Este trabalho tem por objetivo mostrar a atividade meristemática da endoderme em raízes de cinco espécies de Commelinaceae (Commelina erecta. Floscopa glabrata, Dichorisandra tyrsiflora, Tradescantia spathacea e T. zebrina). Nas cinco espécies foi observado que na raiz as iniciais endodérmicas sofrem diversas divisões periclinais e dão origem às camadas radiadas de células do córtex. Observou-se ainda, que o cessar dessas divisões nem sempre ocorre simultaneamente em todas as células iniciais, pois, quando ocorre a diferenciação, células que sofreram uma última divisão aparecem ao lado de células que não apresentaram essa divisão. A geração de células pela endoderme pode ser verificada através da observação das células subseqüentes à mesma. Estas células subseqüentes estão dispostas em fileiras radiadas, onde é possível observar que existe uma progressão do tamanho celular do menor (adjacente à endoderme) para o maior (mais distante da endoderme). Estas fileiras de células que são resultantes da endoderme foram denominadas de derivadas da endoderme meristemática (DEM). No caule, a atividade meristemática do periciclo e da endoderme é limitada à região nodal. Nesta região ocorre a formação de novas raízes, gemas caulinares e saídas de traços. Nos entrenós, os feixes vasculares são colaterais e a endoderme se apresenta, em geral, como bainha amilífera, podendo apresentar estrias de Caspary; o periciclo encontra-se parenquimático. Na região do nó, observa-se uma intensa atividade do periciclo (ou da região pericíclica) formando feixes vasculares colaterais envoltos por fibras e o plexo periférico em direção às raízes adventícias. Verificou-se também uma pequena atividade da endoderme, produzindo duas a três camadas do córtex interno. No primeiro e no segundo entrenós, a endoderme apresenta-se suberificada e o periciclo parenquimático. Corroborando com estudos recentes e autores do século XIX, o espessamento primário é devido à atividade centrípeta do periciclo e à atividade centrífuga da endoderme. Existem poucos relatos na literatura sobre a presença de endoderme e periciclo nas plantas vasculares. A maioria dos autores consideram os tecidos que envolvem os feixes vasculares das folhas como a bainha do feixe. O objetivo deste trabalho é mostrar que a bainha do feixe da folha é composta de endoderme e periciclo, bem como a continuidade destes tecidos entre o caule e a folha. Há uma perfeita continuidade entre os tecidos do caule e da folha quando se observa o traço indo em direção a folha. Nota-se também, que a endoderme que no caule envolve o cilindro vascular também envolve os feixes das folhas, constituindo assim, os monostelos. Não foi observada a presença de estria de Caspary na folha, foi observado somente o espessamento irregular da endoderme e do periciclo. Por se tratarem de monostelos, decidiu-se aceitar a denominação de unidades vasculares no lugar de feixes, nas folhas. Há poucos relatos na literatura sobre anatomia de plantas parasitadas por agentes minadores, os quais promovem escavações ou caminhos através do consumo dos tecidos internos das plantas por larvas de diversos insetos. A proposta deste trabalho foi analisar anatomicamente a ocorrência de minas foliares em Commelina diffusa e Floscopa glabrata causadas por espécies de larvas endofitófagas de dípteros, pertencentes a duas famílias: Agromyzidae e Chironomidae. Em Commelina diffusa foram encontradas larvas da família Agromyzidae e em Floscopa glabrata observaram três exuvias cefálicas de Chironomidae. Os dados anatômicos revelaram que os minadores consumiram apenas os tecidos parenquimáticos do mesofilo, formando minas lineares. Além disso, notou-se que a epiderme e os tecidos vasculares de porte médio foram mantidos intactos em ambas as espécies, não apresentando alterações estruturais, como a neoformação de tecidos. / The aim of this work is to show the meristematic activity of the endodermis in roots of five species of Commelinaceae (Commelina erecta, Floscopa glabrata, Dichorisandra tyrsiflora, Tradescantia spathacea e T. zebrina). In all species was observed the initial endodermis suffered several periclinal divisions, originating the radiate layers of the cortex cells. In addition, these divisions does not stop simultaneously in all initial cells because, when differentiation occurs, cells that had a last division apears beside cells that have not divide yet. The generation of cells by the endodermis can be verified observing its subsequent cells. These subsequent cells are disposed as rows and in a radiate pattern where is possible to observe a progression in cell size, that is, the cells become greater as the distance from the endodermis increases. These rows of cells are derived from the endodermis and are called derivatives of the meristematic endodermis (DME). In the stem, the meristematic activity of pericycle and endodermis is limited to the nodal region. In this region occurs the formation of new adventitious roots, buds and leaf traces. In the internodes the vascular bundles are collateral, the endodermis usually appears as a starch sheath, which may have Caspary strips, and the pericycle is parenchymatous. In the nodal region, there is intensive activity of pericycle (or pericycle region), promoting the formation of vascular bundles with fibers around it, and the peripheral plexus of adventitious roots. Moreover, there was also little activity of endodermis producing only from two to three layers of inner cortex. The endodermis has suberin. In conclusion, the primary thickening is caused by the centripetal activity of pericycle and the centrifugal activity of the endodermis. In specialized literature, reports on anatomy of presence of the endodermis and the pericycle in leaves of vascular plants are few in number. Most authors consider the tissues that involve the leaves bundles as sheath bundle. The aim of this work was to show the sheath bundle is composed by the endodermis and the pericycle, and investigate the continuity of tissues between stem and leaf in Monocotyledons. The results showed that there is a perfect continuity between the tissues of stem and leaf when the leaf trace is observed going in direction to the leaf. In addition, it was observed that the endodermis of stem involve not only the vascular cylinder but also the leaf bundles, that is, the monosteles. The presence of Caspary strip in the endodermis was not observed. It was decided to name the leaf bundle as unity instead of bundle, as proposed for recently researches. In specialized literature, reports on anatomy of miners in host plants are few in number. These agents trigger excavations, or paths, by consumption of plant inner tissues by larvae of several insects. The aim of this work was to investigate leaf miner occurrence in Commelina diffusa and Floscopa glabrata using anatomical techniques. In this case, it was discovered that members of Agromyzidae and Chironomidae families, which are Diptera endophytophagous larvae types, were responsible for the tunnels. Moreover, in Commelina diffusa was found Agromyzidae larvae while in Floscopa glabrata three Chironomidae cephalic exuviae were found. The miners, as shown by anatomical studies, used only parenchymatic tissues of mesophyll for their feeding, causing the formation of linear miners. In addition, the epidermis and the middle-sized vessel bundles, in both species, were kept intact, showing no structural modification, like neoformation of tissues.

Este trabalho tem por objetivo mostrar a atividade meristemática da endoderme em raízes de cinco espécies de Commelinaceae (Commelina erecta. Floscopa glabrata, Dichorisandra tyrsiflora, Tradescantia spathacea e T. zebrina). Nas cinco espécies foi observado que na raiz as iniciais endodérmicas sofrem diversas divisões periclinais e dão origem às camadas radiadas de células do córtex. Observou-se ainda, que o cessar dessas divisões nem sempre ocorre simultaneamente em todas as células iniciais, pois, quando ocorre a diferenciação, células que sofreram uma última divisão aparecem ao lado de células que não apresentaram essa divisão. A geração de células pela endoderme pode ser verificada através da observação das células subseqüentes à mesma. Estas células subseqüentes estão dispostas em fileiras radiadas, onde é possível observar que existe uma progressão do tamanho celular do menor (adjacente à endoderme) para o maior (mais distante da endoderme). Estas fileiras de células que são resultantes da endoderme foram denominadas de derivadas da endoderme meristemática (DEM). No caule, a atividade meristemática do periciclo e da endoderme é limitada à região nodal. Nesta região ocorre a formação de novas raízes, gemas caulinares e saídas de traços. Nos entrenós, os feixes vasculares são colaterais e a endoderme se apresenta, em geral, como bainha amilífera, podendo apresentar estrias de Caspary; o periciclo encontra-se parenquimático. Na região do nó, observa-se uma intensa atividade do periciclo (ou da região pericíclica) formando feixes vasculares colaterais envoltos por fibras e o plexo periférico em direção às raízes adventícias. Verificou-se também uma pequena atividade da endoderme, produzindo duas a três camadas do córtex interno. No primeiro e no segundo entrenós, a endoderme apresenta-se suberificada e o periciclo parenquimático. Corroborando com estudos recentes e autores do século XIX, o espessamento primário é devido à atividade centrípeta do periciclo e à atividade centrífuga da endoderme. Existem poucos relatos na literatura sobre a presença de endoderme e periciclo nas plantas vasculares. A maioria dos autores consideram os tecidos que envolvem os feixes vasculares das folhas como a bainha do feixe. O objetivo deste trabalho é mostrar que a bainha do feixe da folha é composta de endoderme e periciclo, bem como a continuidade destes tecidos entre o caule e a folha. Há uma perfeita continuidade entre os tecidos do caule e da folha quando se observa o traço indo em direção a folha. Nota-se também, que a endoderme que no caule envolve o cilindro vascular também envolve os feixes das folhas, constituindo assim, os monostelos. Não foi observada a presença de estria de Caspary na folha, foi observado somente o espessamento irregular da endoderme e do periciclo. Por se tratarem de monostelos, decidiu-se aceitar a denominação de unidades vasculares no lugar de feixes, nas folhas. Há poucos relatos na literatura sobre anatomia de plantas parasitadas por agentes minadores, os quais promovem escavações ou caminhos através do consumo dos tecidos internos das plantas por larvas de diversos insetos. A proposta deste trabalho foi analisar anatomicamente a ocorrência de minas foliares em Commelina diffusa e Floscopa glabrata causadas por espécies de larvas endofitófagas de dípteros, pertencentes a duas famílias: Agromyzidae e Chironomidae. Em Commelina diffusa foram encontradas larvas da família Agromyzidae e em Floscopa glabrata observaram três exuvias cefálicas de Chironomidae. Os dados anatômicos revelaram que os minadores consumiram apenas os tecidos parenquimáticos do mesofilo, formando minas lineares. Além disso, notou-se que a epiderme e os tecidos vasculares de porte médio foram mantidos intactos em ambas as espécies, não apresentando alterações estruturais, como a neoformação de tecidos. / The aim of this work is to show the meristematic activity of the endodermis in roots of five species of Commelinaceae (Commelina erecta, Floscopa glabrata, Dichorisandra tyrsiflora, Tradescantia spathacea e T. zebrina). In all species was observed the initial endodermis suffered several periclinal divisions, originating the radiate layers of the cortex cells. In addition, these divisions does not stop simultaneously in all initial cells because, when differentiation occurs, cells that had a last division apears beside cells that have not divide yet. The generation of cells by the endodermis can be verified observing its subsequent cells. These subsequent cells are disposed as rows and in a radiate pattern where is possible to observe a progression in cell size, that is, the cells become greater as the distance from the endodermis increases. These rows of cells are derived from the endodermis and are called derivatives of the meristematic endodermis (DME). In the stem, the meristematic activity of pericycle and endodermis is limited to the nodal region. In this region occurs the formation of new adventitious roots, buds and leaf traces. In the internodes the vascular bundles are collateral, the endodermis usually appears as a starch sheath, which may have Caspary strips, and the pericycle is parenchymatous. In the nodal region, there is intensive activity of pericycle (or pericycle region), promoting the formation of vascular bundles with fibers around it, and the peripheral plexus of adventitious roots. Moreover, there was also little activity of endodermis producing only from two to three layers of inner cortex. The endodermis has suberin. In conclusion, the primary thickening is caused by the centripetal activity of pericycle and the centrifugal activity of the endodermis. In specialized literature, reports on anatomy of presence of the endodermis and the pericycle in leaves of vascular plants are few in number. Most authors consider the tissues that involve the leaves bundles as sheath bundle. The aim of this work was to show the sheath bundle is composed by the endodermis and the pericycle, and investigate the continuity of tissues between stem and leaf in Monocotyledons. The results showed that there is a perfect continuity between the tissues of stem and leaf when the leaf trace is observed going in direction to the leaf. In addition, it was observed that the endodermis of stem involve not only the vascular cylinder but also the leaf bundles, that is, the monosteles. The presence of Caspary strip in the endodermis was not observed. It was decided to name the leaf bundle as unity instead of bundle, as proposed for recently researches. In specialized literature, reports on anatomy of miners in host plants are few in number. These agents trigger excavations, or paths, by consumption of plant inner tissues by larvae of several insects. The aim of this work was to investigate leaf miner occurrence in Commelina diffusa and Floscopa glabrata using anatomical techniques. In this case, it was discovered that members of Agromyzidae and Chironomidae families, which are Diptera endophytophagous larvae types, were responsible for the tunnels. Moreover, in Commelina diffusa was found Agromyzidae larvae while in Floscopa glabrata three Chironomidae cephalic exuviae were found. The miners, as shown by anatomical studies, used only parenchymatic tissues of mesophyll for their feeding, causing the formation of linear miners. In addition, the epidermis and the middle-sized vessel bundles, in both species, were kept intact, showing no structural modification, like neoformation of tissues.

Les études menées depuis de nombreuses années sur l'embryogenèse de la souris ont permis sa caractérisation morphologique. Le but de notre équipe est de participer à l'élucidation des facteurs jouant un rôle important dans le développement préimplantatoire murin. Pour cela, nous nous sommes focalisés sur un des 1ers types cellulaires déterminé à ces stades : l'endoderme primitif (EPr). L'EPr est un tissu extra-embryonnaire formant un épithélium indispensable au bon déroulement du développement et à la survie de l'embryon. Afin de mieux caractériser les facteurs génétiques et voies de signalisations impliquées dans la différenciation de l'EPr chez la souris, je me suis intéressée à la signalisation rétinoïque ainsi qu'à la voie Wnt canonique. J'ai tout d'abord analysé l'expression des marqueurs de l'EPr et de l'épiblaste (le tissu embryonnaire) par RT-PCR quantitative au cours de la différenciation de cellules F9 en cellules de l'EPr par l'acide rétinoïque. Ainsi, j'ai montré que les marqueurs endodermiques sont induits séquentiellement et dans le même ordre que in vivo. J'ai également adapté ce protocole aux embryons préimplantatoires. Grâce à l'hybridation in situ et l'immunohistochimie j'ai caractérisé les profils d'expression de différents acteurs de la voie des rétinoïdes ainsi que de Dkk1 un inhibiteur spécifique de la voie Wnt canonique.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mon travail de thèse a été principalement de comprendre comment les voies de signalisations contrôlent la différenciation et la morphogenèse de l'embryon de vertébré. Les communications entre cellules sont à la base du développement des métazoaires et de leurs évolutions et sont souvent impliquées dans les pathologies humaines. J'ai profité de la puissance des approches fonctionnelles chez le xenope pour essayer de comprendre comment les signaux BMP, FGF, Nodal et Notch sont intégrés dans le temps et l'espace afin de coordonnées différentes décisions cellulaires. Premièrement, nous avons montré que la voie Nodal est active avant la transition mid-blastuléene et permet l'induction du mesedoderme à travers l'auto régulation de l'expression de ces ligands Xnr5 et Xnr6 (Skirkanish et al. soumis à Development). Deuxièmement, j'ai montré que différent ligand de la voie Nodal contrôlent séquentiellement l'induction du mesendoderm et les mouvements de gastrulation (Luxardi et al., Development, 2010). Troisièmement, j'ai montré qu'un cinquième ligand de la voie Nodal, Xnr4, contrôle la régionalisation médio latérale de la plaque neurale ouverte et la neurogenèse. Quatrièmement, nous avons montré qu'une famille de microARN, nir449, contrôle la différenciation des cellules multi-ciliées à travers son action sur un ligand de la voie Notch, Delta-1 (Marcet et al. Nature Cell Biology, en révision). Enfin, j'ai découvert une nouvelle fonction des signaux BMP dans le control de la spécification des épithéliums muco cilié. / My PhD work generally addressed how signaling pathways control differentiation and morphogenesis in the vertebrate embryo. intercellular communication is the basis of metazoan development and evolution and is often involved in human pathologies. I take advantage of the power of functional approaches in the Xenopus embryo, to try and understand how BMP, FGF, Nodal and Notch signals are intragrated in time ans space to coordinate vatious cellular decisions. First, we showed that Nodal signaling is activated before the mid blastula transition and allow mesendoderm induction through the auro regulation of the expression of its ligands Xnr5 and Xnr6 (Skirkanish et al., submitted to development). Second, I have demonstrated that in a gastrulation movements (Luxardi et al., Development, 2010). Third, I have demonstrated that a fifth Nodal ligand, Xnr4, control medio-lateral patterning of the open neural plate and neurogenesis. Froth, we showed that a microRNA family, mir449, controls differenciation of multiciliated cells through the regulation of the Notch ligand Delta-1 (Marcet et al. Nature Cell Biology, in revision). Last, I have discovered a novel function of the BMP pathway in the control of cell type specification within the epidermal mucocialiary epithelium

Le gène unr (upstream of N-ras) code une protéine de liaison à l’ARN, Unr, qui régule la stabilité et la traduction d’ARN messagers cibles. L’invalidation du gène unr chez la souris conduit à une létalité embryonnaire à mi-gestation (10,5 jours post-coitum, jpc). Unr est donc essentielle pour le développement de la souris. Le phénotype le plus frappant des embryons unr-/- est leur petite taille qui est déjà visible à 8,5jpc. Ce phénotype pourrait refléter un problème précoce de prolifération/différenciation au cours du développement qu’il est possible d’étudier dans les cellules souches embryonnaires (ES). Les cellules ES sont dérivées des cellules pluripotentes des embryons au stade blastocyste (3,5jpc). Les cellules ES peuvent s’auto-renouveler c’est à dire proliférer indéfiniment sous forme non différenciée ce qui correspond à leur état pluripotent ou différencier en tous les types cellulaires (lignages) adultes dérivés des feuillets primordiaux (endoderme définitif, mésoderme et ectoderme) ce que défini la pluripotence. Ces deux propriétés des cellules ES conditionnent leur devenir et définissent leur identité. Nous avons remarqué que les cellules ES unr-/- ont tendance à différencier spontanément alors qu’elles sont cultivées dans des conditions qui les maintiennent dans un état non différencié et prolifératif (état pluripotent). En routine, les cultures de cellules ES unr-/- contiennent environ 25% de cellules morphologiquement différenciées. Nos travaux montrent en effet, qu’elles différencient en endoderme primitif. Nous avons reproduit ce phénotype dans une autre lignée des cellules ES de fond génétique différent par déplétion stable d’Unr. La restauration de l’expression d’Unr dans les cellules ES unr-/- limite fortement leur engagement en différenciation. Unr contribue donc au maintient de l’état pluripotent des cellules ES en prévenant leur différenciation spontanée vers le lignage endodermique primitif (Epr). Ce tissu au cours du développement va former l’épithélium de la poche embryonnaire ou sac vitellin. Nos données préliminaires montrent que les cellules ES en absence d’Unr maintiennent tout de même leur capacité de différenciation multi-lignages (endoderme définitif, mésoderme et ectoderme) quand celle-ci est induite. Ensuite, nous nous sommes intéressés au(x) mécanisme(s) d’action d’Unr. Nous avons fait l’hypothèse qu’Unr pourrait directement agir en régulant positivement des gènes qui inhibent la différenciation des cellules ES en Epr ou en régulant négativement des gènes qui l’induisent. Nous avons identifié le gène gata6 comme cible potentielle d’Unr. Une augmentation modérée de l’expression du facteur de transcription Gata6 dans les cellules ES conduit à une autorégulation positive du gène gata6 et induit la différenciation des cellules ES en Epr. Nos données suggèrent qu’Unr pourrait directement déstabiliser les ARNms Gata6 dans les cellules ES afin de prévenir leur différenciation spontanée en Endoderme primitif. / Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein with cold shock domains, involved in regulation of messenger RNA stability and translation. Unr is essential to mouse development since Embryos deficient for Unr die at mid-gestation. Here we report that unr knockout ES cells maintained under growth conditions that sustain self-renewal spontaneously differentiate toward the primitive endoderm (PrE) lineage. This phenotype was reproduced in another ES line (E14tg2a) after shRNA-induced Unr depletion. Moreover, Unr rescue in Unr-deficient ES cells limits their PrE differentiation engagement. However, Unr is dispensable for multilineage differentiation, as shown by knockout ES cells capacity to produce differentiated teratomas. We further investigated the molecular mechanisms underlying the differentiation of unr-/- ES to primitive endoderm, and found that Unr acts downstream of Nanog. Our data also show Gata6 mRNAs are more stable in Unr-deficient ES cells as compared to wild-type ES cells. We propose that the possible repression by Unr of this key inducer of PrE differentiation at a post-transcriptional level may contributes to the stabilization of ES cells pluripotent state.

A síndrome Kranz é um conjunto de características anatômicas, ultra-estruturais e bioquímicas que culminam na realização da fotossíntese C4. Tal síndrome apresenta grande diversidade dentre as Angiospermas, tornando-se conveniente seu estudo em todos os níveis acima citados para a completa caracterização da mesma. No presente trabalho foi investigada a síndrome Kranz de Gomphrena arborescens e G. scapigera (Amaranthaceae) com ênfase na origem ontogenética da bainha Kranz, na descrição ultra-estrutural e na confirmação bioquímica sobre o tipo de fotossíntese C4. O desenvolvimento foliar dessas espécies indica que a bainha Kranz é originada da camada mais interna do mesofilo, a endoderme foliar. Uma discussão sobre os termos presentes na literatura para a descrição dessa bainha, todos eles focados em sua função na fotossíntese C4, demonstra a importância de se utilizar termos que informem a origem ontogenética dessa bainha, como endoderme e periciclo. Na análise ultra-estrutural, foram identificados possíveis fatores que interferem na fotossíntese de ambas as espécies, como o espessamento e a composição da parede da bainha Kranz, o posicionamento centrípeto dos cloroplastos e a presença de retículo periférico nos mesmos. Embora a análise bioquímica tenha resultado em informações ainda não conclusivas, o dimorfismo dos cloroplastos sugere a realização da fotossíntese C4 do tipo NADP-ME. O presente trabalho, de uma forma geral, contribui ao conhecimento da síndrome Kranz dentre as Amaranthaceae s.s., um grupo em que a ultra-estrutura e a bioquímica ainda são pouco conhecidas, e ressalta a importância dos estudos anatômicos, principalmente com enfoque ontogenético, para o melhor conhecimento da diversidade da síndrome Kranz dentre as Angiospermas. / The Kranz syndrome is a set of anatomical, ultrastructural and biochemical features that culminate in the C4 photosynthesis. This syndrome has a huge diversity among Angiosperms, so it became suitable to survey all the levels above cited for its complete characterization. In the present work the Kranz syndrome of Gomphrena arborescens and G. scapigera (Amaranthaceae) is studied, with emphasis on the ontogenetic origin of the Kranz sheath, on the ultrastructural description, and on the biochemical confirmation about the C4 photosynthesis kind. The foliar development of these species shows that the Kranz sheath is originated from the inner layer of the mesophyll, the foliar endodermis. A discussion about the literature terms used to describe the Kranz sheath, all of them referring to the function of this layer in C4 photosynthesis, demonstrates the importance of using terms that inform the ontogenetic origin of this layer, such as endodermis and perycicle. The ultrastructural analysis identified possible factors that interfere on the C4 photosynthesis of both species, such as wall thickening and composition of Kranz sheath cells, the centripetal position of chloroplasts and the peripheral reticulum in chloroplasts. Although biochemical analysis has resulted in no conclusive information, the chloroplast dimorphism suggests the NADP-ME C4 photosynthesis. This work, in a general way, contributes to the knowledge of the Kranz syndrome among Amaranthaceae s.s., a group that has the ultrastructure and the biochemistry of C4 photosynthesis poorly known. It also draws attention to the importance of anatomical surveys concerning the ontogenetic origin of Kranz sheath for a better understanding on the diversity of Kranz syndrome among Angiosperms.

Após o estabelecimento do crescimento axial, promovido pelos meristemas apicais, em monocotiledôneas surge abaixo do meristema apical caulinar, uma região entre o córtex e o cilindro central que promove o crescimento em espessura. Este crescimento é promovido através da adição de tecidos vasculares (centripetamente) e de tecidos parenquimáticos (centrifugamente). Durante muitos anos este espessamento foi denominado e interpretado de diferentes formas, sendo demonstrado como um único meristema denominado de meristema de espessamento primário com atividade bidirecional. Recentemente, pesquisas demonstram que o espessamento primário em caule é promovido pela atividade de dois tecidos, a endoderme e o periciclo, ambos em atividade meristemática. Com o intuito de trazer à tona informações detalhadas sobre estes dois tecidos que compõem esta zona meristemática, assim como o seu funcionamento e origem, o Capítulo I traz informações morfológicas e anatômicas detalhadas do caule de 16 espécies de Tillandsioideae (Bromeliaceae). Os representantes escolhidos para esta análise foram os gêneros Alcantarea, Tillandsia e Vriesea que possuem uma ampla variação morfológica permitindo, assim, comparar entre eles o processo de espessamento do caule. Demostrou-se ser a endoderme e o periciclo os tecidos, que juntos, promovem o espessamento e a manutenção do corpo primário dessas plantas. No entanto, mais evidências que suportem a hipótese que o espessamento primário é realizado por dois tecidos são necessárias. Assim o capítulo II aborda a caracterização do espessamento primário sob a luz da expressão gênica do gene SCARECROW (SCR), gene candidato a ser um marcador da atividade endodérmica, permitindo assim separar e caracterizar molecularmente os tecidos que promovem o espessamento primário. Desta forma, analisou-se a expressão do scr ao longo do desenvolvimento do caule em Zea mays (Poceae), avaliando a possibilidade do gene scr ser um marcador de atividade endodérmica. Com a confirmação, o gene ortólogo ao scr de Vriesea gigantea foi clonado e caracterizado. E finalmente, analisou-se o padrão de expressão de scr em morfotipos diferentes, Vriesea gigantea e Tillandisia usneoides espécies escolhidas durante a análise do capitulo I / After the establishment of axial growth, promoted by apical meristems, in monocots appears below the shoot apical meristem, a region between the cortex and central cylinder that promotes the growth in thickness. This growth is promoted by the addition of vascular tissues (centripetally) and parenchyma tissues (centrifugally). During many years this thickening was called and interpreted in different ways and it has been shown as a single meristem called the primary thickening meristem with bidirectional activity. Recently, researches show that the primary thickening in stem is promoted by the activity of two tissues, the endodermis and pericycle, both in meristematic activity. In order to elicit detailed information about these two tissues that compose this meristematic zone, as well as its operation and origin, Chapter I provides detailed anatomical and morphological information about the stems of 16 species of Tillandsioideae (Bromeliaceae). The representatives chosen for this analysis were the genus: Alcantarea, Tillandsia and Vriesea that have a wide morphological variation, thus allowing to compare between the process of stem thickening. It was demonstrated to be the endodermis and pericycle the tissues that together promote the thickening and maintenance of this primary plant body. However, more evidences supporting the hypothesis that the primary thickening is accomplished by two tissues are required. Thus Chapter II deals with the characterization of the primary thickening in the light of gene expression. The SCARECROW (SCR) gene is good candidate to be a marker of endodermal activity, thereby separating and molecularly characterizing the tissues that promote primary thickening. Therefore, it was analyzed the expression of SCR throughout the development of the stem in Zea mays (Poaceae), evaluating the possibility of SCR gene be a marker of endodermal activity throughout the development of a monocot. With the confirmation, the ortholog of SCR gene of Vriesea gigantean was cloned and characterized. And finally, the expression pattern of SCR was analyzed in Vriesea gigantean and Tillandisia usneoides species chosen during the analysis of Chapter I

A E3.5 jours de développement (E3.5), l’embryon murin se compose d’une monocouche de cellules externes correspondant au Trophectoderme (TE) et d’une masse cellulaire interne (MCI), hétérogène, constituée de deux sous-populations de cellules précurseurs : les cellules épiblastiques (Epi) et les cellules d’endoderme primitif (EPr). NANOG, marqueur épiblastique et GATA6, marqueur de l’EPr, sont co-exprimés à E3.5 dans la MCI puis adoptent une expression exclusive au sein de leur lignage respectif. La différenciation du lignage EPr nécessite l’expression de GATA6 et l’activation de la voie Récepteur Tyrosine Kinase (RTK) activée par le FGF (RTK-FGF) pour l’induction de gènes cibles de GATA6 tels que Sox17 et Gata4.Au cours de ma thèse, j’ai, dans un premier temps, étudié la relation GATA6/voie RTK-FGF lors de l’induction de l’expression des gènes de différenciation de l’EPr. J’ai utilisé des cellules souches embryonnaires murines ES sauvages ou mutantes pour Gata6 (ES Gata6-/-), dans lesquelles j’ai surexprimé différentes formes mutantes de Gata6 inactivées sur les différents résidus identifiés comme potentiellement phosphorylables par la voie RTK-FGF. Ainsi, j’ai analysé l’expression protéique des gènes Sox17 et Gata4 ainsi que des expressions ARN de ces cibles et d’autres gènes caractéristiques exprimés dans l’EPr dans les différentes conditions de surexpression des formes de Gata6 en absence ou présence d’inhibiteurs de la voie RTK-FGF. Ainsi, j’ai pu mettre en évidence que la transmission du signal s’effectue au travers de récepteur au FGF et qu’il existe une compensation entre les branches RTK-MEK-ERK et RTK-PI3K ciblant le résidu Sérine 37 de GATA6. Enfin, les résidus S34 et T509 sont nécessaires et les résidus S34, S37 et T509 semblent coopérer, au travers d’un mécanisme pour le moment non détaillé, pour l’induction des gènes cibles exprimés au sein de l’EPr.Dans un second temps, j’ai débuté la caractérisation phénotypique du rôle des facteurs Dickkopf1 (DKK1), un inhibiteur de la voie WNT/β-caténine, et NOGGIN, un inhibiteur de la voie des Bone Morphogenic Protein (BMP) lors de la différentiation de l’EPr en endoderme pariétal (EP) et viscéral (EV). A l’aide de modèles de souris KO pour DKK1 et NOGGIN, croisées en fond C57Bl6 pur, j’ai pu observer que l’expression d’OCT4 était maintenue au sein des embryons homozygotes mutants pour Dkk1 et double homozygotes mutants pour Dkk1 et Noggin. Cependant, le mécanisme potentiel de compensation ou de coopération de ces deux marqueurs n’est pour le moment pas détaillé précisément et mérite l’analyse d’un plus grand nombre d’embryons mutants. / At E3.5 days of development (E3.5), mouse embryo consists of a monolayer of external cells corresponding to Trophectoderme (TE) and of an intern cell mass (ICM), heterogeneous, constituted by two subpopulations of precursory cells: epiblastic cells (Epi) and primitive endoderm cells (EPr). NANOG, an Epi marker and GATA6, a PrE marker, are co-expressed at E3,5 in the MCI and then adopt an exclusive expression within their respective lineage. EPr differentiation requires both expression of GATA6 and RTK pathway, activated by FGF ligand, in order to induce late markers Sox17 and Gata4 expression.First, I studied the relation GATA6/RTK during this process to understand the mechanism of induction of these target genes during final EPr differentiation. I used embryonic stem cells ES WT or Gata6 mutants (ES Gata6-/-), in which I transfected various Gata6 mutant constructions on different residues characterized as potentially phosphorylable by the RTK pathway. So, I analyzed protein expression of Sox17 and Gata4 target genes as well as RNA expression of characteristic genes expressed in the EPr in different inhibition conditions of RTK pathway. So, I was able to highlight that the transmission of the signal is made through the FGF receptor (FGFR1) and that there is compensation between RTK-MEK-ERK and RTK-PI3K pathways highlighted by later Gata6 overexpression of certain mutant forms. Finally, residue S34, S37 and T509 seems to cooperate, through a mechanism not detailed for the moment, for the induction of the EPr target genes.Then, I was interested to phenotypically characterize the role of Dickkopf1 (DKK1), an inhibitor of the WNT/β-catenin pathway, and NOGGIN, an inhibitor of the Bone Morphogenic Protein (BMP) pathway during the EPr differentiation in parietal endoderm (EP) and visceral (EV). Using models of mouse KO for Dkk1 and Noggin, met in pure background C57Bl6, I was able to observe that OCT4 expression was maintained within the Dkk1-/-, and Dkk1-/- Noggin-/- embryos. However, the potential compensation or cooperation mechanism of these two markers is not understanding well for the moment and deserves the analysis of a largest mutant embryos number.

A 3,5 jours de développement (E3.5), l'embryon de souris est composé d'une couche externe de trophectoderme (TE) entourant la cavité blastocélique et la masse cellulaire interne (MCI). La MCI comprend une population hétérogène de précurseurs d'épiblaste (Epi) et d'endoderme primitif (EPr) dans une configuration "poivre et sel". A E4.5, ces cellules ségrégent, les cellules d'EPr migrant vers la surface de la MCI pour former un épithélium. A E4.75 cet épithélium donne naissance à 2 tissus distincts : un épithélium d'endoderme viscéral (EP) et à l'endoderme pariétal (EP) qui migre le long du TE. Une transition épithélium-mésenchyme est impliquée dans la formation de l'EP. Je m'intéresse au rôle de Dkk1, un inhibiteur de la voie Wnt canonique et activateur de la voie Wnt/PCP, et Noggin, un inhibiteur des BMP, dans la différenciation de l'endoderme extraembryonnaire. J'ai montré que Dkk1 est un marqueur de l'EPr qui devient apicalement polarisé à E4.5. Son expression est ensuite restreinte aux cellules de l'EP. J'ai aussi montré que Noggin est exprimé dès la préimplantation puis dans l'EP et au niveau de la charnière EV-Ep. Par des expériences de perte et de gain de fonction des voies Wnt et BMP et en utilisant les souris mutantes j'ai analysé le rôle de ces deux facteurs dans la différenciation de l'endoderme extraembryonnaire. / At 3.5 days of development (E3.5), the mouse embryo consists of an outer layer of trophectoderm (TE) surrounding the blastocelic cavity and the inner cell mass (ICM). The ICM is composed of intermingled populations of epiblast (Epi) and primitive endoderm (PrE) precursors, that sort to form two distinct tissues. At E4.75 this epithelium differentiates into visceral endoderm (VE) and parietal endoderm (PE) that migrates along TE. An epithelium-mesenchyme transition (EMT) is involved in PE formation while the VE is maintained as an epithelium. My work focuses on the role of Dkk1, a Wnt canonical pathway inhibitor and Wnt/PCP pathway activator, and Noggin, a BMP pathway inhibitor, in extraembryonic endoderm differentiation. I have shown that Dkk1 is a marker of PrE precursors and is apically polarised at E4.5. Afterwards, its expression is restricted to PE. Noggin is expressed during preimplantation and then in PE and EV-EP hinge. By gain and loss of fonction experiments of Wnt and BMP pathways and by using mutant mice, I studied the role of these two factors in extraembryonic endoderm differentiation.