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by Sau Cheung Tiu. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 348-388). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

蒲金氏細胞(Purkinje cell)是小腦中的一種主要神經元，其主要作用在於協調身體活動及平衡。蒲金氏細胞之早期分化需要兩個密切相關的LIM同源盒結構域基因Lhx1及Lhx5。在胚胎小腦發育期間，這兩個基因的失活化會導致蒲金氏細胞數量大量減少。但有趣的是，就算在蒲金氏細胞完成分化之後，Lhx1/5之表達依然維持在高水平。這顯示Lhx1/5在產後小腦發育過程中可能有更多作用。為了研究這些可能作用，我把條件性Lhx1/5雙基因剔除小鼠和Pcp2-IRES-Cre轉基因小鼠交配，從而令Lhx1/5在產後第二天的蒲金氏細胞失活化。結果顯示Lhx1/5雙突變體老鼠在出生後兩星期即有顯著但程度不太大的運動失調。但在八星期，牠們出現嚴重的運動協調及身體平衡能力缺失。可是，擁有一個正常的Lhx1或Lhx5等位基因的控制小鼠並沒有這些不正常行為出現。在出生後的三個星期內，缺乏Lhx1/5會導致蒲金氏細胞樹突不正常發展，但小腦的整體細胞結構和分層卻維持正常。另外，這兩個基因對維持蒲金氏細胞已發展的樹突並不起作用，而且在六個月大的成年突變小鼠並沒有蒲金氏細胞退化。利用微陣列及逆轉錄聚合酶鏈式反應，我們在成年突變小鼠的小腦中確定了數個參與在麩胺酸及鈣訊息的突觸基因表達量下降。而這些突觸基因也在其他運動失調小鼠有下降的表達量。研究結果說明了Lhx1及Lhx5對蒲金氏細胞樹突發展有著重要、但功能重疊的作用。 / 在探究Lhx1/5如何控制蒲金氏細胞樹突發展時，我們發現Lhx1/5與Foxp4有蛋白質交互作用。Foxp4屬forkhead家族成員轉錄因子，它表達在小腦原基、遷移中及成熟的蒲金氏細胞。為了初步瞭解Foxp4在蒲金氏細胞發展中的作用，我在產後第十天小腦薄片組織培養中，利用siRNA降低Foxp4基因的表達量。結果發現蒲金氏細胞樹突及關聯的伯格曼膠質細胞支架出現結構性受損。這顯示Foxp4對維持蒲金氏細胞樹突有重要作用。 / 為了進一步研究Foxp4在活體蒲金氏細胞及小腦發育的作用，我把條件性Foxp4基因剔除小鼠和不同的Cre轉基因小鼠交配，從而令Foxp4在不同的發育過程階段中失活化。但是有趣地，我只能在同質結合突變小鼠 (Foxp4Δ/Δ)，即Foxp4在生殖細胞時期已經被剔除的情況下，觀察到小腦發育遲緩。當Foxp4在其他發育過程階段中失活化，我並沒有觀察到任何缺陷表型。這個結果顯示了在活體中發生了功能性的彌補，但在小腦薄片組織培養中卻沒有發生。另外，條件性Lhx1/5雙基因剔除小鼠和條件性Foxp4基因剔除小鼠的不同表型意味著在控制蒲金氏細胞及小腦發育過程中，有其他蛋白質可能參與在Lhx1/5及Foxp4的轉錄複合子中。我們需要更多的研究去明白Foxp和 LIM同源盒結構域蛋白質在功能上的聯系及它們在中樞神經系統發育中的作用。 / Purkinje cells (PCs) are one of the principal neurons in the cerebellum that is essential for the coordination of fine-tuning body movement and balancing. Early differentiation of PCs requires two closely related LIM-homeodomain genes Lhx1 and Lhx5, as inactivation of both genes results in significant reduction of PC number in embryonic cerebellum. Interestingly, high levels of Lhx1/5 expressions persist even after PC differentiation in the postnatal cerebellum. Hence, there may be additional roles for these two genes during postnatal PC development. To address this question, conditional inactivation approach was used to inactivate both Lhx1/5 in postnatal PCs specifically beginning at postnatal day 2 (P2). Lhx1/5 double conditional knockout (DKO) mutants were generated by crossing Lhx1/5 conditional null mutant mice with Pcp2-IRES-Cre mice. The mutants initially showed modest but noticeable ataxic locomotion at around two weeks after birth. However at 8 weeks old, the mutants displayed severe deficits in motor coordination and body balance. The control animals with one functional copy of either Lhx1 or Lhx5 did not show any abnormality. Deficiencies of both genes could lead to abnormal PC dendritogenesis during the first three weeks of life although the general cytoarchitectural lamination of cerebellar cortex was maintained. However, the two genes were dispensable for the maintenance of developed dendrites in adult mouse and no PC degeneration was observed in the 6 month-old double mutant mouse. Further microarray and semi-quantitative RT-PCR analysis identified down-regulation of several synaptic genes that involved in glutamate and/or calcium signaling in our Lhx1/5 DKO mutant and such disturbance had also been found in other ataxic mouse models. Overall, our findings suggest that Lhx1/5 are required but functionally redundant in dendritogenesis of PCs. / During investigation on how Lhx1/5 control the dendritogenesis of PCs, Lhx1/5 proteins were found to physically interact with Foxp4. Foxp4 belongs to the forkhead transcription factor family that is expressed in developing cerebellum primordium, migrating and mature PCs. To initially examine the function of Foxp4 in PC development, Foxp4 was knocked down by siRNA in organotypic cerebellar slice culture at P10. Impaired organization of PC dendritic arbors and associated Bergmann glial scaffold were resulted, suggesting that Foxp4 is essential for the maintenance of PC dendritic arborization. / To further investigate the function of Foxp4 during the cerebellum and PCs development in vivo, a Foxp4 CKO mouse line was generated and crossed with different lines of Cre-deleter mice, including Zp3-Cre, Pax2-Cre, En1-Cre and Pcp2-IRES-Cre, to inactivate Foxp4 at different developmental stages. Intriguingly, although developmental delay of cerebellum was found in germline deletion of Foxp4 homozygous recombined null mutant, no defective phenotype was observed when Foxp4 was inactivated at other stages. Hence, functional compensation might take place in vivo but not in the cerebellar slice culture. The phenotypic difference between Lhx1/5 DKO and Foxp4 CKO mice imply potential involvement of other proteins in the transcription complex between Lhx1/5 and Foxp4 in regulating the cerebellum and/or PCs development. Thus further investigation is required to understand the functional association between Foxp and LIM-homeodomain protein families during the development of central nervous system. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tam, Wing Yip. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 187-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.1 / 摘要 --- p.4 / Acknowledgements --- p.6 / Abbreviations --- p.8 / Figure list --- p.12 / Table list --- p.15 / Chapter Chapter 1 --- General Introduction --- p.16 / Chapter 1.1 --- An overview of cerebellum functions and anatomy --- p.16 / Chapter 1.2 --- Purkinje cell development in the mouse --- p.19 / Chapter 1.2.1 --- Embryonic development of mouse cerebellum --- p.19 / Chapter 1.2.2 --- Postnatal development of mouse cerebellum --- p.21 / Chapter 1.3 --- Degeneration of Purkinje cell leads to spinocerebellar ataxia --- p.22 / Chapter 1.4 --- LIM-homeodomain genes Lhx1 and Lhx5 --- p.24 / Chapter 1.4.1 --- LIM-homeodomain --- p.24 / Chapter 1.4.2 --- Lhx1 and Lhx5 are crucial to Purkinje cell differentiation --- p.25 / Chapter 1.5 --- Hypothesis, aim and strategy of the study --- p.26 / Chapter Chapter 2 --- Generation of Lhx5 conditional knockout allele in the mouse --- p.31 / Chapter 2.1 --- Chapter summary --- p.31 / Chapter 2.2 --- Introduction --- p.32 / Chapter 2.3 --- Materials and methods --- p.35 / Chapter 2.3.1 --- Materials --- p.35 / Chapter 2.3.2 --- Construction of Lhx5-conditional targeting vector by recombineering --- p.39 / Chapter 2.3.3 --- Gene targeting in mouse embryonic stem cells --- p.53 / Chapter 2.3.4 --- Generation of Lhx5 CKO mouse --- p.62 / Chapter 2.3.5 --- Histological examination of Lhx5 CKO mouse brain --- p.63 / Chapter 2.4 --- Results --- p.64 / Chapter 2.4.1 --- Generation of Lhx5 conditional targeting construct --- p.64 / Chapter 2.4.2 --- Screening of targeted ES cell clones --- p.64 / Chapter 2.4.3 --- Karyotyping --- p.65 / Chapter 2.4.4 --- Generation of chimeric mice and maintenance of Lhx5 CKO mice --- p.66 / Chapter 2.4.5 --- Histological examination of Lhx5 recombined null mutant mouse --- p.67 / Chapter 2.4.6 --- Gross anatomical examination of Lhx5 recombined null mutant mouse --- p.69 / Chapter 2.5 --- Discussion --- p.71 / Chapter Chapter 3 --- Generation and characterization of Pcp2-CreER[superscript T]² transgenic mouse --- p.75 / Chapter 3.1 --- Chapter summary --- p.75 / Chapter 3.2 --- Introduction --- p.76 / Chapter 3.3 --- Materials and methods --- p.79 / Chapter 3.3.1 --- Materials --- p.79 / Chapter 3.3.2 --- Construction of pPcp2-IRES-CreER[superscript T]²-FRT-Kan-FRT --- p.81 / Chapter 3.3.3 --- Generation of BAC-Pcp2-IRES-CreER[superscript T]² transgene --- p.85 / Chapter 3.3.4 --- Generation of Pcp2-CreER[superscript T]² transgenic mice --- p.90 / Chapter 3.3.5 --- Characterization of Pcp2-CreER[superscript T]² transgenic mice --- p.91 / Chapter 3.4 --- Results --- p.93 / Chapter 3.4.1 --- Construction of BAC-Pcp2-IRES-CreER[superscript T]² --- p.93 / Chapter 3.4.2 --- Production of Pcp2-CreER[superscript T]² transgenic mice --- p.93 / Chapter 3.4.3 --- Expression of Cre recombinase in Pcp2-CreER[superscript T]² transgenic mice --- p.93 / Chapter 3.4.4 --- Histological examination of Pcp2-CreER[superscript T]² transgenic mice --- p.97 / Chapter 3.4.5 --- Behavioral test of Pcp2-CreER[superscript T]² transgenic mice by rotarod --- p.98 / Chapter 3.5 --- Discussion --- p.100 / Chapter Chapter 4 --- Characterization of Lhx1/5 double conditional knockout mouse --- p.103 / Chapter 4.1 --- Chapter summary --- p.103 / Chapter 4.2 --- Introduction --- p.104 / Chapter 4.3 --- Materials and methods --- p.106 / Chapter 4.3.1 --- Mouse strain --- p.106 / Chapter 4.3.2 --- Behavioral tests --- p.106 / Chapter 4.3.3 --- Histological examination of cerebellum --- p.107 / Chapter 4.3.4 --- CreER[superscript T]² induction by tamoxifen --- p.108 / Chapter 4.3.5 --- Gene expression profiling using microarray --- p.109 / Chapter 4.3.6 --- Transmission electron microscopy --- p.110 / Chapter 4.3.7 --- Statistical analysis --- p.111 / Chapter 4.4 --- Results --- p.112 / Chapter 4.4.1 --- Early postnatal developmental delay in female DKO mutant --- p.112 / Chapter 4.4.2 --- Lhx1/5 DKO mutants displayed significant motor deficit --- p.114 / Chapter 4.4.3 --- Abnormal Purkinje cell dendritic arborization in the adult Lhx1/5 DKO mutant mouse --- p.117 / Chapter 4.4.4 --- Reduction in the number of synaptic vesicles in the adult Lhx1/5 DKO mutant --- p.119 / Chapter 4.4.5 --- Abnormal Purkinje cell dendrite development in the Lhx1/5 DKO mutant mouse --- p.120 / Chapter 4.4.6 --- Lhx1/5 were not required for the maintenance of developed Purkinje cell dendrite --- p.122 / Chapter 4.4.7 --- Comparison of gene expression profiles in the Lhx1/5 DKO mutant and control --- p.128 / Chapter 4.5 --- Discussion --- p.130 / Chapter Chapter 5 --- Foxp4 - a potential interacting partner of Lhx1/5 --- p.137 / Chapter 5.1 --- Chapter summary --- p.137 / Chapter 5.2 --- Introduction --- p.138 / Chapter 5.3 --- Materials and methods --- p.139 / Chapter 5.3.1 --- Co-immunoprecipitation --- p.139 / Chapter 5.3.2 --- Foxp4 expression pattern and knockdown in cerebellar slice culture --- p.140 / Chapter 5.3.3 --- Generation of Foxp4 CKO mouse --- p.146 / Chapter 5.4 --- Results --- p.148 / Chapter 5.4.1 --- Lhx1 and Lhx5 physically interacted with Foxp4 --- p.148 / Chapter 5.4.2 --- Foxp4 expression during mouse cerebellum development --- p.150 / Chapter 5.4.3 --- Effective gene silencing by siRNA in cerebellar slice culture --- p.151 / Chapter 5.4.4 --- Silencing gene expression of Foxp4 at P5 exerted no observable effect on Purkinje cell survival or differentiation --- p.154 / Chapter 5.4.5 --- Developed Purkinje cell dendritic arbors and associated Bergmann glial fibers were impaired when Foxp4 was knockdown at P10 --- p.157 / Chapter 5.4.6 --- Generation of Foxp4 targeting construct and conditional knockout mouse --- p.159 / Chapter 5.4.7 --- Developmental delay of cerebellum in Foxp4 recombined homozygous mutants --- p.163 / Chapter 5.4.8 --- Normal cerebellum development in adult En1-Cre; Foxp4[superscript fx/fx] and Pax2-Cre; Foxp4[superscript fx/fx] mutants --- p.165 / Chapter 5.4.9 --- Purkinje cell-specific knockout of Foxp4 did not impair Purkinje cell maintenance, motor activity and learning --- p.167 / Chapter 5.5 --- Discussion --- p.171 / Chapter 5.6 --- Acknowledgements --- p.177 / Chapter Chapter 6 --- General discussion, future works and conclusion --- p.179 / Chapter 6.1 --- Evolutionary conserved function of Lhx1 and Lhx5 in neurons --- p.180 / Chapter 6.2 --- LHX1 and LHX5 in human diseases --- p.181 / Chapter 6.3 --- Transcription complex between LIM-homeodomain and forkhead domain proteins may be important in the cerebellum development --- p.182 / Chapter 6.4 --- Future works --- p.183 / Chapter 6.5 --- Conclusion --- p.186 / References --- p.187

During development of the cerebral cortex, the various neuronal subtypes have to reach their correct final position in the post mitotic compartment where they complete their maturation and eventually establish functional networks. Precise positioning of individual neurons is acquired through tight regulation of the multiple transitions that neurons undergo on their way to the cortical plate. Neurons of the cerebral cortex are organized in layers and columns. Although several molecular mechanisms have been identified that control the final position of neurons along the radial dimension of the cortex (i.e. layer specificity), much less is known about how their final tangential, or mediolateral, distribution is controlled. However this may have a direct impact on the structural and functional organization of cortical columns, since sister neurons derived from the same progenitor display selective patterns of connectivity with each other and/or share similar functional properties. Here we studied the role of B-ephrins in the control of migration of cortical pyramidal neurons. Gain of function experiments using in utero electroporation of ephrin-B1 revealed a striking alteration of the tangential distribution of pyramidal neurons during the multipolar stage of radial migration, resulting in clustering of the pyramidal neurons in the cortical plate. Conversely, clonal analysis of migrating neurons in ephrin-B1 knockout mice showed a wider mediolateral dispersion of cortical neurons. Static and dynamic analyses of migrating neurons revealed that ephrin-B1 modulates the morphology of pyramidal neurons during their multipolar phase, thereby restricting their tangential migration at that stage. Our results demonstrate that ephrin-B1 is a specific inhibitor of non-radial migration of pyramidal neurons, thereby controlling the pattern of cortical columns. These data shed new light on this important aspect of pyramidal neuronal migration, and illustrate how alterations of patterns of migration can affect cortical column organization. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le cortex cérébral constitue l’une des structures les plus évoluées et complexes de notre cerveau. Sa surface est divisée en de nombreuses aires fonctionnelles. La mise en place des aires corticales dépend à la fois de facteurs intrinsèques comme la sécrétion de morphogènes ou l’expression en gradient de différents facteurs de transcription, mais elle dépend aussi de facteurs extrinsèques au cortex, en particulier l'innervation par le thalamus. <p>Les ephrines et leurs récepteurs Eph constituent une famille multigénique de facteurs de signalisation impliqués dans divers événements clé du développement cortical où ils sont exprimés selon des profils spatio-temporels complexes. Aux stades tardifs du développement, EphA7 et l’ephrine-A5 sont exprimés en gradients complémentaires au sein de chaque territoire des aires présomptives, constituant ainsi les marqueurs les plus précoces de ces aires corticales. <p>Par la combinaison d’approches in-vitro utilisant la technique d’électroporation focale de tranches corticales embryonnaires, puis in-vivo en utilisant la technique de transgénèse d’addition, nous avons identifié une séquence régulatrice de EphA7 appelée pA7, capable de mimer l’expression endogène de EphA7 au sein du télencéphale dorsal en développement. La lignée de souris pA7-GFP ainsi générée exprime la GFP spécifiquement au sein du télencéphale dorsal durant les stades précoces. Aux stades périnataux cette expression se régionalise au sein de la plaque corticale de chacune des aires présomptives selon des gradients récapitulant ceux observés pour EphA7. Nous avons ensuite purifié des neurones exprimant différents niveaux d’EphA7 par la technique de FACS «Fluorescence-Activated Cell Sorting » et l’analyse de leur transcriptome nous a permis de trouver un grand nombre de gènes différentiellement exprimés. Tous ceux testés par la technique d’hybridation in situ sont exprimés selon un gradient latéral fort et médial faible dans le cortex pariétal, similaire à celui d’EphA7. L’examination de leur profil au sein de cortex de souris dépourvus d’afférences thalamiques, nous a permis de conclure que l’expression de ces gènes incluant EphA7 s’établit indépendamment de celles-ci. Ainsi, notre étude a permis d'identifier un répertoire de gènes neuronaux, pouvant agir en amont ou en combinaison avec EphA7 pour contrôler les facteurs intrinsèques essentiels à l’établissement des aires corticales./<p>The cerebral cortex is subdivided into distinct cortical areas characterized by specific patterns of gene expression and neuronal connectivity. The patterning of cortical areas is thought to be controlled by a combination of intrinsic factors that are expressed in the cortex, and external signals such as inputs from the thalamus. EphA7 is a member of the ephrin/Eph family of guidance factors that is involved in key aspects of the development of the cortex, and is expressed in several gradients within developing cortical areas. <p>By combining in vitro transcriptional assays and mouse transgenics, we identified a regulatory element of the EphA7 promoter, named pA7, that can recapitulate salient features of the pattern of expression of EphA7 in the developing forebrain, including gradients in the cortex. Using a mouse reporter line where GFP expression recapitulates EphA7 expression, we developed a GFP-based cell sorting procedure to isolate cortical neuron populations displaying different levels of EphA7 expression. Transcriptome analysis of these populations enabled to identify a specific array of differentially expressed genes. All genes validated further in vivo were confirmed to be expressed along distinct gradients in the developing cortical plate, similarly to EphA7. The expression of these genes was unchanged in mutant mice defective for thalamocortical projections, indicating that their graded pattern is largely intrinsic to the cortex. Our study identifies a novel repertoire of cortical neuron genes that may act upstream of, or together with EphA7, to control the intrinsic patterning of cortical areas. <p> <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Le cortex cérébral est composé d’un grand nombre de types de neurones organisés radialement en couches cellulaires et tangentiellement en aires corticales fonctionnellement distinctes. Son développement est régulé par des signaux fonctionnant comme morphogènes sécrétés par des centres organisateurs situés à la périphérie du télencéphale dorsal. Ces morphogènes contrôlent l’expression dans les progéniteurs corticaux de gènes codant pour des facteurs de transcription qui régulent la prolifération, la différenciation et la spécification des progéniteurs corticaux. Les cascades de gènes impliquées dans la mise en place du cortex cérébral dans lesquelles ces signaux et facteurs de transcription interviennent restent cependant actuellement mal connues.<p>Les gènes Dmrt3 et Dmrt5 appartiennent à une famille de gènes fortement conservée évolutivement codant pour des facteurs de transcription à doigt de zinc connus pour leur rôle dans le développement sexuel. Des résultats obtenus dans le laboratoire ayant montré que Dmrt5 joue un rôle crucial dans la neurogenèse au niveau du système olfactif chez le xénope, j’ai voulu savoir, dans un premier temps, si la fonction de Dmrt5 dans la neurogenèse était une fonction ancestrale. Pour répondre à cette question, j’ai recherché des gènes Dmrts chez une espèce de Cnidaire, Nematostella vectensis, et ai étudié leur expression au cours du développement. L’un d’entre-eux, NvDmrtB, est fortement exprimé dans le système nerveux et s’est avéré être requis pour la différenciation des cellules nerveuses chez N. vectensis, suggérant que les gènes Dmrts avaient déjà un rôle dans la neurogenèse dans l’ancêtre commun des Bilatériens et des Cnidaires.<p>Par ailleurs, d’autres travaux ont montré que chez la souris les gènes Dmrt5 et Dmrt3 sont exprimés dans le cerveau en développement au niveau du télencéphale dorsal. Afin d’approcher leur fonction dans le développement cortical, j’ai analysé leur expression au cours du développement embryonnaire, caractérisé les anomalies de développement du cortex cérébral des souris knockout Dmrt5-/- et Dmrt3-/- ainsi que des souris double knockout Dmrt3-/-;Dmrt5-/- et étudié leur régulation. Mes résultats ont montré que Dmrt3 et Dmrt5 sont coexprimés dans les progéniteurs corticaux en gradient avec un maximum d’expression du côté caudo-médian. J’ai également observé que l’absence de Dmrt5 induit une réduction de la taille des vésicules télencéphaliques et que les structures de la partie caudo-médiane du cortex telles que le plexus choroïde et l’hippocampe sont altérées ainsi que les aires visuelle et somato-sensorielle. Au niveau moléculaire, mes résultats ont montré que Dmrt5 est requis pour l’expression de différents gènes codant pour les signaux Wnt et Bmp sécrétés au niveau de la région caudo-médiane des vésicules télencéphaliques, et qu’il contrôle négativement Pax6 et positivement Emx2, des déterminants respectivement de l’identité rostro-latérale et caudo-médiane du cortex cérébral. Bien que la taille des vésicules télencéphaliques n’apparaisse pas affectée chez les souris Dmrt3-/-, l’expression de différents composants de la voie Wnt et celle d'Emx2 et Pax6 est légèrement altérée, comme chez les souris Dmrt5-/-. Chez les souris double knock-out Dmrt3-/-;Dmrt5-/-, une réduction de la taille du cortex et des altérations de l’expression des gènes similaires et plus sévères que celle des souris Dmrt5-/- ont été observées. Nous avons également mis en évidence que l’expression de Dmrt3 est réduite chez les souris Dmrt5-/- et que inversement celle de Dmrt5 est légèrement augmentée chez les souris Dmrt3-/-. Enfin, nous avons observé que l’expression de ces deux gènes est dépendante du facteur de transcription Gli3 et que seul l’expression de Dmrt3 requiert les facteurs de transcription Pax6 et Emx2.<p>Ensemble, nos résultats indiquent que les facteurs de transcription Dmrt5 et Dmrt3 contrôlent le développement de la partie caudo-médiane du cortex, Dmrt5 agissant en amont de Dmrt3. Ils suggèrent également que ces facteurs y joueraient des rôles partiellement redondants en régulant l’expression de cibles communes tels les gènes Wnt3a et Pax6.! / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

The cerebral cortex is one of the most complex and divergent of all biological structures and is composed of hundreds of different types of highly interconnected neurons. This complexity underlies its ability to perform exceedingly complex neural processes. One of the most important questions in developmental neurobiology is how such a vast degree of diversity and specificity is achieved during embryogenesis. Furthermore, understanding the cellular and genetic basis of cortical development may yield insights into the mechanisms underlying human disorders such as mental retardation, autism, epilepsies and brain tumors. <p>During this Phd-project, we set out to identify novel transcription factors involved in cortical neurogenesis. Therefore, we initially took advantage of a model of in vitro embryonic stem cell (ESC)-derived corticogenesis that was previously established in the lab (Gaspard et al. 2008) and from several previously generated ESC lines that allow overexpression of specific transcription factors potentially involved in corticogenesis (van den Ameele et al. 2012). <p>Among the genes tested, Bcl6, a B-cell lymphoma oncogene known to be expressed during cortical development but without well-characterized function in this context, displayed a strong proneurogenic activity and thus became the main focus of this thesis. <p><p>During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is well known to be important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signalling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition.<p>We showed that Bcl6 starts to be expressed specifically during the transition from progenitors to postmitotic neurons and is required for proper neurogenesis of the mouse cerebral cortex. Bcl6 promotes this neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. Bcl6 triggers exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD+-dependent deacetylase Sirt1, which we showed to be required for Bcl6-dependent neurogenesis in vitro. The resulting epigenetic silencing of Hes5 leads to neuronal differentiation despite active Notch signalling. These findings thus suggest a role for Bcl6 as a novel proneurogenic factor and uncover Notch-Bcl6-Sirt1 interactions that may affect other aspects of physiology and disease (Tiberi et al. 2012a). <p><p>A subsequent yet unpublished part of this Phd-project focused on unraveling roles for Bcl6 in regionalization of the cerebral cortex. In all mammals, the three major areas of the neocortex are the motor, somatosensory and visual areas, each subdivided in secondary domains and complemented with species-specific additional areas. All these domains comprise of neurons with different functionality, molecular profiles, electrical activity and connectivity. Spatial patterning of the cortex is mainly under the control of diffusible molecules produced by organizing centers, but is also regulated by intrinsic, cell-autonomous programs (Tiberi et al. 2012b). <p>Since Bcl6 expression is confined to frontal and parietal regions of the developing cerebral cortex and remains high in postmitotic neurons, also after completion of neurogenesis, we hypothesized it would be involved in acquisition of motor and somatosensory identity. As expected from the neurogenesis defect in these regions, we observed a trend towards a reduced size of the frontal areas in the Bcl6 mutant cortex. Preliminary data from cDNA microarray profiling after gain- and loss-of-function of Bcl6 and from in situ hybridization on mouse cortex however do not show dramatic changes in molecular markers of different cortical areas. Similarly, the coarse-grained pattern of thalamocortical and efferent projections of motor and somatosensory neurons appears to be spared. These preliminary findings thus suggest that Bcl6 is not strictly required for proper acquisition of motor and somatosensory areal identity. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.