tns3 loss at P14, contrary to the corpus callosum for the fimbria, whose effect of Tns3 loss appears only at P21. This could be explained by a difference in the timing of OL differentiation, which differs across the brain regions. Interestingly, Tns3 loss does not seem to affect Nkx2.2 iOLs population, whose density do not show any changes two weeks after

Perspectives

The precise mechanisms by which Tns3 act during OL differentiation need to be further characterized. Interestingly, Tns3 is already known to be involved in pseudopodia formation in various cell types, so one possibility could be that Tns3, the most expressed Tensin in the oligodendroglial lineage, help iOLs to remodel their actin cytoskeleton and extend their cytoplasmic processes in order to find all the axons (around 40-60 axons per OL in the mouse cortex) that they myelinate. Without Tns3, OLs would fail to acquire a more mature oligodendroglial morphology, resulting in the loss of them and thus reduction to half in the density of oligodendrocytes found in the third week postnatal.

To investigate this hypothesis, we are currently performing an analysis of the morphological changes undergone by Tns3-deleted iOLs (Tns3-iKO) during their differentiation and before their increased cell death in OPC differentiating primary cultures. Our preliminary results show a clear decrease of the recombined cell population after three days of differentiation compared to the non-recombined cells from the same Tns3 flox mice, with no differences during the first 24h. Therefore, it seems that Tns3 is required during a precise time-window of OL differentiation, for reasons needing further characterization. We are currently analyzing these time lapse to perform a morphological analysis of recombined cells dying or surviving and nonrecombined cells, in order to identify potential cytoskeletal dysregulation caused by the absence of Tns3. An immunostaining performed after the experiment on the live imaged cells revealed a strong expression of Tns3 in the many of the GFP+ (recombined) surviving OLs. We hypothesize that most Tns3 deficient cells have died during the 3 days of imaging but a time course characterization of Tns3 expression in similar cultures remains to be done in order to confirm or refute this hypothesis. Finally, our present controls are either MACS OPCs from Tns3 flox ; Cre-negative; Rosa26 stop- YFP mice or non-recombined (GFP-) OPCs from Tns3 flox ; Pdgfra-CreER T ; Rosa26 stop- YFP . However, the expression of Cre recombinase in OPC nuclei is already known to impact the oligodendroglial generation. [CP1] In order to avoid this effect, we used exclusively Tns3 WT ; Pdgfra-CreER T ; Rosa26 stop-YFP mice as controls for our in vivo experiments. Thus, we still need to perform similar time lapse experiments using the best control cells before to reinforce our preliminary results showing increase cell death of differentiating Tns3-iKO oligodendrocytes.

Abstract

Multiple sclerosis (MS) is a neurological disease characterized by a loss of oligodendrocytes, the myelinating cell of the Central Nervous System. Despite recent advances leading to the suppression of the auto-immune attack on oligodendrocytes, efficient remyelinating therapies are still lacking. In MS, the spontaneous remyelination from oligodendrocyte precursor cells (OPCs), present all over the brain, is inefficient and diminishes with age. The presence of OPCs within demyelinating MS lesions and their failure to differentiate into myelinating cells suggests that induction of OPC differentiation is a critical event for successful remyelination.

We identified Tns3 (Tensin 3) as a target of key oligodendrogenic factors such as Chd7, Chd8 or Olig2 and showed that Tns3 was strongly induced at the onset of oligodendrocyte differentiation while downregulated in mature oligodendrocytes, constituting a good marker for immature oligodendrocytes. I characterized Tns3 expression in oligodendroglia to be: 1) mainly restricted to the immature OL stage, 2) localized in the cytoplasm and cell processes, and 3) overlapping with known immature oligodendrocytes markers such as Itpr2 and Nkx2.2. Tns3 expression is also found during adult brain remyelination after LPC injection, especially in newly formed OLs, therefore constituting a novel marker for immature OLs.

In vivo