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Summary During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition., Graphical Abstract, Highlights • Bcl6 ensures robust neurogenesis by repressing major extrinsic self-renewal pathways • Bcl6 inhibits the Notch, Wnt, SHH, and FGF signaling pathways at multiple levels • Bcl6 represses transcription through Sirt1 recruitment and histone deacetylation, Bonnefont et al. show that Bcl6 promotes neurogenesis by directly repressing genes belonging to the major signaling pathways promoting cortical progenitor self-renewal. These data indicate that a single cell-intrinsic factor represses multiple extrinsic signaling pathways to ensure irreversible neurogenic commitment.

The development of the cerebral cortex requires complex sequential processes that have to be precisely orchestrated. The localization and timing of neuronal progenitor proliferation and of neuronal migration define the identity, laminar positioning, and specific connectivity of each single cortical neuron. Alterations at any step of this organized series of events-due to genetic mutations or environmental factors-lead to defined brain pathologies collectively known as malformations of cortical development (MCDs), which are now recognized as a leading cause of drug-resistant epilepsy and intellectual disability. In this heterogeneous group of disorders, macroscopic alterations of brain structure (eg, heterotopic nodules, small or absent gyri, double cortex) can be recognized and probably subtend a general reorganization of neuronal circuits. In this review, we provide an overview of the molecular mechanisms that are implicated in the generation of genetic MCDs associated with aberrations at various steps of neurogenesis and cortical development.El desarrollo de la corteza cerebral requiere de una secuencia de complejos procesos que tienen que estar coordinados con precisión. La localización y la cronología de la proliferación de las neuronas precursoras y de la migración neuronal definen la identidad, el posicionamiento laminar y la conectividad específica de cada una de las neuronas corticales. Las alteraciones en cualquier etapa de esta serie organizada de acontecimientos- debidas a mutaciones genéticas o a factores ambientales- llevan a patologías cerebrales definidas que en conjunto se denominan malformaciones del desarrollo cortical (MDC), las cuales son reconocidas actualmente como causa de epilepsia resistente a fármacos e incapacidad intelectual. En este grupo heterogéneo de trastornos, las alteraciones macroscópicas de la estructura cerebral (por ej. nódulos heterotópicos, giros pequeños o ausentes, doble corteza) pueden ser reconocidas y es probable que subtiendan a una reorganización general de los circuitos neuronales. En esta revisión se entrega una panorámica de los mecanismos moleculares que se han involucrado en la generación de las MDC asociadas con aberraciones en varias etapas de la neurogénesis y del desarrollo cortical.Le développement du cortex cérébral fait appel à des processus séquentiels complexes qui doivent être orchestrés précisément. La localisation et la chronologie de la prolifération de neurones précurseurs et celles de la migration neuronale définissent l'identité, le positionnement laminaire et la connectivité spécifique de chaque neurone cortical unique. Toute modification, quel que soit le stade de ces séries organisées d'événements (en raison de mutations génétiques ou de facteurs environnementaux), entraîne des pathologies cérébrales définies, globalement connues sous le terme de malformations du développement cortical (MDC). Ces malformations sont maintenant reconnues comme principalement responsables de la résistance aux médicaments contre l'épilepsie et du déficit intellectuel. Dans ce groupe hétérogène de maladies, les modifications macroscopiques de la structure cérébrale (par exemple, nodules hétérotopiques, gyrus petit ou absent, double cortex) peuvent être identifiées et probablement sous-tendre une réorganisation générale des circuits neuronaux. Cet article présente une vue d'ensemble des mécanismes moléculaires impliqués dans l'apparition de MDC génétiques associées à des aberrations à des stades différents de la neurogenèse et du développement cortical.

Emerging evidence suggests a complex interplay between the endocannabinoid system, omega-3 fatty acids and the immune system in the promotion of brain self-repair. However, it is unknown if all omega 3 fatty acids elicit similar effects on adult neurogenesis and if such effects are mediated or regulated by interactions with the endocannabinoid system. This study investigated the effects of DHA and EPA on neural stem cell (NSC) fate and the role of the endocannabinoid signalling pathways in these effects.EPA, but not DHA, significantly increased proliferation of NSCs compared to controls, an effect associated with enhanced levels of the endocannabinoid 2-arachidonylglycerol (2-AG) and p-p38 MAPK, effects attenuated by pre-treatment with CB1 (AM251) or CB2 (AM630) receptor antagonists. Furthermore, in NSCs derived from IL-1 beta deficient mice, EPA significantly decreased proliferation and p-p38 MAPK levels compared to controls, suggesting a key role for IL-1 beta signalling in the effects observed. Although DHA similarly increased 2-AG levels in wild-type NSCs, there was no concomitant increase in proliferation or p-p38 MAPK activity. In addition, in NSCs from IL-1 beta deficient mice, DHA significantly increased proliferation without effects on p-P38 MAPK, suggesting effects of DHA are mediated via alternative signalling pathways. These results provide crucial new insights into the divergent effects of EPA and DHA in regulating NSC proliferation and the pathways involved, and highlight the therapeutic potential of their interplay with endocannabinoid signalling in brain repair. (C) 2016 Elsevier Ltd. All rights reserved. We gratefully acknowledge support from the Dowager Countess Eleanor Peel Trust (UK) ​grant ref: AJT.1064.2.MJA (11/09) [TH-PRCL.FID2228] and Royal Society International Exchange grant (ref: IE 131551). The authors declare no conflicts of interest. peer-reviewed

Neurodevelopmental disorders, including autism spectrum disorders, are highly male biased, but the underpinnings of this are unknown. Striatal dysfunction has been strongly implicated in the pathophysiology of neurodevelopmental disorders, raising the question of whether there are sex differences in how the striatum is impacted by genetic risk factors linked to neurodevelopmental disorders. Here we report male-specific deficits in striatal function important to reward learning in a mouse model of 16p11.2 hemideletion, a genetic mutation that is strongly associated with the risk of neurodevelopmental disorders, particularly autism and attention-deficit hyperactivity disorder. We find that male, but not female, 16p11.2 deletion animals show impairments in reward-directed learning and maintaining motivation to work for rewards. Male, but not female, deletion animals overexpress mRNA for dopamine receptor 2 and adenosine receptor 2a in the striatum, markers of medium spiny neurons signaling via the indirect pathway, associated with behavioral inhibition. Both sexes show a 50% reduction of mRNA levels of the genes located within the 16p11.2 region in the striatum, including the kinase extracellular-signal related kinase 1 (ERK1). However, hemideletion males show increased activation in the striatum for ERK1, both at baseline and in response to sucrose, a signaling change associated with decreased striatal plasticity. This increase in ERK1 phosphorylation is coupled with a decrease in the abundance of the ERK phosphatase striatum-enriched protein-tyrosine phosphatase in hemideletion males. In contrast, females do not show activation of ERK1 in response to sucrose, but notably hemideletion females show elevated protein levels for ERK1 as well as the related kinase ERK2 over what would be predicted by mRNA levels. These data indicate profound sex differences in the impact of a genetic lesion linked with neurodevelopmental disorders, including mechanisms of male-specific vulnerability and female-specific resilience impacting intracellular signaling in the brain.

The eutherian X-chromosome specific family of Armcx genes has been described as originating by retrotransposition from Armc10/SVH, a single Arm-containing somatic gene. Armcx3 and Armc10/SVH are characterized by high expression in the central nervous system and they play an important role in the regulation of mitochondrial distribution and transport in neurons. In addition, Armcx/Arm10 genes have several Armadillo repeats in their sequence. In this study we address the potential role of this gene family in neural development by using the chick neural tube as a model. We show that Armc10/SVH is expressed in the chicken spinal cord, and knocking-down Armc10/SVH by sh-RNAi electroporation in spinal cord reduces proliferation of neural precursor cells (NPCs). Moreover, we analyzed the effects of murine Armcx3 and Armc10 overexpression, showing that both proteins regulate progenitor proliferation, while Armcx3 overexpression also specifically controls neural maturation. We show that the phenotypes found following Armcx3 overexpression require its mitochondrial localization, suggesting a novel link between mitochondrial dynamics and regulation of neural development. Furthermore, we found that both Armcx3 and Armc10 may act as inhibitors of Wnt-β-catenin signaling. Our results highlight both common and differential functions of Armcx/Armc10 genes in neural development in the spinal cord., This work was supported by grants from Spanish MINECO (SAF2013-42445R), CIBERNED (ISCIII) and La Marató de TV3 Foundation to ES; Spanish MINECO (BFU2010-21507) to FU; BFU2013-46477-P to EM

The Drosophila transcriptional co-activator protein Yorkie and its vertebrate orthologs YAP and TAZ are potent oncogenes, whose activity is normally kept in check by the upstream Hippo kinase module. Upon its translocation into the nucleus, Yorkie forms complexes with several tissue-specific DNA-binding partners, which help to define the tissue-specific target genes of Yorkie. In the progenitor cells of the eye imaginal disc, the DNA-binding transcription factor Homothorax is required for Yorkie-promoted proliferation and survival through regulation of the bantam microRNA (miRNA). The transit from proliferating progenitors to cell cycle quiescent precursors is associated with the progressive loss of Homothorax and gain of Dachshund, a nuclear protein related to the Sno/Ski family of co-repressors. We have identified Dachshund as an inhibitor of Homothorax-Yorkie-mediated cell proliferation. Loss of dachshund induces Yorkie-dependent tissue overgrowth. Conversely, overexpressing dachshund inhibits tissue growth, prevents Yorkie or Homothorax-mediated cell proliferation of disc epithelia and restricts the transcriptional activity of the Yorkie-Homothorax complex on the bantam enhancer in Drosophila cells. In addition, Dachshund collaborates with the Decapentaplegic receptor Thickveins to repress Homothorax and Cyclin B expression in quiescent precursors. The antagonistic roles of Homothorax and Dachshund in Yorkie activity, together with their mutual repression, ensure that progenitor and precursor cells are under distinct proliferation regimes. Based on the crucial role of the human dachshund homolog DACH1 in tumorigenesis, our work suggests that DACH1 might prevent cellular transformation by limiting the oncogenic activity of YAP and/or TAZ., This work was supported by grants from Fundação para a Ciência e Tecnologia [PTDC/BIA-BCM/71674/2006] to F.J.; and from the Spanish Ministry of Economy and Competitiveness (MINECO); and EU Feder Funds [BFU2012-34324] to F.C.; and Consolider ‘From Genes to Shape’ (MICINN) of which F.C. was a participant researcher. C.B.-P. was the recipient of fellowships from Fundação para a Ciência e Tecnologia [SFRH/BPD/46983/2008]. F.J. is the recipient of IF/01031/2012 from Fundação para a Ciência e Tecnologia.

Disrupted-in-Schizophrenia 1 (DISC1) is a gene known as a risk factor for mental illnesses possibly associated with dopamine impairments. DISC1 is a scaffold protein interacting with proteins involved in the dopamine system. Here we summarise the impact of DISC1 disruption on the dopamine system in animal models, considering its effects on presynaptic dopaminergic function (tyrosine hydroxylase levels, dopamine transporter levels, dopamine levels at baseline and after amphetamine administration) and postsynaptic dopaminergic function (dopamine D1 and D2 receptor levels, dopamine receptor-binding potential and locomotor activity after amphetamine administration). Our findings show that many but not all DISC1 models display (1) increased locomotion after amphetamine administration, (2) increased dopamine levels after amphetamine administration in the nucleus accumbens, and (3) inconsistent basal dopamine levels, dopamine receptor levels and binding potentials. There is also limited evidence for decreased tyrosine hydroxylase levels in the frontal cortex and increased dopamine transporter levels in the striatum but not nucleus accumbens, but these conclusions warrant further replication. The main dopaminergic findings are seen across different DISC1 models, providing convergent evidence that DISC1 has a role in regulating dopaminergic function. These results implicate dopaminergic dysregulation as a mechanism underlying the increased rate of schizophrenia seen in DISC1 variant carriers, and provide insights into how DISC1, and potentially DISC1-interacting proteins such as AKT and GSK-3, could be used as novel therapeutic targets for schizophrenia.

The tight regulation of organ size is a prerequisite of unperturbed development of complex organisms. The molecular mechanisms determining and regulating the size of entire organisms and organs are poorly understood. The actual growth process of an organ requires stem cells, which integrate signals from pathways involved in the initiation or termination of pro-proliferative programs. Recently, the Hippo signaling pathway, with its transcriptional effector YAP1, has been discovered as a critical regulator of both organ size control and tumorigenesis, two processes with common cellular signaling mechanisms. Therefore, it is of interest to determine, how precisely Hippo signaling regulates progenitor and stem cell proliferation. The mammalian epidermis is a rapidly regenerating epithelial tissue, whose maintenance depends on the self-renewing ability of epidermal stem cells, which reside in the basal layer. Epidermal stem cells are essential for skin homeostasis and regeneration during wound repair. Interestingly, the epidermal deletion of Sav1 (WW45), a core component of the Hippo signaling protein cascade, was shown to increase the progenitor population in the basal layer of the epidermis. In this work, we provide further evidence, that the Hippo pathway, and its nuclear effector YAP1 are important for the regulation of stem cell proliferation in the epidermis. Using gain- and loss-of-function studies, we show that activation of Yap1 in the basal layer leads to a hyper-thickened epidermis, due to an expansion of the epidermal stem cell compartment, and a decrease of the suprabasal differentiated cell layers. Moreover, we demonstrate that long-term constitutive activation of Yap1 in the epidermis leads to the formation of squamous cell carcinoma-like tumors. Deletion of Yap1 (cKO) in the basal layer of the epidermis, on the other hand, causes a severe skin defect with an abnormally thin epidermis due to decreased epidermal proliferation. Further, we show, that YAP1 regulates epidermal proliferation through interaction with the TEAD transcription factors. The YAP1-S79A mutation has been shown to impair the interaction with the TEAD transcription factors. Mice expressing solely YAP1-S79A in the basal layer phenotypically resemble Yap1 cKO mice. Loss of the MST1/2 kinases, which are considered core components of the Hippo pathway, was previously shown to lead to hepatomegaly in the liver. In this work we show that loss of the MST1/2 kinases in the epidermis does not lead to a hyperproliferative phenotype as observed for epidermal Yap1 activation. This observation suggests that YAP1 might be regulated by other mechanisms in the epidermis. α-catenin is implicated in tumor suppression and cell density sensing in the skin. Our group determined a direct binding of YAP1 and α-catenin. We identify α-catenin as an upstream negative regulator of YAP1. α-catenin controls YAP1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase and, therefore, regulates the proliferative function of YAP1 in keratinocytes. Together, these data identify YAP1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a ‘‘crowd control’’ molecular circuitry in mammalian skin. In collaboration with the Pu laboratory we further elucidate, that activation of Yap1 in the fetal heart induces cardiomyocyte proliferation. Correspondingly, Yap1 deletion causes lethal myocardial hypoplasia and impaired cardiomyocyte proliferation. The findings presented in this thesis show that in the epidermis and the heart, YAP1 is essential for proliferation and normal tissue development. Moreover, this work demonstrates the diversity of the Hippo signaling machinery in different tissues, showing that in the epidermis the core kinases MST1/2 do not regulate YAP1-mediated proliferation. Thus, it provides novel insights about the Hippo signaling pathway and may ultimately contribute to a better understanding of this pathway in both, normal physiological and disease conditions., Die normale Entwicklung von komplexen Organismen setzt eine präzise Regulation der Organgröße vorraus. Jedoch sind die molekularen Mechanismen, welche die Größe von gesamten Organismen und einzelnen Organen bestimmen, weitgehend unbekannt. Stammzellen sind für den Wachstumsprozess eines Organs essentiell. Sie verarbeiten Signale, die pro-proliferative Expressionsprogramme aktivieren oder beenden. Es wurde kürzlich entdeckt, dass der Hippo Signalweg, einschliesslich seines transkriptionellen Coaktivators YAP1, sowohl bei der Regulation der Organgröße als auch in der Karzinogenese involviert ist, zwei Prozessen mit gemeinsamen zellulären Signalmechanismen. Daher ist es von großem Interesse herauszufinden, wie genau der Hippo Signalweg die Progenitor- und Stammzellproliferation steuert. Die Epidermis ist ein sich fortlaufend erneuerndes Epithelgewebe, welches für seinen Erhalt Stammzellen mit der Fähigkeit zur Selbsterneuerung benötigt. Epidermale Stammzellen sind sowohl für die Homöostase der Haut, als auch für die Wundheilung essentiell. Interessanterweise wurde gezeigt, dass in der Epidermis der Verlust von Sav1 (WW45), einem Kernbestandteil der Hippo Signalkaskade, zu einer Vermehrung der Progenitorpopulation in der Basalschicht führt. Die Ergebnisse dieser Arbeit zeigen, dass der Hippo Signalweg und im Speziellen sein Effektor YAP1, wichtig für die Regulation der Stammzellproliferation in der Epidermis sind. Anhand von Gain-of function- und Loss-of-function-Mausmodellen, zeigen wir, dass die Aktivierung von Yap1 in der Basalschicht der Epidermis eine Hautverdickung basierend auf der Vermehrung der epidermalen Stammzellen hervorruft. Zusätzlich kommt es zu einem Verlust an suprabasalen differenzierten Schichten der Epidermis. Eine andauernde konstitutive Aktivierung von Yap1 in der Epidermis ruft zudem die Bildung von Plattenepithelkarzinom (SCC)-ähnlichen Tumoren hervor. Im Gegensatz dazu verursacht der Verlust von Yap1 (cKO) in der Basalschicht der Epidermis einen schweren Defekt der Haut. Aufgrund einer verringerten Proliferationsrate kommt es zu der Bildung einer ungewöhnlich dünnen Epidermis. Wir zeigen, dass der Co-Aktivator YAP1 die Proliferation der Epidermis durch eine Interaktion mit den TEAD Transkriptionsfaktoren reguliert. Es wurde für die YAP1-S79A Mutationsform gezeigt, dass eine Interaktion mit den TEAD Transkriptionsfaktoren nicht mehr stattfinden kann. Wir zeigen, dass der Phenotyp von Mäusen, die lediglich YAP1-S79A in der Basalschicht der Epidermis exprimieren, dem der Yap1 cKO Mäuse gleicht und einen Proliferationsdefekt aufweist. Zuvor wurde in der Leber gezeigt, dass der Verlust der MST1/2 Kinasen, die zu den zentralen Kinasen des Signalwegs gehören, zu Hepatomegalie führt. Hier präsentieren wir Ergebnisse, die belegen, dass der Verlust der MST1/2 Kinasen in der Epidermis nicht zu dem durch epidermale Yap1 Aktivierung hervorgerufenen hyperproliferativen Phenotypen führt. Diese Ergebnisse legen nahe, dass YAP1 in der Epidermis durch andere Mechanismen reguliert wird. Tatsächlich stellt unsere Arbeitsgruppe eine Interaktion zwischen YAP1 und α-Catenin fest. α-Catenin fungiert als Tumorsuppressor und gibt Informationen über Zelldichte weiter. Wir identifizieren α-Catenin als negativen Regulator von YAP1. α-Catenin reguliert dabei die Aktivität und die Phosphorylierung von YAP1, indem es die Interaktion von YAP1 mit den 14-3-3 Proteinen und der Phosphatase PP2A moduliert. Hierdurch reguliert α-Catenin die proliferativen Eigenschaften von YAP1 in Keratinozyten. Zusammengefasst identifizieren diese Ergebnisse YAP1 als einen entscheidenden Faktor der proliferativen Eigenschaften von epidermalen Stammzellen, der somit bedeutend für die Homöostase der Haut ist und auch bei Krankheitsprozessen wie Krebs eine entscheidende Rolle spielen könnte. In Zusammenarbeit mit dem Pu Labor zeigen wir zudem, dass die Aktivierung von Yap1 im embryonalen Herzen zu einer vermehrten Proliferation der Kardiomyozyten führt. Entsprechend verursacht der Verlust von Yap1 eine lethale Hypoplasie des Myokards und beeinträchtigt die Proliferation der Kardiomyozyten. Die Ergebnisse dieser Arbeit in Epidermis und Herz demonstrieren die Bedeutung von YAP1 für zelluläre Proliferation und normale Gewebeentwicklung. Darüberhinaus gelingt es uns auch die gewebespezifische Vielfalt des Hippo Signalweges weiter aufzuschlüsseln. Wir zeigen, dass die Kinasen MST1/2 die YAP1-vermittelte Proliferation in der Epidermis nicht regulieren. Die Ergebnisse liefern neue Erkenntnisse über den Hippo Signalweg und tragen zu einem besseren Verständnis des Signalwegs in physiologischen und pathologischen Zuständen bei.

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulationin vitro. Here we report its rolein vivousing mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3β and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3β inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.