L’hippocampe est un composant majeur du cerveau des mammifères et joue d'importants rôles dans la mémoire, l’apprentissage et la navigation spatiale. Il comprend deux régions distinctes: les champs ammoniens et le gyrus denté (DG). Pendant ma thèse, je me suis intéressée au facteur de transcription COUP- TFI, jouant des rôles clefs dans la spécification et migration neocorticale. Peu de choses sont connues sur son rôle dans l’hippocampe. COUP-TFI y est exprimé en gradient dans les progéniteurs et dans les neurones différentiés, et est fortement localisé dans le neuroépithelium du DG. Le but de ma thèse était de déchiffrer le rôle de COUP-TFI dans le développement de l’hippocampe, au cours de la différentiation et migration des granules, population principale du DG. À l’aide de lignées de souris dans lesquelles COUP-TFI est soit inactivé dans les progéniteurs soit seulement dans les cellules différentiées, j’ai montré que l’absence de COUP-TFI induit différents degrés d’altérations. En l’absence de COUPTFI dans les progéniteurs, les précurseurs du DG se différentient précocement, ont une prolifération réduite et leur migration est altérée. De plus, les afférences du cortex n’innervent pas le DG septal et l’apoptose y est accrue. Le DG en résulte fortement réduit chez adulte, particulièrement dans la région septal. La perte de COUP-TFI dans les cellules différentiées n’entraine que des anomalies mineures et transitoires. Ainsi, mes résultats indiquent que COUP-TFI régule la différentiation et migration des granules, particulièrement au niveau des progéniteurs, et propose COUP-TFI comme un nouveau facteur requis dans le développement et le fonctionnement de l’hippocampe. / The hippocampus is a major component of the mammalian brain and plays important roles in memory, learning, and spatial navigation. It comprises two distinct regions: the hippocampus proper and the dentate gyrus (DG). During my thesis, I have challenged the role of the strong transcriptional regulator COUP-TFI, playing key roles during neocortical specification and migration. Yet, little is known about its involvement in the hippocampus. COUP-TFI is expressed in a gradient fashion in both proliferating progenitors and differentiated neurons in the hippocampus, and is highly localized in the DG neuroepithelium. The aim of my thesis was thus to decipher the role of COUP-TFI in the developing hippocampus, and specifically in cell differentiation and migration of granule cells, the major DG cell population. Using two mutant mouse lines, in which COUP-TFI is either ablated in progenitors, or solely in post-mitotic cells, I have shown that absence of COUP-TFI induces different degrees of growth impairments. In the absence of COUP-TFI in progenitors, DG precursors tend to differentiate precociously, exhibit reduced proliferation and granule cells migration is impaired. Postnatally, inputs from the cortex fail to innervate the septal DG and apoptosis is abnormally increased. The DG results strongly reduced in adult, particularly in the septal region. Loss of COUP-TFI in differentiated cells leads only to minor and transient defects. Together, my results indicate that COUP-TFI is involved in regulating granule cell differentiation and migration predominantly in progenitors, and propose COUPTFI as a novel transcriptional regulator required in hippocampal development and functions.

ABSTRACT Retinoic acid (RA) is a positive regulator of P19 EC cell differentiation. Pre-B cell leukemia transcription factors (PBXs) act in conjunction with homeobox genes during cell differentiation. PBX mRNA and protein levels are increased rapidly in P19 cells during RA-induced differentiation. However, silencing of PBX expression in P19 cells (AS cells) results in a failure of these cells to differentiate upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) and Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) are orphan members of the steroid-thyroid hormone superfamily. The mRNA and protein levels of both COUP-TFI and COUP-TFII are low in proliferating wild type P19 EC cells. However, when wild type P19 cells are induced to differentiate upon RA treatment, COUP-TFI and COUP-TFII mRNA and protein levels are dramatically increased while the levels of pluripotency associated gene products are strikingly reduced. Conversely, COUP-TFI and COUP-TFII mRNA levels fail to be elevated upon RA treatment in PBX AS P19 EC cells. Therefore it was hypothesized that COUP-TFs may be downstream targets of PBX and required factors mediating the RA-dependent differentiation cascade in P19 cells. To determine the role of COUP-TFI during differentiation of P19 cells, PBX AS cells that inducibly express V5 tagged COUP-TFI using the Tet-Off® Advanced Inducible Gene Expression system were prepared. Using this system, we demonstrate that exogenous COUP-TFI expression, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption and early apoptosis. Furthermore, using this cell model which inherently is incapable of undergoing RA-mediated differentiation due to blockage of PBX induction, we demonstrate that a supraphysiological level of COUP-TFI expression can overcome the blockage of RA-dependent differentiation in PBX AS cells. However, AS cells expressing a physiological level of COUP-TFI differentiate to endodermal cells only upon treatment with RA. Additionally, gene expression studies indicate that the reductions of pluripotency maintenance genes observed in the COUP-TFI expressing cells are similar to that of wild type P19 cells (upon RA treatment) suggesting that COUP-TFI expression is a driving force towards loss of pluripotency. Moreover, gene expression studies indicate COUP-TFI is involved in the regulatory modulation of at least two RA response genes, CYP26A1 and HoxA1, indicating that COUP-TFI may have some effect on either maintaining or reducing these genes expression levels when COUP-TFI becomes expressed. COUP-TFII is expressed as two distinct variants, Variant 1(V1) and Variant 2 (V2). V1 is the variant that functions as a classical nuclear receptor by binding target DNA sequences and affecting gene transcription whereas V2 is a truncated form of V1 lacking the ability to bind DNA. We therefore hypothesized that V2 could serve as a dominant negative receptor by limiting the amount of functional V1 in the cell. Unexpectedly, we found using P19 cells that overexpress V2 that RA-mediated differentiation proceeded normally suggesting V2 does not function as a dominant negative repressor. Taken together, these studies demonstrate for the first time (i) that COUP-TFI functions as a physiologically relevant regulator during RA-mediated endodermal differentiation of P19 cells and (ii) COUP-TFII V2 is endogenously expressed in P19 cells; however its role during RA-mediated differentiation remains unclear. / Biochemistry

Le néocortex est une région du cerveau qui traite toutes les entrées sensorielles et créé des réponses comportementales. Il est subdivisé en zones fonctionnelles, chacune ayant une cytoarchitecture, un motif d’expression génique et un profil de connectivité spécifiques. L'organisation en zones est pré-modelée par des gènes organisateurs, et ensuite affinée par l’activité sensorielle. Dans cette étude, j'ai étudié d'abord si ce pré-modelage est établi dans les progéniteurs et/ou les cellules post-mitotiques, et si l'activité neuronale spontanée est nécessaire pour l’établissement de la connectivité correcte entre néocortex et thalamus, station relais principale des données sensorielles. Avec l'aide d'une série de souris transgéniques, j’ai montré que la fonction du gène organisateur COUP-TFI est suffisante et nécessaire pour organiser l'identité sensorielle dans les cellules post-mitotiques, et que COUP-TFI régule l'activité intrinsèque des neurones corticaux, influençant la bonne intégration des entrées thalamiques dans le cortex somatosensoriel. J’ai montré que COUP-TFI contrôle directement l'expression du gène Egr1, qui dépend fortement de l'activité neuronale. COUP-TFI et Egr1 agissent sur l'acquisition de la morphologie des cellules étoilées dans les neurones de la couche 4, cibles principales des axones thalamiques et trait typique des zones somatosensoriels primaires. En conclusion, ce travail montre que le pré-modelage cortical dépend primordialement d’un programme génétique établi dans les cellules post-mitotiques et que l'activité intrinsèque et les propriétés génétiques agissent ensemble pour façonner l'organisation des premiers circuits dans le néocortex. / The neocortex is a region of the brain that processes all sensory inputs creating appropriate behavioral responses. It is subdivided into functional areas, each with a specific cytoarchitecture, gene expression pattern and connectivity profile. The organization into areas is pre-patterned by the action of areal patterning genes, and subsequently refined by sensory evoked activity. In this study, I have first investigated whether early areal patterning is committed in progenitor and/or post-mitotic cells, and then assessed whether spontaneous neuronal activity is required in establishing correct connectivity between the neocortex and the thalamus, the principal relay station of peripheral sensory inputs. With the help of a series of transgenic mice, my work showed that the function of the areal patterning gene COUP-TFI is sufficient and necessary to organize sensory identity in post-mitotic cells, and that COUP-TFI regulates intrinsic activity properties of cortical neurons, and thus proper integration of thalamic inputs into the somatosensory cortex. In particular, I found that COUP-TFI directly controls the expression of the immediate early gene Egr1, which expression levels strongly depend on neuronal activity. Both COUP-TFI and Egr1 act on the acquisition of the stellate cell morphology of layer 4 neurons, the main targets of thalamic axons and a typical trait of primary somatosensory areas. In conclusion, this work demonstrates that cortical area patterning primordially depends on a genetic program established in post-mitotic cells and that intrinsic genetic and activity properties act together to shape the organization of early circuits in the neocortex.

名古屋大学博士学位論文 学位の種類:博士（医療技術学） (課程) 学位授与年月日　平成22年3月25日

Nuclear receptors (NRs) are important transcription factors involved in the regulation of a variety of physiological processes such as embryonic development, cell differentiation and homeostasis (for review, see Mangelsdorf et al., 1995 TenBaum and Baniahrned, 1997). In contrast to membrane bound receptors, they bind small lipophilic ligands and function in the nucleus as ligand-modulated transcription factors. The ligands for nuclear receptors include steroids (glucocorticoids, progestins, mineralocorticoids, androgens and estrogens), vitamin D3, retinoids, thyroid hormone, prostaglandins, farnesoids etc. Several other nuclear receptors are classified as orphan receptors for which no ligand has yet been identified. More than 300 nuclear receptors have now been identified and together these proteins comprise the single largest family of metazoan transcription factors, the nuclear receptor superfamily. Recently, a unified nomenclature has been evolved (nuclear receptor nomenclature committee, 1999), a summary of which is presented in Table 1.