Contrairement aux mammifères adultes, la rétine des amphibiens possède la particularité de croître durant toute la vie de l'animal grâce à l'activité continue d'une population de cellules souches localisée au sein d'une niche bien délimitée, la zone marginale ciliaire (ZMC). Ce modèle offre ainsi la possibilité d'étudier in vivo les mécanismes moléculaires à l'origine du maintien et de la prolifération des cellules souches neurales à des stades post-embryonnaires. Dans ce but, l'identification et la caractérisation des différentes voies de signalisation présentes au sein de la niche biologique des cellules souches rétiniennes est une première étape indispensable. Mon projet de thèse a été divisé en deux objectifs principaux: l'étude des interactions entre les voies Wnt et Hedgehog au sein de la ZMC chez le xénope et la réalisation de l'étude fonctionnelle de Yap, l'effecteur principal de la voie de signalisation Hippo dans ce modèle. Par des approches génétiques et pharmacologiques, la première partie de ce projet a permis de mettre en évidence un antagonisme inattendu entre les signaux Wnt et Hedgehog au sein de la ZMC qui régule l'activité proliférative des cellules souches et des progéniteurs rétiniens. Ce travail nous a conduit à proposer un modèle dans lequel ces deux voies réguleraient la balance prolifération/différenciation dans la rétine post-embryonnaire. Dans un deuxième temps, les expériences de gain et de perte de fonction du gène Yap ont montré que ce dernier joue un rôle essentiel dans la régulation du programme temporel de la phase de réplication de l'ADN des cellules souches rétiniennes. En effet, l'inhibition de Yap entraîne une importante réduction de la durée de la phase S du cycle cellulaire associée à une instabilité génomique. Une surexpression de c-Myc et de la voie p53-p21 semble impliquée dans ce phénotype. Nos travaux nous ont également permis d'identifier un nouveau partenaire de YAP, le facteur de transcription PKNOX1. L'ensemble de ces données nous a ainsi conduit à proposer un modèle selon lequel le complexe YAP/PKNOX1 pourrait être nécessaire au bon déroulement de la phase de réplication des cellules souches, indispensable à la maintenance de l'intégrité du génome de ces cellules et de leur descendance. / In contrast to the adult mammals, the retina of amphibians shows continuous growth during adulthood through active neural stem cells localized in the defined niche called ciliary marginal zone (CMZ). This model offers an exceptional tool to study in vivo the molecular mechanisms involved in the maintenance and proliferation of neural stem cells during post-embryonic stages. In this order, the identification and the characterization of the signaling pathways acting in biological retinal stem cell niche is an essential step.My PhD research was divided in two main parts: the study of the interaction between the Wnt and Hedgehog pathways within the CMZ and the functional study of Yap, the downstream effector of the Hippo pathway in this model. By using genetic and pharmacological tools, the first part of this project demonstrated an unexpected antagonism between the Wnt and the Hedgehog signaling in the CMZ that regulates proliferative activity of retinal stem and progenitor cells. In this article, we propose a model in which an antagonistic interplay of Wnt and Hedgehog pathways may regulate the balance proliferation/differentiation in the post-embryonic retina. Second, gain and loss of function experiments of Yap have shown that this factor plays a key role in the regulation of temporal replication of DNA retinal stem cells. Indeed, inhibition of Yap leads to strong reduction of the S-phase length during the cell cycle associated with genomic instability. c-Myc and p53-p21 overactivation seems to be involved in this phenotype. This work also allowed us to identify a novel YAP partner, the transcriptional factor PKNOX1. We indeed propose a model in which the YAP/PKNOX1 complex may be required for the successful convening of the replication phase on stem cells, essential for the maintenance of genome integrity on the cells and their progeny.

Neuromesodermal progenitors (NMPs) are bipotent progenitors, located at the caudal end of the embryo and are essential for axis formation. These stem cell-like progenitors possess the ability to self-renew and differentiate to both mesodermal and neural lineages, such as skeletal muscle and spinal cord derivatives. These progenitors arise at E8.5 and are localised in the caudal lateral epiblast (CLE), a posterior region of the embryo near the primitive streak. Later in development, they reside in the tail bud until cessation of axial elongation at E13.5. Throughout these stages NMPs are characteristically marked by co-expression of T(Bra) (Brachyury) and Sox2. This characteristic is also present in in vitro NMPs, which can be derived from Epiblast Stem Cells (EpiSCs) through treatment with Wnt/β-catenin signalling agonists and Fgf2, which simulates their in vivo environment. Protein and mRNA profiling of NMPs and mutant phenotypes in vivo supports the hypothesis that a non-canonical Wnt pathway, the Wnt/Planar Cell Polarity pathway (PCP) could be involved in NMP fate decision and/or maintenance. This thesis focuses on understanding more about the role of PCP by aiming to identify the spatio-temporal profile of Wnt/PCP pathway components in NMP regions during axial elongation, as well as determining its role in NMP behaviour through manipulation of this pathway via in vivo and in vitro assays Employing in situ hybridisation and immunohistochemistry techniques, key Wnt/PCP components, including Pk1, Vangl2 and Ptk7, were confirmed to be present in in vivo and in vitro NMPs, thus, providing strong evidence that Wnt/PCP may be involved regulating NMP behaviour. Disruption of Wnt/PCP signalling through overexpression of Wnt/PCP components was tested in refined in vivo and in vitro assays. Overexpression of Vangl2 and Ptk7, but not Pk1 in NMPs regions in vivo resulted in loss of contribution to neural lineages, as well as lower contribution to NMP regions themselves. Similarly, Wnt/PCP components were disrupted in vitro through generation of dox-inducible overexpression cells lines for Wnt/PCP components. These lines were used to generate NMPs from an optimised novel alternative source Epiblast-Like Cells (EpiLCs), however no clear affect to lineage was observed. Overall this work has successfully advanced our knowledge of Wnt/PCP mediated control of NMP differentiation and maintenance, and provided a finer grained description of the relationships between them.

A conclusão do seqüênciamento do genoma de múltiplos vertebrados trouxe um importante desafio para entender e predizer função, particularmente para seqüências não-codificantes, a partir de seqüências primarias de DNA. A hipótese de que a conservação evolutiva prediz seqüências funcionais é comumente aceita, inclusive para seqüências envolvidas na regulação da transcrição gênica, mesmo que a conservação de seqüências tenha gerado imperfeitas predições de enhancers (acentuadores) funcionais. O colágeno tipo XVIII é um componente da maioria das membranas basais; mutações no gene COL18A1 levam a síndrome de Knobloch, uma doença autossômica recessiva caracterizada por degeneração vitreoretiniana e macular e encefalocele occipital. O COL18A1 tem 43 exons que transcrevem três isoformas a partir de dois promotores diferentes. As três isoformas apresentam um complexo modelo de expressão tecido-específico, incluindo expressão em rim, pulmão, cérebro e retina. Os níveis de expressão do COL18A1 são considerados clinicamente importantes na vasculogenese, e em predisposição para o hepatocarcinoma e diabetes tipo 2. Dessa forma, a identificação de regiões regulatórias fornecerá indícios sobre a regulação da expressão do COL18A1 em estados normal e patogênico. Além disso, a endostatina e o FRZC18 são fragmentos proteolíticos do colágeno XVIII envolvidos na sinalização Wnt. No entanto, o papel in vivo do FRZC18 ainda não foi estudado. Uma profunda investigação do papel deste domínio na via de sinalização Wnt é indubitavelmente necessária, bem como a compreensão da regulação do COL18A1 pela via de Wnt. Empregamos um sistema eficiente de teste em zebrafish para analisar o potencial funcional de elementos enhancers na regulação transcricional do COL18A1. Identificamos quatro elementos enhancers que controlam a transcrição consistente com o COL18A1 endógeno de zebrafish, em tecidos incluindo retina, rim, vasos sanguíneos, intestino, cartilagem e fígado. Apesar dos algoritmos utilizados não tenham detectado conservação em seqüências não-codificantes de humanos à teleósteos no lócus do COL18A1 estudado, as seqüências humanas funcionaram apropriadamente em zebrafish transgênicos. Adicionais análises computacionais post hoc revelaram similaridade entre seqüência humana e de zebrafish dentro ou próximo das quatro regiões enhancers. Testamos funcionalmente o FRZC18 com superexpressão de seu RNAm em embriões de zebrafish. Este experimento resultou em embriões com fenótipos que assemelharam à mutantes da via não-canônica de Wnt (slb e ppt). Este resultado aponta o FRZC18 como um antagonista da via de sinalização não-canônica de Wnt, possivelmente por interação com Wnt11 e Wnt5. Dissecamos o promotor 1 do COL18A1, o qual mostrou características de genes housekeeping e similaridades com o promotor 2 do COL18A1. Também mostramos possível ligação de TCF/LEF aos promotores do COL18A1. A via de Wnt levou à redução de atividade dos promotores do COL18A1 e também redução dos níveis de expressão através de superexpressão de β-catenina. Este trabalho elucidou de forma geral, os elementos regulatórios em cis do COL18A1 e melhor caracterizou o seu papel na via de sinalização Wnt como um antagonista também da via não-canônica e como um alvo da via canônica. / The completion of multiple vertebrate genome sequences has presented an important challenge to understand and predict function from primary DNA sequence, particularly for noncoding sequence. It is commonly hypothesized that evolutionary conservation predicts functional DNA sequences, including those involved in regulating gene transcription, although sequence conservation has proven to be an imperfect predictor of enhancer function. Type XVIII collagen is a component of most basement membranes; mutations in the COL18A1 gene lead to Knobloch Syndrome, an autosomal recessive disease characterized by vitreoretinal and macular degeneration and occipital encephalocele. COL18A1 has 43 exons that transcribe three isoforms from two different promoters. The three isoforms display complex patterns of tissue-specific expression, including in kidney, lung, brain, and retina. Expression levels of COL18A1 are thought to be clinically important in vasculogenesis, and in predisposition to hepatocarcinoma and diabetes type 2. Therefore, identification of the regulatory regions will provide insight into normal and pathogenic regulation of COL18A1 expression. Furthermore, endostatin and FRZC18 are cleaved fragments from collagen XVIII that are involved in Wnt signaling, however in vivo role of FRZC18 has not been investigated yet in any model organism. Thus, a deeper investigation of FRZC18 role in Wnt signaling is indubitable necessary, as well as a comprehension of COL18A1 regulation by Wnt signaling. We have employed an efficient system of transgenesis in the zebrafish to functionally evaluate potential enhancer elements regulating COL18A1 transcription. We identified four enhancer elements that control transcription consistent with zebrafish endogenous COL18A1, in tissues including retina, kidney, blood vessels, gut, cartilage and liver. Although the algorithms we used did not detect noncoding conservation from human to teleosts at the COL18A1 locus, the human sequences functioned appropriately in zebrafish transgenics. Additional post hoc computational analysis revealed detectable sequence similarities between human and zebrafish in or near two of the four enhancer regions. We tested one of these zebrafish regions and confirmed orthologous enhancer activity. We functionally tested FRZC18 with its mRNA overexpression in zebrafish embryos. This experiment resulted in embryos with phenotype remaining slb and ppt, mutants of non-canonical wnt components. This result points FRZC18 as an antagonist of non-canonincal Wnt signaling possibly by interaction with Wnt11 and Wnt5. We dissected COL18A1 promoter 1 and it showed characteristics of a housekeeping gene and similarities with promoter 2 and we also showed possible TCF/LEF binding to COL18A1 promoters. Wnt signaling responded to downregulate promoter activity of COL18A1 and also decrease its expression by overexpression of s-catenin. This work broadly elucidated COL18A1 cis regulatory elements and better characterized its role in Wnt signaling as an antagonist of noncanonical and also as a target of canonial signaling.

No description available.

Das seit 1957 als essentiell klassifizierte Spurenelement Selen vermittelt seine Funktion hauptsächlich durch seinen Einbau in Selenoproteine in Form der 21. proteinogenen Aminosäure Selenocystein. Insgesamt wurden 25 humane Gene für Selenoproteine identifiziert, deren genaue Funktion häufig noch nicht bekannt ist. Selen ist das einzige Mitglied aus der Gruppe der Mikronährstoffe, für das nach wie vor eine antikanzerogene Funktion vor allem in Bezug auf Darmkrebs postuliert wird. Die Grundlage dafür liefert eine Interventionsstudie, bei der 1.312 Probanden für 4,5 Jahre mit 200 μg Selen/Tag supplementiert wurden. Dies resultierte in einer Senkung der Gesamtkrebsmortalität um 50 %. Die Fragen einer optimalen Selenzufuhr, die nicht nur den Bedarf deckt, sondern auch die Entfaltung der antikanzerogenen Wirkung von Selen gewährleistet und die zugrunde liegenden molekularen Mechanismen sind noch ungeklärt. Zudem liegt die Selenzufuhr bei einem Großteil der europäischen Bevölkerung unter den Empfehlungen. Deshalb wurden in der vorliegenden Arbeit vier Wochen alte Mäuse für sechs Wochen marginal defizient (0,086 mg/kg Futter) bzw. selenadäquat (0,15 mg/kg Futter) gefüttert. Dieser geringe Unterschied im Selengehalt resultierte in einer Senkung des Plasmaselenspiegels der selenarmen Tiere auf 13 % und der GPx-Aktivität in der Leber auf 35 %. Zunächst wurde der Einfluss von Selen auf die globale Genexpression im murinen Colon mittels Microarray untersucht. Von den im Colon exprimierten Selenoproteinen reagierte die mRNA von SelW, SelH, GPx1 und SelM im Selenmangel besonders deutlich mit Expressionsverlust. Da diese Selenoproteine nicht nur im Colon, sondern auch in Leukozyten reguliert waren, sind sie auch als humane Biomarker für die in dieser Studie gewählte Schwankung des Selengehalts geeignet. Des Weiteren wurde auf Basis der Microarraydaten eine Signalweganalyse durchgeführt, die der Identifizierung krebsrelevanter Signalwege diente, um mögliche molekularbiologische Erklärungsansätze für die Rolle von Selen im Krebsgeschehen zu finden. Es zeigte sich, dass die mRNA von Schlüsselgenen des Wnt-Signalwegs wie β-Catenin, Gsk3β, Dvl2, Tle2, Lef1 und c-Myc auf Schwankungen des Selengehalts reagiert. Vor allem die Induktion von c-Myc, einem Zielgen des Wnt-Signalwegs, deutet darauf hin, dass dieser im Selenmangel tatsächlich aktiver ist als bei selenadäquater Versorgung. Ein weiterer möglicher Erklärungsansatz für die postulierte präventive Funktion von Selen gegenüber Darmkrebs ist die gastrointestinale Glutathionperoxidase (GPx2), die physiologisch in den proliferierenden Zellen des Kryptengrunds exprimiert wird. Die Regulation dieses Enzyms durch den Wnt-Signalweg, der ebenfalls in proliferierenden Zellen aktiv ist, konnte mittels Reportergenanalyse und endogen auf mRNA- und Proteinebene in Zellkultur gezeigt werden. Die Aktivierung verkürzter Promotorkonstrukte und die Mutation eines potentiellen Bindeelements identifizierten den für die Bindung von TCF und β-Catenin verantwortlichen Bereich. Als Zielgen des Wnt-Signalwegs scheint GPx2 zu den an Proliferationsprozessen beteiligten Genen zu gehören, was unter physiologischen Bedingungen die Aufrechterhaltung des intestinalen Epithels gewährleistet. Bei der Entstehung intestinaler Tumore, die in der Initiationsphase zu über 90 % mit einer konstitutiven Aktivierung des Wnt-Signalwegs einhergeht, wirkt GPx2 möglicherweise prokanzerogen. Die genaue Funktion von GPx2 während der Kanzerogenese bleibt weiter zu untersuchen. / Suboptimal selenium (Se) status has been suggested to be associated with a higher risk of developing various cancers, especially colon cancer. In mammals, Se exerts its functions through selenoproteins into which it is incorporated as selenocysteine. Since the function of many selenoproteins has not been identified the underlying mechanisms of the anti-carcinogenic function of Se remains unclear. Therefore, mice were fed either a marginal Se-deficient diet (0.086 mg Se/kg) or a Se-adequate diet (0.15 mg Se/kg) for six weeks. The plasma Se level was reduced to 13 % in the Se-deficient group while GPx activity in the liver was reduced to 35 %. The influence of Se on the global gene expression pattern was analysed using microarray technology. Among selenoproteins SelW, GPx1, SelH and SelM were consistently lower expressed in animals fed with the Se-deficient diet. As the mRNA of these genes was regulated in leucocytes as well, they are possible new biomarkers for the Se status in human studies. In addition, pathway analysis revealed that the cancer-relevant Wnt pathway was affected by the Se status, indicated by changes in the mRNA expression of key proteins like β-catenin, Gsk3β, Dvl2, Tle2, Lef1 and the Wnt target gene c-Myc. The regulation of these genes by Se points to a slightly increased basal activity level of the Wnt pathway in the Se poor state and may therefore contribute to the higher cancer risk in a marginal Se deficiency. Another possible explanation for anti-carcinogenic effects of Se is the gastrointestinal glutathione peroxidase GPx2, a selenoprotein predominantly expressed in proliferating cells at the crypt grounds of the intestine. The regulation of GPx2 via the Wnt pathway was confirmed by reporter gene experiments and by analysing endogenous GPx2 expression on the mRNA as well as on the protein level in different cell culture systems. Shortened promoter constructs and the mutation of a potential TCF binding element identified the area responsible for β-catenin/TCF binding. GPx2 is the first selenoprotein identified as a target of the Wnt pathway. This finding suggests a function of GPx2 in the maintenance of normal renewal of the intestinal epithelium as well as in cancer development.

Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Die evolutionär konservierten morphogenen Signalwege Wnt/β-Catenin und Hedgehog (Hh) spielen vor allem in der Embryogenese, Zelldifferenzierung und Kanzerogenese eine große Rolle. Es ist bekannt, dass es zwischen Komponenten der beiden Signalkaskaden zu verschiedensten Wechselwirkungen und Hintergrundreaktionen in unterschiedlichsten Organismen und Geweben kommt. Ziel dieser Arbeit war die Aufklärung einzelner mole- kularer, transkriptioneller Prozesse hinter diesen Kreuzreaktionen in primären Hepatozyten und dem Hepatozellulären Karzinom. Dafür sind die beiden Signalwege durch verschiedenste Einflüsse, wie dem Einsatz von siRNAs, transgenen Mausmodellen und rekombinanten Proteinen gegen einzelne Faktoren der Hedgehog, aber auch der Wnt/β-Catenin Kaskade in ihrer Genexpression verändert und die Reaktionen der Signalwegskomponenten mittels der quantitativen Real-Time-PCR untersucht worden. Neben dem Organismus Maus haben einzelne vergleichende Experimente auch auf der humanen Ebene zum Erkenntnisgewinn beigetragen. Durch die Einflussnahme auf den Hedgehog Signalweg in murinen Hepatozyten wird deutlich, dass die Antworten auf die einzelnen Veränderungen in den Signalkaskaden sehr vielschichtig und umfangreich sind. Abhängig vom induzierten bzw. reprimierten Gen, aber auch vom gelenkten Signalweg variieren die Genexpressionen auf unterschiedlichste, und zum Teil gegenläufige Weise. Ferner wird deutlich, dass es zu Unterschieden in der Genantwort bezüglich der verschiedenen Organismen Maus und Mensch, aber auch zu Variationen der Interaktionen in den diversen Gewebe bzw. Zelltypen kommt.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 99-118) Also available in print.