Part I abstract: Genetic methods for inducibly and reversibly inhibiting neuronal activity of specific neurons are critical for exploring the functions of neuronal circuits. The engineered human glycine receptor, called ivermectin (IVM)-gated silencing receptor (IVMR), has been shown to possess this ability in vitro, which abolish the binding with endogenous glycine and improve the sensitivity to ivermectin. Based on that, we constructed the knock-in plasmid which put IVMR in the downstream of a loxP-flanked STOP cassette. The Rosa26-IVMR mouse line was generated by inserting the plasmid into the Rosa26 locus though homologous recombination. IVMR expression can be induced by crossing with specific Cre mouse line or stereotactic injection of Cre-expressing virus. When expressing IVMR in unilateral striatal by injecting Cre-expressing virus, APO-induced rotation behavior was observed after inactivation of unilateral striatal neurons by administering ivermectin. Furthermore, 10mg/kg and 5mg/kg IVM are the effective ligand concentrations as they are able to induce obvious rotational behavior, but the effect last longer for 10mg/kg IVM than that for 5mg/kg. The physiological recording in vivo exhibited that neuron firing dramatically decreased in IVM-treated freely moving Rosa26-IVMR; Emx1-Cre mice and neuronal excitability in brain slice showed a substantial reduction as shown by increased threshold of the current needed to evoke the action potential and the reduced frequency of the action potential. In conclusion, our mouse line can inactivate the neuronal activity effectively in an inducible and reversible way with systemic administration of the ligand. So it provides a powerful tool for exploring selective circuit functions in freely behaving mice. Part II abstract: Schizophrenia is a chronic and severe mental disease which affects around 0.5%-1% population. However, the underlying cause are complex and remain unclear. Genetic abnormalities are considered to be the main risky factor. Although the typical symptoms start to occur between 18 and 30 age, the disturbance of neurodevelopmental process at earlier age is believed to be involved. To date, only a few of susceptibility genes are confirmed in human patients. Previously, through a meta-analysis of copy number variants (CNV) data from the International Schizophrenia Consortium and in vitro studies, we found a novel serine/threonine kinase gene, unc-51-like kinase 4 (Ulk4), as a risk factor for major mental disorders including schizophrenia. To investigate the Ulk4's roles in corticogenesis, Ulk4 knockout mice was employed. Though analyzing a series of developmental process during corticogenesis including laminar specification, neuronal migration in Ulk4 deficient mice, we found that Ulk4 loss led to the thinner layer II-IV, delayed neuronal migration and increased cell death in layer II-IV but did not affect the proliferation of progenitors which later give rise to the projection neurons in layer II-IV. Meantime the influence of Ulk4 deficiency on the deep layer (layer V and layer VI) development was limited. In conclusion, Ulk4 plays a crucial role on corticogenesis and regulates a variety of neurodevelopmental processes. When defective, this will lead to the increased risk of neurodevelopment disorders and also might be involved in the onset of mental disease including schizophrenia at early adolescence.

MicroRNA (miRNA) function is required for normal animal development, in particular in stem cell and precursor populations. I hypothesize that miRNAs are similarly required for stem cell maintenance and appropriate fate commitment in the brain. To test the requirement for global microRNA production, I depleted the microRNA biosynthetic enzyme DICER in the developing mouse brain. I found that DICER loss in embryonic neural progenitor cells leads to embryonic lethality with microcephaly. By histological analysis, I found defects in both neural progenitor cell maintenance and cell differentiation. I also identified new candidate microRNAs for this phenotype by profiling miRNAs in DICER-depleted and control cells. Three microRNAs which are good candidates to modulate nervous differentiation are miR-23b, -182, and -34a. I describe the expression pattern and functional characterization of these candidates. In particular, miR-34a depletes neuron production after progenitor cell differentiation in culture, likely by modulating cell cycling and Notch pathway genes.

No description available.

No description available.

La différenciation et le développement requièrent une régulation fine de l’expression desgènes, médiée en partie par les modifications épigénétiques. Parmi les modificationsd’histones, la chromatine bivalente, signature chromatinienne atypique associant lesmarques permissive H3K4me2/3 et répressive H3K27me3, est de par sa plasticité, pressentiepour jouer un rôle décisionnel dans l’acquisition d’une identité cellulaire. Pour étudier le rôlede la chromatine bivalente au cours du développement, nous avons choisi d’utiliserl’empreinte parentale. Ce cadre développemental bien caractérisé, conduit à l’expression decertains gènes à partir d’un seul des deux allèles selon son origine parentale. La méthylationdifférentielle de l’ADN d’une région clé, appelée ICR (Imprinting Control Region), bienqu’absolument requise pour l’expression mono-allélique de ces gènes, n’est pas suffisantepour rendre compte de la complexité du profil d’expression de ces gènes suggérantl’implication d’autres mécanismes. Sur 15 ICR méthylés sur l’allèle maternel, nous avonsprécisément mis en évidence que la chromatine bivalente est présente par défaut sur l’allèlenon-méthylé lorsque celui-ci est transcriptionnellement inactif, quel que soit le stadedéveloppemental ou le tissu étudié, participant ainsi à la régulation fine de l’expressiontissu-spécifique à partir de ces régions. Dans leur ensemble, nos données révèlent que lachromatine bivalente joue un rôle moins dynamique que pressentie. Ainsi, au niveau del’empreinte parentale, sa fonction principale serait de protéger l’allèle non-méthylé des ICRcontre l’acquisition de méthylation tout en aidant à le maintenir réprimé dans certainstissus. Nous proposons que la chromatine bivalente joue un rôle similaire sur l’ensemble desîlots CpG du génome, contribuant ainsi à la protection de l’identité cellulaire. Afin decompléter cette première étude, j’ai étudié la régulation de l’expression d’un candidat de larégulation de la dynamique de la chromatine bivalente, l’histone déméthylase pourH3K27me3, JMJD3. Les résultats obtenus suggèrent que l’induction d’expression observéeau cours de la différenciation neurale s’appuie sur une dynamique de la structuretridimensionnelle de la chromatine qui pourrait elle-même être régulée par la transcriptiond’un eARN (enhancer ARN) et l’hydroxyméthylation. Ce modèle souligne un mode derégulation complexe de ce nouvel acteur épigénétique, impliquant des régionsintragéniques, et pourrait notamment permettre de comprendre les mécanismes impliquésdans sa dérégulation dans les cancers. / Fine-tuned regulation of gene expression is required for cell fate determination anddevelopment. Epigenetics modifications are well documented to be instrumental in thisprocess. Among them, bivalent chromatin, an unusual chromatin signature, which associatesthe permissive mark H3K4me2/3 and the repressive mark H3K27me3, is believed to arbitrategene expression during cell commitment. To study its precise role in development, we haveundertaken to study bivalency in the context of genomic imprinting. This well-defineddevelopmental frame is a process restricting expression of some genes to one parental alleleonly. The constitutive differential DNA methylation at the key region called ICR (ImprintingControl Region), is absolutely required but not sufficient to explain the complexity of themono-allelic expression pattern of imprinted genes, indicating that other mechanisms couldbe involved. Specifically, on 15 maternally methylated ICR, we showed that bivalentchromatin is acquired by default on the unmethylated allele of ICR when it istranscriptionally inactive whatever the developmental stage or the tissue studied and thuscontribute to tissue-specific expression from these regions. Altogether, our results revealthat chromatin bivalency is much less dynamic than proposed. In the context of genomicimprinting, it seems to plays more a safeguard function at ICR by protecting theunmethylated allele against DNA methylation acquisition while keeping it silent in a subsetof tissues. To complete this study, I studied the regulation of JMJD3, a histone demethylasefor H3K27me3, candidate to regulate bivalency dynamic. Our results suggest that theinduction of Jmjd3 expression observed during neural differentiation rely on the dynamic ofthe tridimensional architecture at the locus which could be regulated by the transcription ofan eRNA (enhancer RNA) and by hydroxymethylation. This model highlight a complex way ofregulation for this new epigenetics actor, involving intragenic regions and could help tounderstand how Jmjd3 expression is deregulated in a pathological context such as in cancer.

Murine neural precursor cells have been a well studied model for neural cell fate determination and stem cell function both in vivo and in primary culture. However, factors such as cell number, the presence of multiple cell populations and of niche intrinsic factors made it difficult to dissect the mechanisms regulating cortical development. To overcome this issue, we have developed a culture system where mouse embryonic stem cells (ES) are differentiated to cortical radial precursors through retinoic acid treatment of embryoid bodies. One day after plating in neural differentiation conditions, ~70% of cells in the culture are cortical radial precursors (RPs) as indicated by the definitive cortical marker Emx1, and over 8 days in culture, these RPs differentiate to pyramidal glutamatergic neurons of the cortex mimicking in vivo development. Astrocyte differentiation can be observed later as the culture progresses, which again mimics the typical timed genesis of cells in the cortex. The stem cell properties and cell fate of these RPs can be manipulated with growth factors in culture as they are in vivo. In particular, FGF2 promotes proliferation and survival, while ciliary neurotrophic factor (CNTF) induces precocious astrocyte formation. Thus, our ES-derived cortical RP cultures can serve as an alternate and complementary in vitro model to examine neural precursor biology during early development.

Murine neural precursor cells have been a well studied model for neural cell fate determination and stem cell function both in vivo and in primary culture. However, factors such as cell number, the presence of multiple cell populations and of niche intrinsic factors made it difficult to dissect the mechanisms regulating cortical development. To overcome this issue, we have developed a culture system where mouse embryonic stem cells (ES) are differentiated to cortical radial precursors through retinoic acid treatment of embryoid bodies. One day after plating in neural differentiation conditions, ~70% of cells in the culture are cortical radial precursors (RPs) as indicated by the definitive cortical marker Emx1, and over 8 days in culture, these RPs differentiate to pyramidal glutamatergic neurons of the cortex mimicking in vivo development. Astrocyte differentiation can be observed later as the culture progresses, which again mimics the typical timed genesis of cells in the cortex. The stem cell properties and cell fate of these RPs can be manipulated with growth factors in culture as they are in vivo. In particular, FGF2 promotes proliferation and survival, while ciliary neurotrophic factor (CNTF) induces precocious astrocyte formation. Thus, our ES-derived cortical RP cultures can serve as an alternate and complementary in vitro model to examine neural precursor biology during early development.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18183号 / 医博第3903号 / 新制||医||1004(附属図書館) / 31041 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 髙橋 良輔, 教授 髙橋 淳, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les gliomes sont les tumeurs primitives les plus fréquentes du système nerveux central. Cette dernière décennie, l’apport de la biologie moléculaire a permis de mieux appréhender leurs comportements et de mieux préciser leur origine. Nous avons montré que le profil moléculaire des glioblastomes (grade IV dans la classification de l’OMS) et celui des astrocytomes pilocytiques (grade I dans la classification de l’OMS) différait notamment par l’expression d’un gène nommé KIAA 0510. La caractérisation de sa séquence nous a mené à nous intéresser à la Ténascine R, une glycoprotéine de la matrice extracellulaire impliquée dans les processus de migration et de différenciation cellulaire. Par ailleurs, l’expression de la Ténascine R pendant le développement, suggère son implication au cours de la corticogenèse.Dans le but de mieux comprendre l’origine des astrocytomes pilocytiques, notamment ceux de la région des voies optiques, nous avons mis en évidence au niveau du chiasma optique en développement des cellules de la glie radiaire à partir desquelles les astrocytomes pilocytiques des voies optiques pourraient dériver. / Gliomas are the most frequently occurring primary tumors in the central nervous system. These last years, molecular biology technics allowed a better understanding of the gliomagenesis as well as behaviour of these tumors. We have previously shown that molecular profiling of glioblastomes (WHO grade IV) and pilocytic astrocytomas (WHO grade I) differed for KIAA 0510 gene expression. This sequence was fully characterized and shown to be part of the tenascin R gene encoding for an extracellular matrix glycoprotein involved in migration and cell differentiation. In addition, during development, Tenascin-R may be involved in corticogenesis.In parallel, in the developing optic chiasm, we evidenced cells with radial glial characteristics from which the hypothalamo-chiasmatic pilocytic astrocytomas could derive.

Les mécanismes développementaux qui spécifient le nombre et le phénotype laminaire des neurones du cortex cérébral jouent un rôle essentiel dans l’établissement de la cytoarchitecture corticale. Le nombre de neurones dans chaque couche d'une aire donnée est déterminé par le taux de production neuronale, qui dépend étroitement de l'équilibre entre les divisions prolifératives et différenciatives. Des observations clés suggèrent que la durée de la phase G1 (TG1) ferait partie intégrante d'un mécanisme cellulaire régulant le mode de division des précurseurs du cortex. Nous avons testé cette hypothèse par l'accélération expérimentale de la progression dans la phase G1 de précurseurs corticaux de souris in vivo, via la surexpression des cyclines E1 et D1. A E15, la réduction de TG1 promeut la rentrée dans le cycle cellulaire aux dépens de la différenciation neuronale, résultant en une modification de la cytoarchitecture du cortex adulte. Des données de modélisation confirment que les effets induits par la réduction de TG1 sont médiés par des changements du mode de division. Les effets de la surexpression des cyclines E1 et D2 à E13 sont plus modérés qu'à E15, indiquant des différences intrinsèques entre les précurseurs corticaux précoces et tardifs. La mesure des phases du cycle cellulaire des populations de précurseurs corticaux à l’aide de différentes techniques révèle un niveau important d’hétérogénéité et souligne la nécessité de prendre en compte la diversité des précurseurs co‐existant dans les zones germinales du télencéphale. / In the cerebral cortex, area‐specific differences in neuron number and phenotype are distinguishing features both within and across species. The developmental mechanisms that specify the number of neurons and their laminar fate are instrumental in specifying cortical cytoarchitecture. Neuron number in layers and areas correlate with changes in the rate of neuron production, largely determined by the balance between proliferative and differentiative divisions in cortical precursors. Key observations suggest a concerted regulation between the duration of the G1 phase (TG1) and mode of division and have led to the hypothesis that TG1 could be an integral part of a cellular mechanism regulating the mode of division of cortical precursors. To test this hypothesis we experimentally accelerated TG1 in mouse cortical precursors in vivo, via the forced expression of cyclinE1 and cyclinD1. At E15, TG1 reduction promoted cell‐cycle re‐entry at the expense of differentiation and led to cytoarchitectural modifications. Modeling confirms that the TG1‐induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. Forced expression of G1 cyclins was also applied to early cortical precursors. The effects of cyclinD1 and cyclinE1 up‐regulation at E13 were milder than those observed at E15, pointing to intrinsic differences between early and late cortical precursors. The used of various techniques to measure cell‐cycle kinetics in distinct precursor populations underlined the necessity of taking the full diversity of neural precursors co‐existing in the GZ of the telencephalon into account when performing cellcycle kinetics analysis.