Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growthpromoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.

Les métalloproteases matricielles (MMPs) appartiennent à une famille d'endopéptidases dépendantes du zinc, présentent sous forme secrétée ou membranaire (MT-MMP) et qui jouent un rôle fondamental dans la signalisation cellulaire. L'activité des MMPs est régulée par leur inhibiteurs endogènes, les inhibiteurs tissulaires des MMPs (TIMPs). Le système MMP/TIMP régule les interactions cellule-cellule et cellule-matrice extra cellulaire et module la motilité cellulaire par clivage protéolytique des composants de la matrice extra cellulaire aussi bien lors de processus physiologiques que dans des situations pathologiques.Dans un premier temps, nous avons mis en évidence le rôle de TIMP-1 dans la modulation de la croissance neuritique et la morphologie neuronale, via l'inhibition de MMP-2 et non de MMP-9. souches de la muqueuse olfactive (OE-MSCs). Nous montrons dans cette étude que les gélatinases MMP-2 et MMP-9 ainsi que la MMP membranaire MT1-MMP, sont impliquées dans la migration des OE-MSCs. Nous montrons également que les gélatinases sont probablement impliquées dans les propriétés neurotrophiques des OE-MSCs et des cellules engainantes olfactives.L'ensemble de ces résultats apporte de nouveaux éléments fondamentaux, dans la compréhension du rôle du système MMP/TIMP dans les processus post-lésionnels qui ont lieu au sein du système nerveux central. / The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), which play a fundamental role in the cell signalling. The activity of the MMPs is regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). The MMP / TIMP system regulates the cell-cell and cell-extracellular matrix interactions and modulates the cellular motility through the cleavage of protein components of the extracellular matrix, as well during physiological and pathological conditions.Our results suggest that TIMP-1 is implicated in the modulation of the neurite outgrowth and morphology of cortical neurons through the inhibition at least in part, of MMP-2 and not MMP-9. Afterward, we study of the system MMP / TIMP in the migration of the stem cells of olfactory ectomesenchymal stem cells (OE-MSCs). We show that gelatinases MMP-2 and MMP-9 as well as MT1-MMP, are involved in OE-MSCs migration. We also show that gelatinases are probably involved in neurotrophic properties of the OE-MSCs and olfactory ensheathing cells.Altogether, these results provide new evidences on the role of MMP/TIMP system in central nervous system post-lesional processes.

Experimentos anteriores realizados em nosso laboratório indicam que o sucesso gestacional é afetado pela poluição atmosférica. O presente estudo teve como objetivo avaliar os efeitos biológicos associados a uma curva dose resposta das partículas de exaustão do diesel (PED) sobre o desenvolvimento embrionário inicial e o potencial de implantação, utilizando-se como modelo a fertilização in vitro e o cultivo embrionário de camundongos. No Experimento 1, encontrou-se um efeito negativo dose-dependente sobre o desenvolvimento embrionário inicial, o processo de eclosão, a alocação das células e a morfologia da massa celular interna (MCI) dos blastocistos. A análise post-hoc revelou que o desenvolvimento precoce do embrião não foi afetado pelas concentrações de 0,2 µg/cm2 ou 2 µg/cm2, mas foi significativamente afetado pela concentração de 20 µg/cm2 de PED. O processo de eclosão foi prejudicado pelas concentrações de 2 µg/cm2 e 20 µg/cm2. A alocação das células da MCI e a relação entre as células da MCI e do trofectoderma foram significativamente afetadas por todas as concentrações. Adicionalmente, observou-se um efeito negativo sobre a morfologia da MCI para as concentrações de 2 µg/cm2 e 20 µg/cm2. O Experimento 2, apesar de não mostrar efeito significativo sobre o potencial de implantação, evidenciado pela capacidade de adesão dos blastocistos e crescimento trofoblástico, revelou que a morfologia da MCI no dia 8 de cultivo, as taxas de viabilidade e de apoptose celular e a expressão de Oct4 e Cdx2 foram significativamente afetadas. O teste HSD-Tukey demonstrou que a presença de PED (0,2 µg/cm2 e 2 µg/cm2) durante o desenvolvimento embrionário aumentou significativamente a taxa de células em apoptose dos embriões tanto no dia 5 quanto no dia 8 de cultivo e, embora a proporção de células viáveis no dia 8 tenha sido prejudicada por ambas as concentrações, apenas a exposição a 2 µg/cm2 de PED diminuiu a viabilidade celular no dia 5. Por outro lado, tanto a concentração de 0,2 µg/cm2 como a de 2 µg/cm2 tiveram um efeito negativo significativo sobre a qualidade da MCI no dia 8 e a taxa de expressão de Oct4 nos blastocistos e aumentaram a porcentagem de células desses blastocistos expressando Cdx2, adicionalmente, a razão Oct4/Cdx2 dos embriões expostos a 0,2 µg/cm2 e 2 µg/cm2 foi significativamente menor. Frente a esses resultados, presumi-se que as PED poderiam estar envolvidas nos mecanismos que levariam à diminuição do sucesso reprodutivo observado em camundongos expostos à poluição atmosférica ambiental / Previous experiments conducted in our laboratory demonstrate that successful pregnancy is affected by air pollution. The aim of this study was to evaluate the biological effects associated with a dose-response curve of the diesel exhaust particles (DEP) on early embryonic development and implantation potential, using mice in vitro fertilization and culture embryo as model. In Experiment 1, we found a negative dose-dependent effect on the embryonic development, hatching process, cell allocation and morphology of inner cell mass (ICM) of blastocysts. A post-hoc analysis revealed that the early development of the embryo was not affected by concentrations of 0.2 g/cm2 or 2g/cm2, but was significantly affected by the concentration of 20 g/cm2 of DEP. The hatching process was impaired by concentrations of 2 g/cm2 and 20 g/cm2. Cell allocation of ICM and the ratio between cells of ICM and trophectoderm were significantly affected by all concentrations. Addicionaly, we observed a negative effect on ICM morphology was observed for the 2 µg/cm2 and the 20 µg/cm2 concentrations. Experiment 2, despite showing no significant effect on implantation potential, as evidenced by the adhesion ability and trophoblast outgrowth, revealed that ICM morphology on day 8 of culture, rates of cell viability and apoptosis, and expression of Oct4 and Cdx2 were significantly affected. The Tukey HSD test showed that presence of DEP (0.2 g/cm2 and 2 g/cm2) during embryonic development increased significantly the rate of apoptotic cells in embryos as on day 5 as on day 8 of culture, although the proportion of viable cells on day 8 was impaired by both concentrations, only exposure to 2 g/cm2 PED decreased cell viability on day 5. On the other hand, both the concentration of 0.2 g/cm2 such as 2 g/cm2 had a significant negative effect on the quality of ICM on the day 8 and the rate of expression of Oct4 on blastocysts, and increased the percentage of cells from these embryos expressing Cdx2, also, Oct4/Cdx2 ratio were significantly lower in the blastocysts derived from embryos exposed to 0.2 g/cm2 and 2 g/cm2¬ concentrations. Given these results, the suggestion is that DEP could be involved in the mechanisms that lead to decreased reproductive success observed in mice exposed to environmental pollution

Die vorliegende Arbeit behandelte den Einfluss von membran-assoziierten Faktoren im Hippocampus auf das axonale Wachstum, zum einen während der Entwicklung des entorhino-hippocampalen Systems und zum anderen nach Deafferenzierung des adulten Hippocampus. Mit Hilfe des Streifenassays und des Längenauswachsassays wurden zuerst die maturationsabhängigen Eigenschaften von membran-assoziierten Faktoren im Hippocampus getestet. Es zeigte sich, dass entorhinale Axone zwischen ihrem normotypischen Zielgebiet und Kontrollregionen diskrimieren können und bevorzugt auf Membranen ihres Zielgebiets wachsen. Im Folgenden wurden dann Axonen hippocampale Membranen unterschiedlicher Entwicklungsstadien im Streifenassay angeboten. In diesem experimentellen Ansatz wuchsen entorhinale Axone präferenziell auf jenen hippocampalen Membranen, die aus dem Entwicklungsstadium stammen, in den die entorhinalen Fasern in vivo in den Hippocampus einwachsen. Diese Experimente ergaben, dass das in vivo zeitlich genau regulierte Einwachsen entorhinaler Fasern in den Hippocampus von membran-assoziierten Faktoren determiniert ist und ein Zeitfenster für das Vorhandensein dieser Faktoren im Hippocampus existiert. Eines der wesentlichen Charakteristika der Maturation des zentralen Nervensystem ist die Bildung von Myelin und die Myelinisierung von Fasertrakten. Immunozytochemische Analysen mit Myelin-spezifischen Markern ergaben, dass dieses maturationsabhängige Auswachsverhalten zeitlich gut mit der Myelinisierung dieser Hirnregion korreliert. Eine Reihe von in vivo und in vitro Experimenten verschiedener Arbeitsgruppen demonstrierten, dass Myelin starke auswachsinhibitorische Eigenschaften hat, die sogar den Kollaps von Wachstumskolben induzieren können. In Längenauswachsassays zeigte sich, dass Myelin einen starken inhibitorischen Effekt auf das Längenwachstum von entorhinalen Axonen hat. Mit physikalischen Separationstechniken und unter Verwendung des funktionellen Antikörpers gegen inhibitorische Myelinproteine (IN-1) konnte dieser Effekt neutralisiert werden und das neuronale Längenwachstum war wieder vergleichbar zur Kontrollsituation. Untersuchungen im Streifenassay ergaben zusätzlich, dass das wachstumsinhibitorische Myelin und seine Komponenten keine axonalen Lenkungseigenschaften hatte und die gerichtete Zielfindung axonalen Auswachsens nicht beeinflusst. In weiteren Experimenten wurden die membran-assoziierten Faktoren im deafferenzierten Hippocampus untersucht. Dabei zeigte sich, dass nach einer Läsion wachstumsfördernde Faktoren in hippocampalen Membranen vorliegen. Zusätzlich liegen in einem engen Zeitfenster axonale Lenkungsmoleküle vor mit vergleichbarer Attraktivität für entorhinale Axone, wie sie aus entsprechenden Entwicklungsstadien bekannt sind. Die Experimente lassen den Schluss zu, dass im deafferenzierten Hippocampus Faktoren läsionsinduziert werden, und dass diese Faktoren membran-assoziiert sind. Es wird seit langem angenommen, dass das ZNS von adulten Vertebraten in seinem zellulären Zustand determiniert ist und zu keinen grösseren plastischen Veränderungen fähig ist. Gerade nach einer Schädigung von adultem ZNS ist die Regenerationsfähigkeit im Unterschied zu jungen, postnatalen ZNS sehr eingeschränkt. Die beschränkte Regenerationsfähigkeit des adulten ZNS ist wesentlich determiniert durch die Präsenz des auswachsinhibitorischen Myelins. Nichtsdestotrotz gibt es kompensatorisches Sprouting im Hippocampus, die verlorengegangene synaptische Kontakte ersetzen. Die Identifizierung der Faktoren, die das schichten-spezifische Einwachsen aussprossender Axone kontrollieren, trägt wesentlich zum Verständnis dieses Phänomens bei. Weiterhin wird die Aufklärung der zugrundeliegenden molekularen Faktoren für die spezifische Zielerkennung und deren Charakterisierung uns helfen, das Potential und die Limitation der Regeneration im ZNS besser zu verstehen und die Möglichkeit eröffnen, einmal verlorengegangene neuronale Verbindungen durch therapeutische Intervention wieder spezifisch aufzubauen. / In this study, the impact of membrane-associated factors on axonal outgrowth during development and following lesion was examined. We studied the maturation-dependent features of membrane-associated molecules in the hippocampus with the stripe assay for guidance activity and with the outgrowth assay for outgrowth-supporting activity. We could show that entorhinal axons discriminate between their proper target area, the hippocampus, and control regions which do not receive synaptic connections from the entorhinal cortex, and preferred to grow on hippocampal membranes. Further, we examined guidance preferences of entorhinal neurites on hippocampal membranes in different developmental stages. The choice behavior of entorhinal neurites for hippocampal membranes temporally correlates with the ingrowth of the perforant path into the hippocampus and with the stabilization of this brain area in vivo, and further indicate the transient presence of membrane-associated guidance cues in the hippocampus. One of the characteristics of maturational processes in the central nervous system is the developmentally regulated myelination of fiber tracts. Comparison of the stripe assay data with immunohistochemical analysis for MBP and MAG as representative myelin markers revealed a correlation between the changes in axonal choice behavior and increasing myelination. It is known that myelin itself is a strong axonal outgrowth inhibitor and that myelin can also induce growth cone collapse. In outgrowth assays, we could show that myelin has a strong outgrowth inhibitory influence on entorhinal axons which can be neutralized by the monoclonal antibody IN-1. However, in the stripe assay, myelin did not influence the choice behavior of outgrowing axons and this indicates that myelin does not govern information for directed growth. Furthermore, stripe assays were performed with membranes obtained from deafferented hippocampi at various lesion stages. In these experiments, we could show that outgrowth-promoting factors are present in the lesioned hippocampus. Moreover, data from the stripe assay revealed the timely restricted presence of membrane-bound guidance factors which are equally as attractive as neonatal hippocampal membranes. These experiments indicate the lesion-induced expression of outgrowth-promoting factors in the hippocampus, which correlates temporally with the sprouting reaction in vivo. It is suggested that the central nervous system of adult vertebrates is determined in its cellular condition and not capable of structural changes. This is most evident following lesion of adult brain, where the ability for regeneration is highly restricted in comparison to young, postnatal neural tissue. This restricted ability for regeneration in the adult brain is essentially determined by the presence of outgrowth-inhibitory myelin. However, a compensatory sprouting response exists in the adult hippocampus following lesion, which leads to a layer-specific replacement of lost synaptic contacts. The identification of these factors will lead to a deeper understanding of layer-specific axonal sprouting and synaptic replacement. Further, the identification and characterization of the underlying factors will help us to understand the potential and limitations of regeneration in the central nervous system.

<p>The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.</p><p>This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.</p>

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

No description available.

Limb outgrowth is controlled by a specialized group of cells called the apical ectodermal ridge (AER), a thickening of the limb epithelium, at its distal tip. This specialized thickening of ectodermal cells is responsible for maintaining the underlying mesenchymal cells in an undifferentiated and proliferative state, and its structure is preserved through a fine-tuned balance between proliferation and apoptosis. This equilibrium is genetically controlled but little is known about the molecules involved in this process. Several authors have been shown that both fibroblast growth factor (FGF) and Erk pathway activation are crucial for AER function. Recently, FLRT3, a transmembrane protein able to interact with FGF receptors, has been implicated in the triggering of ERK activity by FGFs. In this thesis, we show that flrt3 expression is restricted to the AER, co-localizing its expression with fgf8 and pERK activity. Loss-of-function studies demonstrate that silencing of flrt3 affects the integrity of the AER and, subsequently, its proper function during limb bud outgrowth. Our data also indicate that flrt3 expression is not regulated by FGF activity in the AER, whereas ectopic WNT3A is able to induce flrt3 expression. Overall, our findings confirm flrt3 as a key player during chicken limb development, being necessary but not sufficient for proper AER formation and maintenance under the control of BMP and WNT signalling. During limb bud development, AER structure is maintained through a fine-tuned balance between proliferation and programmed cell death and this equilibrium is genetically controlled, although little is known about the molecules involved in that process. In this thesis we present evidences involving oct4, required to establish and maintain the pluripotent cell population necessary for embryogenesis in mouse and human, in the control of the proliferative balance within the AER cells. Overexpression of otc4 in the limb ectoderm disrupts the ratio apoptosis/proliferation and, moreover, oct4 expression is under the control of wnt-canonical pathway. We also describe a special localization and behaviour of proliferating cells in the AER in response to oct4 activity. We, therefore, describe a role for oct4 as a factor able to maintain a niche of cells that is responsible for the renewal of the AER. / El crecimiento del esbozo de la extremidad está controlado por un grupo especializado de células denominado Cresta Ectodérmica Apical (CEA), un engrosamiento del epitelio del miembro en su borde más distal. Este engrosamiento es responsable del mantenimiento de las células del mesodermo distal en un estado indiferenciado y proliferativo. Diferentes estudios muestran que la actividad de los factores de crecimiento fibroblástico (FCF) y de la vía Erk son cruciales para la correcta funcionalidad de la CEA. Recientemente se ha implicado a FLRT3, una proteína transmembranal capaz de interaccionar con los receptores de los FCF, en la activación de la vía Erk por los mismos. En esta tesis describimos cómo la expresión de flrt3 se restringe a la CEA, colocalizándose su expresión con fgf8 y la actividad de la vía Erk. Los experimentos de pérdida de función demuestran que la inhibición de flrt3 afecta la integridad de la CEA y, consecuentemente, a su función durante el desarrollo del esbozo del miembro. Nuestros datos también indican que la expresión de flrt3 no está regulada a través de los FCF en la CEA, sin embargo, la activación ectópica de WNT3A es capaz de inducir la expresión de flrt3. En conjunto, nuestros resultados demuestran que flrt3 es una molécula clave durante el desarrollo de las extremidades de pollo, siendo necesaria, pero no suficiente, para la correcta formación y mantenimiento de la CEA bajo el control de la señalización a través de BMP y WNT. Durante el desarrollo de las extremidades, la estructura de la CEA se mantiene a través de un fino control del balance entre la proliferación y apoptosis. Este equilibrio se encuentra genéticamente controlado aunque se sabe muy poco acerca de las moléculas involucradas en este proceso. En esta tesis presentamos evidencias en las que oct4, molécula necesaria para establecer y mantener la población de células pluripotentes necesarias durante la embriogénesis en ratón y humanos, controla la tasa de proliferación en las células de la CEA. La expresión ectópica de oct4 en el ectodermo del esbozo de la extremidad perturba la razón entre la apoptosis y la proliferación y, además, su expresión está controlada por la actividad de la vía canónica de los Wnt. También describimos en este trabajo la localización y comportamiento especiales de las células de la CEA en proliferación como respuesta a la actividad de oct4. Por consiguiente, podemos inferir que el rol de oct4 será el de un factor necesario para mantener un nicho celular responsable por la renovación de la CEA.

Experimentos anteriores realizados em nosso laboratório indicam que o sucesso gestacional é afetado pela poluição atmosférica. O presente estudo teve como objetivo avaliar os efeitos biológicos associados a uma curva dose resposta das partículas de exaustão do diesel (PED) sobre o desenvolvimento embrionário inicial e o potencial de implantação, utilizando-se como modelo a fertilização in vitro e o cultivo embrionário de camundongos. No Experimento 1, encontrou-se um efeito negativo dose-dependente sobre o desenvolvimento embrionário inicial, o processo de eclosão, a alocação das células e a morfologia da massa celular interna (MCI) dos blastocistos. A análise post-hoc revelou que o desenvolvimento precoce do embrião não foi afetado pelas concentrações de 0,2 µg/cm2 ou 2 µg/cm2, mas foi significativamente afetado pela concentração de 20 µg/cm2 de PED. O processo de eclosão foi prejudicado pelas concentrações de 2 µg/cm2 e 20 µg/cm2. A alocação das células da MCI e a relação entre as células da MCI e do trofectoderma foram significativamente afetadas por todas as concentrações. Adicionalmente, observou-se um efeito negativo sobre a morfologia da MCI para as concentrações de 2 µg/cm2 e 20 µg/cm2. O Experimento 2, apesar de não mostrar efeito significativo sobre o potencial de implantação, evidenciado pela capacidade de adesão dos blastocistos e crescimento trofoblástico, revelou que a morfologia da MCI no dia 8 de cultivo, as taxas de viabilidade e de apoptose celular e a expressão de Oct4 e Cdx2 foram significativamente afetadas. O teste HSD-Tukey demonstrou que a presença de PED (0,2 µg/cm2 e 2 µg/cm2) durante o desenvolvimento embrionário aumentou significativamente a taxa de células em apoptose dos embriões tanto no dia 5 quanto no dia 8 de cultivo e, embora a proporção de células viáveis no dia 8 tenha sido prejudicada por ambas as concentrações, apenas a exposição a 2 µg/cm2 de PED diminuiu a viabilidade celular no dia 5. Por outro lado, tanto a concentração de 0,2 µg/cm2 como a de 2 µg/cm2 tiveram um efeito negativo significativo sobre a qualidade da MCI no dia 8 e a taxa de expressão de Oct4 nos blastocistos e aumentaram a porcentagem de células desses blastocistos expressando Cdx2, adicionalmente, a razão Oct4/Cdx2 dos embriões expostos a 0,2 µg/cm2 e 2 µg/cm2 foi significativamente menor. Frente a esses resultados, presumi-se que as PED poderiam estar envolvidas nos mecanismos que levariam à diminuição do sucesso reprodutivo observado em camundongos expostos à poluição atmosférica ambiental / Previous experiments conducted in our laboratory demonstrate that successful pregnancy is affected by air pollution. The aim of this study was to evaluate the biological effects associated with a dose-response curve of the diesel exhaust particles (DEP) on early embryonic development and implantation potential, using mice in vitro fertilization and culture embryo as model. In Experiment 1, we found a negative dose-dependent effect on the embryonic development, hatching process, cell allocation and morphology of inner cell mass (ICM) of blastocysts. A post-hoc analysis revealed that the early development of the embryo was not affected by concentrations of 0.2 g/cm2 or 2g/cm2, but was significantly affected by the concentration of 20 g/cm2 of DEP. The hatching process was impaired by concentrations of 2 g/cm2 and 20 g/cm2. Cell allocation of ICM and the ratio between cells of ICM and trophectoderm were significantly affected by all concentrations. Addicionaly, we observed a negative effect on ICM morphology was observed for the 2 µg/cm2 and the 20 µg/cm2 concentrations. Experiment 2, despite showing no significant effect on implantation potential, as evidenced by the adhesion ability and trophoblast outgrowth, revealed that ICM morphology on day 8 of culture, rates of cell viability and apoptosis, and expression of Oct4 and Cdx2 were significantly affected. The Tukey HSD test showed that presence of DEP (0.2 g/cm2 and 2 g/cm2) during embryonic development increased significantly the rate of apoptotic cells in embryos as on day 5 as on day 8 of culture, although the proportion of viable cells on day 8 was impaired by both concentrations, only exposure to 2 g/cm2 PED decreased cell viability on day 5. On the other hand, both the concentration of 0.2 g/cm2 such as 2 g/cm2 had a significant negative effect on the quality of ICM on the day 8 and the rate of expression of Oct4 on blastocysts, and increased the percentage of cells from these embryos expressing Cdx2, also, Oct4/Cdx2 ratio were significantly lower in the blastocysts derived from embryos exposed to 0.2 g/cm2 and 2 g/cm2¬ concentrations. Given these results, the suggestion is that DEP could be involved in the mechanisms that lead to decreased reproductive success observed in mice exposed to environmental pollution

La Polarité Cellulaire Planaire (PCP) est une voie de signalisation originellement identifiée chez les invertébrés pour son rôle dans l’établissement d’une asymétrie cellulaire perpendiculaire à l’axe apico‐basal. Elle définit une polarité dans le plan d’un épithélium et coordonne cette polarité dans tout l'épithélium. L'activation de la voie PCP conduit à une réorganisation ducyto squelette en passant par une modulation des zones d'adhésion, régulant ainsi la forme et les mouvements des cellules. La voie de signalisation de la PCP est conservée tout au long de l'évolution jusqu'au mammifères, et contrôle la morphogénèse de divers tissus dont les tissus épithéliaux et mésenchymateux, ainsi que pour les tissues cardiaques, osseux, pulmonaire ou encore rénaux, mais aussi le système nerveux pour n'en citer que quelques‐uns.Afin d'identifier le rôle de vangl2, un des gènes centraux de la PCP, dans la mise en place de la circuiterie hippocampale, nous avons créé un modèle murin où vangl2 est supprimé de façon conditionnelle (cKO) dans le télencéphale à des stades précoces de l’embryogénèse. J’ai d'abord montré que Vangl2 est enrichi dans les neurones immatures de la zone sous granulaire du DG, ainsi que dans l’arborisation des neurites (axones et dendrites) des cellules granulaires (CG) du gyrus denté (DG) de l’hippocampe. Ainsi, Vangl2 est enrichi dans le stratum lucidum (sl), une région dense en contacts synaptiques entre le DG et le CA3. Dans cette région a lieu une synapse très particulière entre l'axone des CG, la fibre moussue (Mf) qui forme des boutons géants (MfB) et les excroissances épineuse (TE) issues de la partie proximale des dendrites apicaux. L'analyse structurale et ultra structurale de ces épines démontre que l'élargissement et la complexification de la synapse MfB/TE est bloquée dans nos mutants, alors que les zones actives (PSD) des épines sont présentes, mais réorganisées. De façon intéressante,dans une zone plus distale des dendrites des neurones du CA3 (sl), les épines sont, elles, plus grosses, suggérant un remodelage complexe du réseau en l'absence de vangl2. Enfin, j’ai pu montrer que ces défauts morphologiques étaient corrélés à des problèmes de mémoire complexe (mémoire déclarative) qui dépendent de l’hippocampe mais aussi du cortex. Cette étude montre pour la première fois l’importance du signal PCP dans maturation in vivo d’un circuit hippocampique spécifique ainsi que ces conséquences cognitives. D'autres résultats in vitro montrent que la suppression de vangl2 augmente la vitesse de déplacement des cônes de croissance sur des substrats de N‐cadhérine. J’ai utilisé la microscopie en super résolution spt‐PALM‐TIRF pour montrer que cette augmentation de croissance est inversement proportionnelle à la vitesse du flux rétrograde d’actine. Des expériences de FRAP permettent de suggérer que les molécules de N‐cadhérine engagées dans des interactions hémophiliques (adhésion) est plus importante dans les mutants vangl2 Je propose que Vangl2 contrôle le recyclage et la stabilité des protéines N‐cadhérine dans les sites d’adhésion afin de réguler localement les dynamiques d’actine et par conséquent la croissance neuronale. / Planar Cell Polarity (PCP) is a signaling pathway originally known for its role in the establishment of cellular asymmetry perpendicular to the apico‐basal axis, in the plane of an epithelium. PCPsignaling has been shown to be crucial for many tissue patterning, including epithelial and mesenchymal tissue, but also cardiac, lung, bone, or kidney tissues, to cite a few. PCP signaling controls the regulation of cellular movement via the control of adhesion turnover and cytoskeleton reorganization. Vangl2 is one of the most upstream core PCP proteins that has been implicated in the recent years in various neuronal mechanisms, such as axonal guidance, dendrite morphogenesis or synaptogenesis. However, most of these studies rely on acute downregulation of the gene in vitro or in the use of a mouse presenting a spontaneous mutation of this gene, called Loop‐tail (Vangl2Lp) which causes the death of the embryo at birth. Moreover, the Vangl2Lp form of this protein has been described has a dominant‐negative form, making it difficult to untangle the molecular mechanism leading to the many phenotypes (included neuronal ones) reported inhomozygotes Looptail mice. To bypass this problem we created a conditional knockout (cKO) mouse in which vangl2 is deleted in the telencephalon during early embryogenesis. First, I analyzed the profile of expression of the protein during the first 3 weeks after birth, and I show that Vangl2 is specifically targeted to the arborization of granular cells (GC) of the dentate gyrus (DG) of the hippocampus, and excluded from cell bodies. Also, the protein was highly enriched in immature neurons of the subgranular zone of the DG, and in the stratum lucidum, a region of high‐density contacts between the GC and the CA3. In this region, a special type of synapse is formed: the Mossy Fiber Bouton (MfB) / Thorny Excrescence (TE) synapse. These synapses are bigger and more complex than conventional synapses. I then performed a structural and ultrastructural analysis of the DG/CA3 circuit in the Vangl2 cKO mice in order to understand the role of Vangl2 in the hippocampus maturation. For this, I used stereotaxic mice infection viruses, and Serial block face scanning electron microscopy (SBFsEM) with 3D reconstruction. Results show that in cKO mice, Mfs fasciculation is mildly impacted, and that the enlargement and complexification of the MfB/TE synapse is arrested, with TEs almost absent. I was able to link these morphological abnormalities to deficits in complex hippocampal‐dependent learning tasks. This work demonstrates for the first time the importance of PCP signaling for the in vivo maturation of a specific hippocampal circuit and its specific cognitive consequences. Next, I attempted to identify the functional consequences of vangl2 deletion on young hippocampal neuron maturation. My results confirm that Vangl2 is expressed in young hippocampal neurons and that the deletion of the gene affected neurite outgrowth on Ncadherin substrate. I used spt‐PALM‐TIRF super‐resolution microscopy to show that this increased neurite outgrowth was inversely proportional to a decrease in actin retrograde flowand to a decrease in the number of directed actin trajectories. These results strongly suggest that N‐cadherin adhesions are affected by Vangl2 deletion. FRAP experiments demonstratedthat in Vangl2 cKO neurons the recovery of N‐cadherin molecules engaged in homophilicbindings (adhesion) was decreased, suggesting that the turnover of N‐cadherin involved inadhesion is reduced. Altogether, I propose that Vangl2 controls the turnover/stability of Ncadherin proteins at adhesion sites to regulate local actin dynamics and consequently neuronal outgrowth