Dissertations / Theses on the topic 'Eph and ephrin interactions'

For an organism to develop and maintain homeostasis, cell types with distinct functions must often be separated by physical boundaries. The formation and maintenance of such boundaries is commonly attributed to local mechanisms restricted to the cells lining the boundary. Here we show that, besides these local subcellular mechanisms, the formation and maintenance of tissue boundaries involves long-lived, long-ranged mechanical patterns. We analyzed the formation of repulsive epithelial boundaries between two epithelial monolayers, one expressing the receptor tyrosine kinase EphB2 and one expressing its ligand ephrinB1. Upon contact, both monolayers exhibited oscillatory patterns of traction forces and intercellular stresses that spanned several cell rows and tended to pull cell-matrix adhesions away from the boundary. With time, monolayers jammed and supracellular mechanical patterns became long-lived, thereby permanently contributing to sustain tissue segregation. Jamming was paralleled by the emergence of soliton-like deformation waves that propagated away from the boundary. This phenomenon was not specific to EphB2-ephrinB1 repulsion but was also present during the formation of boundaries with an inert interface. Our findings thus unveil a global physical mechanism that sustains tissue separation independently of the biochemical and mechanical features of the local tissue boundary.
Para que un organismo desarrolle y mantenga la homeostasis, a menudo los tipos celulares con distintas funciones deben estar separados por barreras físicas. La formación y mantenimiento de dichas barreras se suele atribuir a mecanismos locales restringidos a las células que las bordean. En este trabajo mostramos que, además de estos mecanismos subcelulares locales, la formación y el mantenimiento de las barreras físicas entre tejidos implica patrones mecánicos de largo alcance y larga duración. En particular, hemos analizado la formación de barreras epiteliales repulsivas entre dos monocapas epiteliales, una que expresa el receptor tirosina quinasa EphB2 y otra que expresa su ligando ephrinB1. Tras el contacto, ambas monocapas exhibieron patrones oscilatorios de fuerzas de tracción y tensiones intercelulares que involucraban varias hileras de células y que tendían a retirar las adhesiones célula-matriz hacia fuera de la barrera. Con el paso del tiempo, las monocapas se densificaron y los patrones mecánicos supracelulares se volvieron estables, contribuyendo así a mantener la segregación tisular permanentemente. La aglomeración de células fue acompañada por la aparición de ondas de deformación, similares a solitones, que se propagaron hasta más allá del campo de visión. Este fenómeno no es específico de las barreras repulsivas controladas por el par EphB2-ephrinB1, sino que también aparecen cuando una única monocapa interfiere con una interfaz inerte. Nuestros hallazgos revelan un mecanismo físico global que mantiene la separación entre tejidos independientemente de las características bioquímicas y mecánicas del límite tisular local.

Au sein d'un organisme les cellules se divisent et assurent la croissance, la différentiation et l'homéostasie des tissus. Des travaux récents proposent qu'elles communiquent activement entre voisines au sein des organes solides pour coordonner leur propre division et la préservation de l'intégrité tissulaire. Nous proposons que la signalisation Eph-éphrine, acteur de la communication cellulaire locale, participe à cette coordination entre division cellulaire et cohésion du tissu. Au cours de ma thèse, j'ai démontré dans plusieurs modèles cellulaires que la signalisation Eph-éphrine contrôle la division cellulaire et peut induire des retards dans l'abscission et de la polyploïdie. J'ai prouvé par vidéomicrosocpie que ces défauts d'abscission dépendent du domaine catalytique du récepteur EphB2 et de l'activation de la protéine tyrosine kinase relais c-Src. En cascade, c-Src phosphoryle un régulateur clé de la stabilité du pont intercellulaire, la protéine citron kinase (CitK). J'ai également observé que CitK était anormalement localisé durant la cytocinése en aval de la voie Eph. Par des essais kinase in vitro, j'ai exclu une phosphorylation directe de CitK par le récepteur Eph et identifié c-Src comme capable de phosphoryler directement CitK. J'ai identifié les résidus tyrosines de CitK phosphorylés par c-Src, mutés deux d'entre eux et à l'aide d'analyses de sauvetage phénotypique, démontré que ces résidus étaient nécessaires et suffisants pour induire des défauts d'abscission. J'ai ensuite validé in vivo ce rôle original de la voie Eph-éphrine, dans le contexte du développement neuronal chez la souris. Plusieurs membres de la famille des Eph-éphrines sont exprimés dans les progéniteurs neuraux à l'origine des neurones corticaux et des auteurs ont montrés que CitK contrôle la cytocinèse de ces cellules. En utilisant un système Cre-lox, j'ai spécifiquement éteint la signalisation Eph dans ces progéniteurs et observé une modification de la ploïdie neuronale dans ces animaux. J'ai également observé dans les progéniteurs neuraux une co-localisation physiologique de résidus tyrosines phosphorylés et de la protéine CitK, qui adopte un enrichissement apical caractéristique. Ces résultats suggèrent notamment que la signalisation Eph-éphrine pourrait contrôler l'abscission des progéniteurs neuraux via la phosphorylation de CitK. La cytocinèse est aujourd'hui décrite comme un processus cellulaire autonome orchestré par la machinerie intracellulaire. Les résultats obtenus durant mon doctorat suggèrent que la cytocinèse est également régulée par l'environnement local de la cellule comme j'en ai fait la démonstration avec la signalisation Eph-éphrine. D'autre part, mes travaux suggèrent que la phosphorylation de CitK sert d'interrupteur moléculaire durant la progression à travers la division cellulaire et le contrôle de la ploïdie des neurones
Cells within an organism successfully divide to ensure growth, differentiation and homeostasie. Recent work suggests that dividing cells actively communicate with neighbours thus spatially and temporally coordinating cell division while maintaining tissue cohesiveness. We hypothesized that Eph-ephrin signalling, a local cell-cell signalling pathway, could participate in coordinating cell division within a tissue. Using vertebrate and invertebrate cell culture models I showed that Eph-signalling controls cell division and induces delay in the abscission of nascent daughter cells as well as polyploidy. Using time-lapse imaging I proved that the Eph-mediated abscission failure depends on the catalytic activity of the receptor via the non receptor tyrosine kinase relay molecule c-Src. Downstream of Eph signalling c-Src phosphorylates the protein citron kinase (CitK) a well known regulator of intercellular bridge stability. I also observed that CitK was abnormally localized during cytokinesis when Eph signalling was active. Further, using in vitro kinase assays, I demonstrated that Eph does not directly phosphorylate CitK but that c-Src could do so. In addition, using Mass Spectrometry I mapped all tyrosine residues directly phosphorylated by c-Src. I mutated two of them located in the Rho binding domain of CitK and demonstrated that phosphorylation of those residues are necessary and sufficient to induce cytokinesis failure. I validated in vivo this novel role of Eph-ephrin signalling in a physiological context in the developing mouse neocortex. Members of the Eph/ephrin family are expressed in neural progenitors that give rise to neurons of the cortex upon neurogenic division. Importantly, CitK has been shown by others to control cytokinesis of these progenitor cells. Using the Cre-lox system, I specifically turned off Eph forward signalling in neural progenitor cells and observed an alteration of neuronal ploidy in these mutant animals. Further, I also observed that CitK which adopts a particular apical localisation in neural progenitors physiologically co-localized with phosphorylated tyrosine residues. Altogether, these results suggest that Eph-ephrin signalling controls abscission of neural progenitors by promoting phosphorylation of CitK. The textbook view of cytokinesis is that it is a cell autonomous event orchestrated by the intracellular machinery. Data obtained during my PhD suggest that cytokinesis is also regulated by local environment, here Eph/ephrin signalling, and that phosphorylation of CitK may represent a molecular switch in the normal progression of cell division and in the control of neuronal ploidy

An important problem in developmental biology is to understand how precise patterns of cell types are maintained during development. Eph receptor tyrosine kinases and ephrins have key roles in stabilising these patterns of cell organisation and segregation during development and can restrict the movement of cells by promoting cell repulsion. Previous work by Alexei Poliakov in the Wilkinson lab has shown that Eph-ephrin signalling leads to directional persistence of migration, and modelling suggests that this can contribute to cell segregation. In order to test experimentally the contribution of directional persistence in cell segregation, I have used and developed in vitro assays to dissect the roles of EphB2-ephrinB1 signalling in cell segregation, boundary sharpening and directional persistence. In these assays, stable HEK293 cell lines expressing EphB2 or ephrinB1 are mixed in cell culture and this leads to segregation of the two cell populations. Plating these cells either side of a removable barrier and allowing migration of cells towards each other leads to the formation of a sharp boundary on interaction. Analysis of cell behaviour shows EphB2 cells to move more persistently after interaction with ephrinB1 cells. To analyse how EphB2-ephrinB1 interactions lead to directional persistence of migration, my studies have focussed on the role of components potentially involved in directional persistence that act downstream of EphB2-ephrinB1 signalling, including the planar cell polarity (PCP) pathway (Dishevelled and Daam1) and core polarity components such as the PAR proteins (PAR-3 and PAR-6B). The PCP and PAR components were all found to have roles in cell segregation, as siRNA-mediated knockdown of each of these components disrupted EphB2-ephrinB1 mediated cell segregation and boundary sharpening. However, cell behaviour studies showed that only Dishevelled and PAR-6B have roles in EphB2-ephrinB1 mediated directional persistence, whilst Daam1 knockdown has no effect on the migratory response of cells. PAR-3 knockdown affects the basal ability of cells to migrate, potentially due to its role in establishing front-rear polarity. Taken together, these findings can be explained by a model in which Dishevelled and PAR-6B have a role in EphB2-ephrinB1 mediated directional persistence required for cell segregation and boundary sharpening. I propose that Daam1 may function in the contact inhibition of locomotion between cells also required for segregation.

Metastatic prostate cancer cells display EphB receptor-mediated attraction when they contact stromal fibroblasts but EphA-driven repulsion and contact inhibition of locomotion (CIL) when they contact one another. The impact of these social interactions between cells during cancer cell invasion and the signalling mechanisms downstream of Eph receptors are unclear. Here, I show that EphA receptors drive prostate cancer cell dissemination in a 20 dispersal assay and a 3D cancer cell" spheroid assay by activating repulsive interactions and CIL between contacting prostate cancer cells. I show that EphA receptors interact with the exchange factor Vav2 to activate RhoA, and that both Vav2 and RhoA are required for prostate cancer cell-cell repulsion. Using pharmacological inhibitors I show actomyosin contractility is not a key driver of CIL. I find instead that microtubule dynamics are important for generating the front-rear polarity switch required during CIL, and that EphA2/EphA4, Vav2 and RhoA affect microtubule stability in prostate cancer cells. Furthermore, I find that in EphA2/EphA4, Vav2 or RhoA knockdown cells, contact repulsion can be restored by partial microtubule destabilisation. I propose that EphAVav2- RhoA-mediated repulsion between contacting cancer cells at the tumour edge could enhance their local metastatic invasion and dissemination from the primary tumour. Subsequently, EphB-mediated attractive migration and failure of CIL, between prostate cancer cells contacting ephrin-B2· expressing fibroblasts, could facilitate cancer cell invasion through the surrounding stroma. Stimulation of prostate cancer cells with ephrin-B2lFc leads to filopodia formation and activation of Cdc42. I show that Cdc42-silenced PC-3 cells have significantly impaired migration towards surface coated ephrin-B2 compared with control siRNA-treated cells. Furthermore, Cdc42 is required for attractive migration and defective CIL during collisions b~tween advanced prostate cancer cells and ephrin-B2-expressing fibroblasts. Using organotypic 3D gel invasion assays, I show that ephrin-B2 expressing fibroblasts enhance prostate cancer cell invasion. These data suggest that EphB-Cdc42-mediated attractive interactions with fibroblasts and defective CIL might facilitate prostate cancer cell invasion through the surrounding stroma.

Cell migration and invasion are essential aspects of normal cellular behaviour, however abnormal cell migration can lead to defects in essential cell processes, such as wound healing, or can also promote diseases such as cancer. Cell migration can be regulated by many factors and, importantly, can depend upon a cells interaction with its surrounding cellular microenvironment. Eph receptors are the largest family of receptor tyrosine kinases and are essential in transmitting signals between cells as, uniquely to this family, the ephrin ligands are cell surface bound, therefore signalling is cell-cell contact dependent. Although Eph and ephrin functions have been studied for many years, much of the mechanisms by which they signal are still unclear. In this thesis, I have investigated the role of Eph-ephrin signalling in regulating different aspects of cell migration and invasion in prostate cancer cells and during keratinocyte cell wound healing. I find that the activity of EphB4 is regulated by the expression of PTEN phosphatase both in DU145 and PC-3 prostate cancer cells. This regulation leads to altered heterotypic cell contact inhibition of locomotion, in a co-culture assay between DU145 cells and fibroblasts. I also find that PTEN expression regulates the number of DU145 cells invading from a tumour cell spheroid, towards fibroblasts, in a 3D collagen gel. I suggest these cell behaviours may be as a consequence of altered Rac activity, downstream of EphB4 and PTEN signalling. I also use SILAC based phosphoproteomics to investigate some of the proteins regulated downstream of PTEN expression ilnd EphB4 activity. Furthermore I find that Ephrin-Bs are essential in regulating keratinocyte cell reepithelialisation during tissue culture wound healing, by regulating the actin cytoskeleton structure and E-cadherin processing. Finally, I have attempted to investigate the role of EphB2 in regulating prostate cancer cell migration and invasion, and find that EphB2 depletion does not alter PC-3 cell migration velocity in two dimensions.

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer related deaths in Australian men. Treatment in the early stages of the disease involves surgery, radiation and/or hormone therapy. However, in late stages of the disease these treatments are no longer effective and only palliative care is available. Therefore, there is a focus on exploration of novel therapies to increase survival and treatment efficacy. Advanced prostate cancer is characterised by bone or other distant metastasis. Spreading of the primary tumour to a secondary location is a complex process requiring an initial loss in cell-cell adhesion followed by increased cell migration and invasion. One gene family that has been known to affect cell-to-cell contact in other model systems are the Eph receptor tyrosine kinases. They are the largest family of receptor tyrosine kinases made up of 14 vertebrate Eph receptors that bind to nine cell membrane bound ephrin ligands. Eph-ephrin interaction is crucial in regulating cell behaviour in developmental processes and it is now thought that the underlying mechanisms involved in development may also be involved in cancer. Aberrant expression has been reported in many human malignancies including prostate cancer. Furthermore, expression has been linked with metastasis and poor prognosis in other tumour models. This study explores the potential role of the Eph receptor family in prostate cancer, in particular the roles of EphA2, EphA3 and ephrin-A5. Gene expression profiles were established for the Eph family in a series of prostate cancer cell lines using quantitative real time RT-PCR. A smaller subset of the most prominently expressed genes was chosen to screen a cohort of clinical samples. Elevated levels of EphA2, EphA3 and their ligands, ephrin-A1 and ephrin-A5 were observed in individual cell lines. Interestingly high EphA3 expression was observed in the androgen responsive cell lines while EphA2 was more prominent in the androgen independent cell lines. However, studies using 5-dihydrotestosterone suggest that EphA3 expression in not regulated by androgen. Cells expressing EphA2 showed a greater ability for migration and invasion while cells expressing EphA3 showed poor migration and invasion. Forced expression of EphA2 in the LNCaP cell line resulted in a more invasive phenotype while forced expression of EphA3 in the PC-3 cell line suggests a possible negative effect for EphA3 on cell migration and invasion. Cell signalling studies show activation of EphA2 decreases activity of proteins thought to be involved in pathways regulating cell movement including Akt, Src and FAK. Changes to the activation status of Rho family members, including RhoA and Rac1, associated with reorganisation of the actin cytoskeleton, an important part of cell migration was also observed. As a result, activation of EphA2 in PC-3 cells resulted in a less invasive phenotype. A novel finding in this study was the discovery of a combination of two EphA2 Mabs able to activate EphA2. Preliminary results show a potential for this antibody combination to reduce prostate cancer invasion in vitro. A unique aspect of Eph-ephrin interaction is the resulting bi-directional signalling that occurs through both the receptor and ligand. In this study a potential role for ephrin-A5 mediated signalling in prostate cancer was observed. LNCaP cells express high levels of EphA3 and its high affinity ligand ephrin-A5. In stripe assays, used to study guidance cues, LNCaP cells show strong attraction/migration to EphA3-Fc stripes but not ephrin-A5-Fc stripes suggesting ephrin-A5 mediated reverse cell signalling is involved. Knockdown of ephrin-A5 using shRNA resulted in a decrease in attraction/migration to EphA3-Fc stripes. Furthermore a reduction in proliferation was also observed in vitro. A subcutaneous xenograft model using ephrin-A5 shRNA cells versus controls showed a decrease in tumour formation. This study demonstrates a difference in EphA2 and EphA3 function in prostate cancer migration/invasion and a potential role for ephrin-A5 in prostate cancer cell adhesion and growth.

In vertebrates, there is a large family of Eph receptor tyrosine kinases and their ephrin ligands, which have complex and varied roles during development and in adult homeostasis. The most researched role of Eph receptors and ephrins is in control of cell migration through the regulation of the actin cytoskeleton and cell adhesion. More recently, it has been found that in some tissues Eph-ephrin signalling also leads to changes in gene transcription, for example to control cell differentiation. In the zebrafish hindbrain, Eph receptors and ephrins are expressed segmentally in the rhombomeres in a complementary pattern with respect to their binding partner. Signalling via this pathway induces a unique cell population to arise at rhombomere borders, known as the boundary cells. In order to understand more about Eph receptor and ephrin function in the hindbrain, RNA-sequencing was carried out on dissected hindbrains of zebrafish with endogenous Eph-ephrin signalling and fish that lack Eph-ephrin signalling. The transcriptional profiles were then compared to identify potential downstream targets, which were verified using RT-qPCR and in situ hybridisation. This identified four genes regulated downstream of Eph-ephrin signalling that are markers of progenitor cells and neural differentiation. When Eph-ephrin signalling is disrupted the expression of these genes alters, and the expression pattern of one gene, mdka, was consistent with loss of hindbrain boundary cells. To investigate this observation further, the expression of progenitor and neurogenic markers was determined when Eph-ephrin signalling was disrupted. This supported previous studies which found that Eph-ephrin signalling is required for formation of boundary cells and that boundary cell loss results in ectopic neurogenesis. In addition, it was found that ectopic neurogenesis was accompanied by the depletion of nestin-expressing neural progenitor cells at later stages of development. Together these findings support previous work showing that hindbrain boundary cells are essential for restricting neurogenesis to neurogenic zones adjacent to the boundaries.

As they acquire distinct fates, embryonic cell populations become separated bysharp boundaries. Several theories have been proposed to explain this astonishingphenomenon, which remains very poorly understood. This study investigatestissue separation at the cellular and molecular level, using the gastrulatingXenopus embryo as a model. At this developmental stage, boundaries divide theembryo into its basic body structures, starting with separation of the germ layers,followed by the separation of presomitic mesoderm from notochord. Live cellmicroscopy at these boundaries revealed a contact-mediated repulsion mechanismthat is controlled by a combination of several Eph receptors and their ephrinligands. Loss and gain of function approaches suggest that ephrin-Ephs interactselectively and not promiscuously. They are expressed in a partiallycomplementary pattern on both sides of the boundary. Integration of signals fromthose pairs reaches maximum intensity at the tissue interface. This phenomenonwas simulated computationally based on relative ephrin and Eph concentrations aswell as binding affinities. Despite differences in the expression profile of ephrinsand Ephs at various boundaries, our model holds true. The underlying cytoskeletalmechanism downstream of ephrin-Eph-mediated cell contraction was investigatedfurther. We found that repulsive events and membrane contraction made the cellmembranes less permissive to form stable adhesive bonds, resulting in a state ofloose adhesion – as indicated by the smooth appearance of Cadherins at theboundary. This thesis provides a novel understanding of ephrin-Eph control oftissue boundaries. It also has a direct impact on deciphering their intricate role intumor cell metastasis and invasion.
Au cours du développement embryonnaire, la différenciation cellulaire entraîne lamise en place de frontières nettes afin de séparer les différentes populations decellules qui se dessinent. Plusieurs théories ont tenté d'expliquer ce phénomènefascinant, qui demeure mal compris. La présente étude s'appuie sur la gastrula deXénope afin d'examiner le processus de séparation des tissus aux niveauxcellulaire et moléculaire. A ce stade du développement, des frontières divisentd'abord l'embryon en trois couches de feuillets embryonnaires, puis séparent lanotocorde du mésoderme présomitique. L'imagerie de ces frontières sur descellules vivantes a révélé un mécanisme de répulsion cellulaire, qui serait contrôlépar une combinaison de plusieurs types de récepteurs Eph et de leurs ligands, leséphrines. Nous montrons à la fois par perte et par gain de fonction que leséphrines interagissent avec les récepteurs Eph de manière sélective, et non pasaléatoire. L'expression de ces protéines de part et d'autre des frontières suit unschéma de complémentarité partielle. L'intégration des signaux générés par lespaires d'éphrine-Eph produit une réaction d'amplitude maximale aux interfacesentre les tissus. Nous proposons une simulation numérique de ce phénomène designalisation intégrée, sur la base des concentrations relatives d'éphrines etd'Ephs, ainsi que de leurs affinités de liaison. Cette simulation semble valide pourmodéliser de nombreuses frontières embryonnaires, et ce malgré la variabilité desprofils d'expression des éphrines et Ephs à ces différentes frontières. Nous avonségalement étudié les modifications du cytosquelette en aval de la voie designalisation éphrine-Eph, qui entraîne la contraction cellulaire. Nos résultatsindiquent que les instances de répulsions cellulaires, en conjonction avec lacontraction membrannaire, entravent la formation de liaisons adhésives stablesentre les cellules. Ceci engendre un état d'adhérance faible, se traduisant parl'apparance lisse des Cadhérines le long des frontières embryonnaires. Laprésente thèse apporte une compréhension nouvelle du rôle de la voie designalisation éphrine-Eph dans la régulation des frontières entre différents tissus.Ces trouvailles promettent également d'éclairer l'étude du rôle de ces moléculesdans l'invasion et la métastase de tumeurs.

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis-inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans (Falivelli et al. 2013).

EINLEITUNG Das maligne Melanom stellt aufgrund seiner frühen Metastasierung und der Resistenz gegenüber den bisher bekannten Therapieansätzen eine der aggressivsten Tumorentitäten dar. Allerdings handelt es sich beim Melanom um einen antigenen und immunogenen Tumor. Dies schürt die Hoffnung, dass durch das bessere Verständnis der Mechanismen, die der Metastasierung, aber auch der Dysregulation des Immunsystems zugrunde liegen, Rückschlüsse auf neue Therapieansätze, beispielsweise unter Einbeziehung der Immunabwehr, gezogen werden können. Darüber hinaus würde die Entwicklung von Radiotracern, die eine frühzeitige Diagnose und möglicherweise auch die Auswahl von Patienten für eine personalisierte Tumortherapie ermöglichen, die Heilungschancen des malignen Melanoms wesentlich verbessern. Das Eph-Ephrin-System wiederum stellt ein vielfältiges Zellkommunikations-System dar, das sowohl in lebenswichtige als auch in pathologische Prozesse involviert ist. Beispielsweise nehmen Eph-Rezeptoren und Ephrine Einfluss auf die gerichtete Bewegung von neuronalen, endothelialen und inflammatorischen Zellen. Zudem beeinflussen sie die Bewegung von Tumorzellen und tragen so zur Tumorprogression bei. Ausgehend von diesem Hintergrund wurde die Hypothese formuliert, dass die Eph-Ephrin-vermittelte Interaktion von Melanomzellen und Tumor-assoziierten inflammatorischen Zellen die Progression und Metastasierung des malignen Melanoms beeinflusst. Im Speziellen sollte im Rahmen der vorliegenden Arbeit geprüft werden, ob die Rezeptor-Tyrosinkinase EphB4 im Zusammenspiel mit seinem Liganden EphrinB2 die Progression und Metastasierung des malignen Melanoms fördert. Darüber hinaus sollte getestet werden, ob der Rezeptor EphB6, der ebenfalls zur Bindung von EphrinB2 fähig ist, aber über eine mutierte und damit funktionsunfähige Kinasedomäne verfügt, eine regulative Rolle übernimmt und antitumorigen wirkt. Aufbauend auf den Erkenntnissen zur Bedeutung von EphB4, EphB6 und EphrinB2 beim malignen Melanom sollten zudem verschiedene Ansätze zur Bildgebung der oben genannten Eph-Rezeptoren und Ephrine mittels PET etabliert und geprüft werden. ERGEBNISSE UND DISKUSSION Im Rahmen der vorliegenden Arbeit wurde erstmalig gezeigt, dass die Membranproteine EphB4, EphB6 und EphrinB2 bei den ausgewählten humanen Melanomzellen und den inflammatorischen Zellen exprimiert werden und somit potentielle Interaktionsmöglichkeiten dieser Zellen darstellen. Infolge der Kokultur mit HL-60(M)-Zellen, die als Modell für Tumor-assoziierte Makrophagen dienten, kam es zu einer verminderten Adhäsion und/oder Migration der Melanomzellen sowie im Falle der A375- und A2058-Melanomzellen zu einer verstärkten Sekretion des proinflammatorischen Zytokins IL-6. Aufgrund der breiten Wirkung von IL-6 ergeben sich daraus vielfältige Einflussmöglichkeiten auf das Tumormikromilieu. Diese wurden im Rahmen der vorliegenden Arbeit jedoch nicht näher charakterisiert, da erste Ergebnisse eine Beteiligung des Eph-Ephrin-Systems ausschlossen. Während die Überexpression von EphB6 keinen Einfluss auf die Metastasierungs-relevanten Eigenschaften der A375-Melanomzellen hatte, führte die erhöhte Proteinbiosynthese von EphB4 zu einer verminderten Migration der Zellen im intakten Zellverband. Des Weiteren bewirkte EphB4 eine verstärkte Adhäsion der A375-Zellen an das Extrazellularmatrix-Protein Fibronektin, wodurch die Migration dieser Melanomzellen, im Sinne einer Metastasierung, zusätzlich beeinträchtigt wird. Die erhöhte mRNA-Expression des Liganden EphrinB2 in den A375-Melanomzellen führte zu einer verminderten chemotaktischen Migration der Zellen. Um den Einfluss von EphB4 auf die Tumorprogression und Tumorangiogenese beim malignen Melanom in vivo untersuchen zu können, wurde im Rahmen der vorliegenden Arbeit ein murines Xenograft-Modell mit subkutanen A375 pIRES- bzw. A375 EphB4-Tumoren etabliert. Die Auswertung der Tumorvolumina sowie der [18F]FDG-, [18F]FMISO- und Hoechst 33342-Anreicherung in den Tumoren ergab, dass die erhöhte EphB4-Proteinbiosynthese zu tendenziell kleineren Tumoren führte. Diese waren zudem signifikant schwächer perfundiert und wiesen im Inneren größere hypoxische Areale auf als die A375 pIRES-Tumoren. Somit zeigte EphB4 neben seiner antimetastasischen Wirkung in vitro auch eine antitumorigene Wirkung in vivo, wobei letztere möglicherweise auf eine Störung der Gefäßbildung zurückzuführen ist. Da eine adäquate Blutversorgung der Tumoren für die Metastasierung von Tumorzellen von Bedeutung ist, könnte dies auch auf eine antimetastatische Wirkung in vivo hinweisen. Des Weiteren wurde im Rahmen der vorliegenden Arbeit ein neuer, 18F-markierter EphB4 Kinaseinhibitor (Verbindung [18F]2) getestet. Dieser zeigte im A375-pIRES/EphB4-Tumor-Xenograft-Modell eine geringe Tumoranreicherung, die von der EphB4-Proteinbiosynthese in den Tumoren unabhängig war. Darüber hinaus kam es zur schnellen hepatobiliären Ausscheidung von Verbindung [18F]2, was deren radiopharmazeutischer Anwendung im Wege steht. SCHLUSSFOLGERUNG UND AUSBLICK Insbesondere die erhöhte Proteinbiosynthese von EphB4 hatte im Falle der untersuchten A375-Melanomzellen zu einer verminderten Migration und zu einer erhöhten Adhäsion der Zellen geführt. Somit konnte im Rahmen der vorliegenden Arbeit gezeigt werden, dass EphB4 die Metastasierungs-relevanten Eigenschaften dieser Zellen in vitro beeinträchtigt. Darüber hinaus deuteten Untersuchungen am A375-pIRES/EphB4-Tumor-Xenograft-Modell auf eine antitumorigene Wirkung des EphB4-Rezeptors in vivo hin. Aufgrund dessen muss der anfänglich formulierten Hypothese, dass EphB4 im Zusammenspiel mit seinem Liganden EphrinB2 die Progression und Metastasierung des malignen Melanoms fördert, widersprochen werden. Eine regulative Beteiligung des Kinase-defizienten Rezeptors EphB6, der ebenfalls zur Bindung von EphrinB2 fähig ist, konnte im Rahmen der vorliegenden Arbeit nicht sicher nachgewiesen werden. Allerdings ergeben sich aufgrund der Expression der Rezeptoren EphB4 und EphB6 sowie deren Ligand EphrinB2 sowohl auf den untersuchten Melanomzellen als auch auf den verschiedenen Tumor-assoziierten inflammatorischen Zellen interessante Interaktionsmöglichkeiten dieser Zellen. Deren Einfluss auf die Progression und Metastasierung des malignen Melanoms sollte in weiterführenden Experimenten untersucht werden. Das im Rahmen der vorliegenden Arbeit etablierte A375-pIRES/EphB4-Tumor-Xenograft-Modell ermöglicht die In vivo-Charakterisierung von Radiotracer, die gegen Rezeptor-Tyrosinkinasen im Allgemeinen oder aber selektiv gegen EphB4 gerichtet sind. Da Verbindung [18F]2 eine ungünstige Pharmakokinetik zeigte, was wahrscheinlich auf die hohe Lipophilie des Radiotracers zurückzuführen ist, sollten sich zukünftige Untersuchungen mit der chemischen Modifikation dieser Verbindung beschäftigen, mit dem Ziel die Lipophilie und damit die biologische Halbwertszeit des Radiotracers zu verbessern. Zusätzlich sollte die Entwicklung von Radiotracern auf der Basis von löslichen Eph-Rezeptoren und Ephrinen (sEph bzw. sEphrin) weiter vorangetrieben werden.

During development of the vertebrate hindbrain, the neuroepithelium becomes subdivided into seven morphological units, known as rhombomeres. It is necessary that rhombomeres have sharp, well-defined boundaries, which are established from initially rough gene expression domains during early hindbrain segmentation. The mechanisms involved in early hindbrain segmentation that create sharp segment borders are not well understood. There is evidence to suggest that both regulation of cell identity and Eph/ephrin-mediated cell sorting are required for establishing sharp interfaces between rhombomeres. This thesis investigates the extent to which identity regulation contributes to hindbrain border sharpening in zebrafish. I created a new zebrafish reporter line by CRISPR/Cas9-mediated reporter integration at the egr2b locus, which enables cell identity and cell intermingling to be visualised in live embryos during border sharpening. This new reporter line indicates a contribution of cell identity regulation to border sharpening. I also demonstrate that the contribution of cell identity switching to border refinement is greater in cases where cell intermingling is increased by perturbed Eph/ephrin signalling. To help study the role of Eph/ephrin signalling in border sharpening, I have also created a novel EphrinB3b mutant. The thesis also investigates the mechanisms of identity regulation by community effects and discusses their contribution to border refinement by identity respecification; community effects are suspected to help overcome noise in early gene induction through spatial averaging and thus help establish regions of homogeneous gene expression. The ability of candidate genes to non cell-autonomously regulate the identity of neighbouring cells in the hindbrain is investigated. Of particular focus is the potential involvement of retinoic acid (a morphogen involved in specification of anteroposterior identity) and segmentally-expressed Cyp26 enzymes involved in its metabolism. Analysis of mosaic embryos is used to compare the ability of isolated cells and clustered groups of cells to maintain a different identity to their surroundings. Results presented here are consistent with segmental regulation of retinoic acid signalling contributing to border sharpening.

Gastrulation is a key early developmental event that generates the three primary germ layers (ectoderm, mesoderm, and endoderm) from which organ systems subsequently develop. The physical mechanisms of germ layer formation differ significantly in amniotes (reptiles, birds, and mammals) and anamniotes (e.g. frog and fish), as amniote gastrulation includes an epithelial-mesenchymal transition (EMT) that is absent from anamniote gastrulation. Despite this striking difference, much of our knowledge regarding the mechanisms underlying gastrulation is derived from frog and fish studies. To better understand amniote gastrulation, the work herein investigates three signaling pathways that regulate amniote gastrulation with distinct and overlapping functions. The central hypothesis is that multiple signaling pathways function cooperatively to precisely modulate cell migration through the primitive streak during avian gastrulation.First, I describe a novel function of Fibroblast Growth Factor (FGF) signaling in the preingression epiblast adjacent to the avian primitive streak, where it governs the expression of molecules from diverse signaling pathways and transcription factor families, and which is mediated largely through the Ras/MAPK pathway. Importantly, FGF signaling also regulates cell migration during avian gastrulation.Next, I report the isolation of a novel chicken non-canonical Wnt ligand (Wnt11b) that is specifically expressed in the primitive streak and adjacent preingression epiblast during gastrula stages. In gain and loss of function studies, Wnt11b and Wnt5a/b participate in regulating cell migration through the streak in a largely redundant fashion. Signaling specifically targets the non-canonical pathway, as similar cell migration defects are observed with a non-canonical mutant of Dishevelled, and activating the canonical pathway has no effect on cell migration.Finally, I investigate the function of A-class Eph-ephrin signaling during avian gastrulation, and describe that Eph receptor forward signaling negatively regulates the migration of cells through the primitive streak. This modulation of cell migration occurs independently of the EMT that accompanies avian gastrulation, as cells are able to undergo the normal cadherin transition and the basal lamina is unaffected.Altogether, the work presented herein provides a significant contribution to our understanding of signaling pathways that modulate gene expression and ongoing cell migration during germ layer formation in amniote gastrulation.

The correct development of multicellular organisms is tightly regulated by intrinsic and extrinsic factors at specific time points. Disturbance on any level of these multiple processes may result in drastic phenotypes or eventually death of the organism. The midbrain-hindbrain boundary (also termed isthmic organizer) is a region of high interest as well in early as also in later development. The isthmic region carries organizer identity by the expression and subsequent release of FGF8. False patterning events of this region in early developmental stages would therefore display dramatic results over time. As it has been shown that the midbrain-hindbrain boundary (mhb) in the zebrafish is a compartment (or lineage restriction) boundary I tried to understand the underlying molecular mechanism for its correct establishment. In this work I focused both on embryological, molecular and genetic means to characterize involved molecules and mechanisms. In the first part of the thesis I followed in vivo cell transplantation assays, having started with an unbiased one. Cells of either side the mhb were challenged with this boundary by bringing them into direct cell contact with their ectopic counterpart. In a biased approach, cells overexpressing mRNA of specific candidate genes were transplanted and their clonal distribution in host embryos was analyzed. In the second part of the thesis I started interfering with specific candidate genes by transiently knocking down their protein translation. The adhesion molecules of the Eph/ephrin class had been shown to restrict cell mixing and thereby creating compartment boundaries in other tissues, such as the hindbrain, in the zebrafish and other organisms. Additionally, we generated several stable genetic mutant lines in cooperation with the Tilling facility at the Max-Planck-Institute. The only acquired potential null mutant ephrinB2bhu2971 was analyzed and characterized further. I observed that a knock down or knock out of only one of the ephrinB2 ligands does not seem to be sufficient for a loss of compartment boundary formation. The combinatory approach of blocking translation of EphrinB2a in ephrinB2bhu2971 mutants gave very complex and interesting phenotypes, which need to be investigated further.

Das maligne Melanom ist die aggressivste Form von Hautkrebs und verschiedene Familien von Rezeptortyrosinkinasen sind an der Entwicklung und der verstärkten Malignität beteiligt. Eph-Rezeptoren stellen die größte Klasse der Rezeptortyrosinkinasen dar und spielen eine wichtige Rolle bei der Tumorangiogenese und -progression. Die Genexpression und Proteinsynthese verschiedener Eph-Rezeptoren und Ephrin-Liganden ist bei vielen Tumorentitäten erhöht. Aus diesem Grund sollten sie sich als Zielproteine für die Entwicklung neuer Radiopharmaka eignen. Zudem zeigen Literaturbefunde einen Einfluss der hypoxischen Zellumgebung auf die Genexpression und die Proteinsynthese verschiedener Eph-Rezeptoren und Ephrin-Liganden. Das Ziel dieser Arbeit war es, Regulationsmechanismen bei verschiedenen Eph-Rezeptoren und Ephrin-Liganden aufzuklären, welche durch ein hypoxisches Umfeld hervorgerufen werden. Dazu wurde neben einem extrinsischen Hypoxiemodell an Monolayerzellkulturen auch ein intrinsisches Hypoxiemodell in Form von Tumorsphäroiden untersucht. Da die Genexpression und die Proteinsynthese von EphA2, EphB4, EphrinA1 und EphrinB2 laut Literatur vom Malignitätsgrad abhängig sind, wurden die metastatischen Melanomzelllinien A375, A2058 und MeWo und die prämetastatische Melanomzelllinie MEL-JUSO verwendet. Die Verifizierung der experimentellen Hypoxie erfolgte durch den etablierten Hypoxiemarker [18F]Fluormisonidazol, sowie dem Nachweis der VEGF-Genexpression unter den verwendeten Kulturbedingungen. Damit konnte die Eignung der hypoxischen Systeme gezeigt werden. Unabhängig vom Hypoxiemodell war in keiner der untersuchten Zelllinien ein Einfluss der Hypoxie auf die Genexpression und Proteinsynthese von EphA2, EphB4, EphrinA1 und EphrinB2 nachweisbar. Ein gesteigerter EphA2-Gehalt in Melanomzellen ist laut Literatur mit einer Erhöhung des Metastasierungspotentials verbunden. Um diesen Einfluss innerhalb einer Zelllinie zu untersuchen, wurden transgene A375-Zellen generiert. Mit dieser Zelllinie fanden Untersuchungen zu verschiedenen Metastasierungseigenschaften statt. Dabei wurde festgestellt, dass sich die Migration der Zellen durch den erhöhten EphA2-Gehalt verringerte, dabei war die hypoxische Umgebung ohne Einfluss. Weiterhin wurde festgestellt, dass der EphA2-Rezeptor das Adhäsionsverhalten von A375-Zellen nicht beeinflusst. Auch ein Einfluss auf das invasive Verhalten konnte nicht festgestellt werden. Eine hypoxische Umgebung war in beiden Fällen nicht von Bedeutung. Aus den Ergebnissen der vorliegenden Arbeit kann geschlussfolgert werden, dass bei den untersuchten Melanomzelllinien keine Regulation der Eph-Rezeptoren und Ephrin-Liganden durch ein hypoxisches Umfeld erfolgt. Durch die ausführliche Charakterisierung des EphA2-Rezeptors in der Arbeit kann jedoch geschlussfolgert werden, dass sich der Rezeptor als potentielles Zielmolekül für die Entwicklung neuer Radiotherapeutika und Radiodiagnostika eignet, nicht jedoch für die Detektion hypoxischer Bereiche in Tumoren. Durch die nunmehr etablierte Generierung von Sphäroiden und einer Zelllinie, welche den Rezeptor verstärkt exprimiert und synthetisiert, stehen nun Zellmodelle für die weiterführende Charakterisierung und Analyse neuer Radiodiagnostika und Radiotherapeutika auf der Basis von Inhibitoren und Antikörper gegen EphA2 zur Verfügung.

L'embryogénèse précoce est un processus complexe au cours duquel divers types cellulaires sont spécifiés de manière contrôlée dans l'espace et dans le temps. Les embryons présentant un lignage cellulaire fixe permettent de suivre la ségrégation des destinées cellulaires à un niveau de résolution cellulaire individuel. Les embryons d'ascidie représentent de ce point de vue un modèlé idéal et nous utilisons l'ascidie Ciona intestinalis pour étudier la manière dont les destinées notochorde et neurale sont ségrégées à partir des progéniteurs communs. A la fin du stade 32 cellules dans ces embryons, deux blastomères végétatifs (A6. 2 et A6. 4) subissent une division cellulaire au cours de laquelle ils donnent chacun naissance à un précurseur de la notochorde et un précurseur neural. L'événement central dans la ségrégation des destinées neurale et notochorde est une activation différentielle de la MAP kinase ERK1/2 entre les deux cellules filles : les cellules qui présentent une activation de ERK1/2 adoptent une destinée notochorde tandis que les cellules qui ne présentent pas d'activation ERK1/2 adoptent une destinée neurale. L'activation de ERK1/2 est sous contrôle du ligand FGF9/16/20 exprimé dans tout l'hémisphère végétatif. Au cours de ma thèse, j'ai pu montrer qu'un ligand membranaire de type ephrin, appelé ephrin-Ad, est exprimé de manière localisée dans les cellules de l'hémisphère animal faisant contact avec la lignée neurale mais pas avec la lignée notochorde. Ce ligand, au travers d'un récepteur à activité tyrosine kinase EPH, est responsable de l'inhibition spécifique de ERK1/2 dans les cellules de destinée neurale. De plus, nous avons démontré que cette voie de signalisation ephrin/EPH est responsable de la polarisation de la cellule mère notochorde/neurale qui acquière ainsi une capacité intrinsèque à se diviser de manière asymétrique avant la cytocinèse. Enfin, j'ai montré que la protéine Ras-GAP est vraisemblablement impliquée dans l'inhibition de la voie des MAPK en aval de la signalisation ephrin/EPH
Early embryogenesis is a complex process during which diverse cell type precursors are specified in a defined spatio-temporal pattern. Embryos with a fixed cell lineage allow the segregation of cell fates to be precisely followed during embryogenesis. The invertebrate chordate embryos of ascidians are, from that point of view, an ideal model and we are using the ascidian Ciona intestinalis to study how notochord and neural fates become segregated. Using a morpholino knockdown strategy, we have shown that inhibition of ERK1/2 in the neural lineage is mediated by an ephrin membrane-bound ligand, namely ephrin-Ad. Ephrin-Ad is expressed in the animal cells, which are in contact with cells of the neural lineage but not those of the notochord lineage. Using dominant negative forms, we have also shown that this ephrin is mediated via an Eph receptor and the Ci-p120-Ras-GAP, leading to the selective inhibition of ERK1/2 in the neural lineage

This dissertation investigates possible roles of the Eph family receptor tyrosine kinases and their ligand ephrins in the developing primary olfactory pathway in the moth Manduca sexta. The Manduca homologs of the Eph receptor (MsEph) and ephrin ligand (MsEphrin) are most closely related to Drosophila Eph and ephrin, respectively, and biochemical assays establishes MsEphrin as a functional ligand for MsEph. In situ labeling with Fc-fusion probes in which IgG Fc was linked to the extracellular domain of MsEph and MsEphrin reveals that both Eph receptors and ephrins are expressed on olfactory receptor cell (ORC) axons during their ingrowth to the primary brain center, the antennal lobe (AL). Eph receptors and ephrins are differentially distributed among identifiable glomeruli such that glomeruli with high receptor staining show little ligand staining and vice versa, indicating a complementary Eph/ephrin expression on subsets of ORC axons innervating particular set of glomeruli. Their expression appears upregulated once ORC axons reach the region where axon sorting occurs. In contrast, neither Eph receptors nor ephrins are detectable in intrinsic components of the AL. In vitro, MsEph and MsEphrin proteins, when present homogeneously in the substratum, inhibit neurite outgrowth from olfactory epithelial explants. Moreover, in patterned substrata, neurites growing on the standard substratum avoid extending onto the substratum containing MsEphrin proteins, with behaviors characterized by turning or stopping at the border. These in vitro observations indicate that MsEphrin can act as an inhibitory/repulsive cue for ORC axons. Based on these in situ and in vitro results, I hypothesize that Eph-ephrin signaling mediates segregation of Eph-positive axons from ephrin-positive axons through repulsive inter-axonal interactions. Simultaneous labeling of ALs for Eph/ephrin and fasciclin II, a homophilic cell adhesion molecule, reveals that fasciclin-positive glomeruli are distributed in a partially overlapping pattern with Eph- or ephrin-positive glomeruli. Thus, Eph receptors and ephrins, together with fasciclin II and other adhesive/repulsive cues, might constitute a combinatorial molecular system in which sorting of ORC axons is determined by the balance of adhesive and repulsive forces.

EphB4 is a protein increased in cancers including prostate cancer. This work explored the role of EphB4 in processes important to metastatic spread of disease using models of the blood vessel wall. Prostate cancer cells with increased EphB4 move more easily through endothelial cell barriers when the endothelial cells make the EphB4 ligand ephrin-B2. This implicates the EphB4-ephrin-B2 interaction in site-specific metastatic dissemination of prostate cancer.

O fator derivado do epitélio pigmentado (PEDF) é um fator neurotrófico que possui um grande potencial trófico nos neurônios motores da medula espinal, bem como é capaz de modular o microambiente da lesão. Desta forma, analisamos a capacidade do tratamento com PEDF em promover a neuroplasticidade da medula espinal após lesão isquêmica. Ratos Wistar adultos foram submetidos à lesão medular isquêmica do tipo fototrombótica, segundo o método de Rose Bengal, na altura do 11° segmento torácico e foram imediatamente tratados com inoculação local de PEDF (grupo PEDF) ou solvente (grupo Salina). Ratos submetidos à cirurgia simulada (grupo Sham) receberam a injeção do solvente. Ao término do procedimento cirúrgico, os ratos foram submetidos a testes neurofuncionais durante 6 semanas. Após esse período, os animais sofreram eutanásia e o tecido medular foi dividido entre as técnicas de imunoistoquímica, western blot e PCR em tempo real. Foi analisada na região lombar anterior da medula espinal a modulação das CSPGs, a expressão dos fatores neurotróficos NT-3, GDNF, BDNF e FGF-2, bem como os níveis das moléculas associadas à angiogênese e apoptose (laminina e Bcl-2), das proteínas relacionadas à neuroplasticidade (MAP-2, GAP-43 e sinaptofisina) e do sistema Eph/efrina e a RhoA, que são capazes de modular o crescimento de fibras. Os resultados mostraram uma recuperação parcial e espontânea do comportamento sensório-motor dos animais que foram submetidos à lesão fototrombótica, onde o tratamento com PEDF foi capaz de potencializar alguns desses parâmetros. A análise da região lombar anterior da medula espinal, caudal à lesão, mostrou uma diminuição das CSPGs nos dois grupos lesados, o que pode ter favorecido os eventos de neuroplasticidade. O tratamento com PEDF foi capaz de promover a regulação dos fatores neurotróficos NT-3 e GDNF, diminuir a angiogênese local (diminuição da laminina) e potencializar o processo de neuroplasticidade (aumento da MAP-2) nessa região. A lesão medular isquêmica foi capaz de modular a expressão do receptor EphA4 e da efrina-B1 e o tratamento com PEDF possivelmente regulou o estado de ativação da efrina-A2 e da efrina-B3 e certamente modulou a ativação da efrina-B2. Ainda, os receptores Eph e as efrinas foram observados diferentemente nos neurônios e astrócitos. Nossos resultados confirmam a capacidade plástica da medula espinal após lesão e mostra que o tratamento com PEDF foi capaz de potencializar esse processo
The pigment epithelium derived factor (PEDF) is a neurotrophic factor that has a great trophic potential in the motor neurons of the spinal cord, and is able to modulate the lesion microenvironment. We analyzed the capacity of the treatment with PEDF to promote the neuroplasticity after ischemic spinal cord injury. Adult male Wistar rats were underwent to photothrombotic ischemic spinal cord injury, according to the Rose Bengal method, at the level of 11° thoracic segment, and were immediately treated with local injection of PEDF (PEDF group) or solvent (Saline group). Rats underwent to a sham surgery (Sham group) received solvent injection. At the end of surgery, the rats were submitted to neurofunctional tests during 6 weeks. After this period, the animals were euthanized, and the anterior lumbar region of the spinal cord tissue was submitted to immunohistochemistry, western blot and real-time PCR analyses. The inhibitory response of CSPGs, the expression of neurotrophic factors (NT-3, GDNF, BDNF and FGF-2), the molecules associated with angiogenisis and apoptosis (laminin and Bcl-2), the proteins related to neuroplasticity (MAP-2, GAP-43 and synaptophysin), as well the Eph/ephrin system and the RhoA, which is able to modulate the fibers growth, were evaluated. The results showed a spontaneous, and parcial, recovery of the sensory motor behavior of the animals that were underwent to a photothrombotic injury, and the treatment with PEDF was able to potentiate some of these parameters. The analysis of the anterior lumbar region of the spinal cord, caudally to the lesion, showed a decrease of CSPGs, which may have favored the neuroplasticity events. The treatment with PEDF was able to promote the regulation of NT-3 and GDNF, as well the reduction of laminin and the increase of MAP-2 in that region. In relation to the Eph/ephrin system, the ischemic spinal cord injury was able to modulate the EphA4 receptor and ephrin-B1 expression, and the treatment with PEDF possibly regulated the activation state of ephrin-A2 and ephrin-B3 and certainly modulated the ephrin-B2 activation. Eph receptors and ephrins have been found specifically in neurons and astrocytes. Our results confirmed the plastic capacity of the spinal cord after injury and showed that the treatment with PEDF was able to enhance this process

This body of work is concerned with the genetics of craniofacial morphology and specifically with that of the cranial sutures which form fibrous articulations between the calvarial bones. The premature fusion of these sutures, known as craniosynostosis, is a common developmental abnormality and has been extensively utilised here as a tool through which to study the genetics of suture morphogenesis and craniofacial diversity. Investigations began with a search for polymorphisms associated with normal variation in human craniofacial characteristics. Denaturing High-Performance Liquid chromatography was used to identify polymorphisms in two genes causative for craniosynostosis by analysing DNA from a large cohort of individuals from four ethnogeographic populations. A single nucleotide polymorphism in fibroblast growth factor receptor 1 was identified as being associated with variation in the cephalic index, a common measure of cranial shape. To further, and specifically, investigate the molecular processes of suture morphogenesis gene expression was compared between unfused and prematurely fusing/fused suture tissues isolated from patients with craniosynostosis. Two approaches, both utilising Affymetrix gene expression microarrays, were used to identify genes differentially expressed during premature suture fusion. The first was a novel method which utilised the observation that explant cells from both fused and unfused suture tissue, cultured in minimal medium, produce a gene expression profile characteristic of minimally differentiated osteoblastic cells. Consequently, gene expression was compared between prematurely fused suture tissues and their corresponding in vitro de-differentiated cells. In addition to those genes known to be involved in suture morphogenesis, a large number of novel genes were identified which were up-regulated in the differentiated in vivo state and are thus implicated in premature suture fusion and in vivo osteoblast differentiation. The second microarray study involved an extensive analysis of 16 suture tissues and compared gene expression between unfused (n=9) and fusing/fused sutures (n=7). Again, both known genes and a substantially large number of novel genes were identified as being differentially expressed. Some of these novel genes included retinol binding protein 4 (RBP4), glypican 3 (GPC3), C1q tumour necrosis factor 3 (C1QTNF3), and WNT inhibitory factor 1 (WIF1). The known functions of these genes are suggestive of potential roles in suture morphogenesis. Realtime quantitative RT PCR (QRT-PCR) was used to verify the differential expression patterns observed for 11 genes and Western blot analysis and confocal microscopy was used to investigate the protein expression for 3 genes of interest. RBP4 was found to be localised on the ectocranial surface of unfused sutures and in cells lining the osteogenic fronts while GPC3 was localised to suture mesenchyme of unfused sutures. A comparison between each unfused suture (coronal, sagittal, metopic, and lambdoid) demonstrated that gene expression profiles are suture-specific which, based on the identification of differentially expressed genes, suggests possible molecular bases for the differential timing of normal fusion and the response of each suture to different craniosynostosis mutations. One observation of particular interest was the presence of cartilage in unfused lambdoid sutures, suggesting a role for chondrogenesis in posterior skull sutures which have generally been thought to develop by intramembranous ossification without a cartilage precursor. Finally, the effects of common media supplements used in in vitro experiments to stimulate differentiation of calvarial suture-derived cells were investigated with respect to their ability to induce in vivo-like gene expression. The response to standard differentiation medium (ascorbic acid + β-glycerophosphate) with and without dexamethasone was measured by both mineralisation and matrix formation assays and QRT-PCR of genes identified in the above described microarray studies. Both media induced collagen matrix and bone nodule formation indicative of differentiating osteoblasts. However, the genes expression profiles induced by both media differed and neither recapitulated the levels and profiles of gene expression observed in vivo for cells isolated from both fused and unfused suture tissues. This study has implications for translating results from in vitro work to the in vivo situation. Significantly, the dedifferentiation microarray study identified differentially expressed genes whose products may be considered candidates as more appropriate osteogenic supplements that may be used during in vitro experiments to better induce in vivo-like osteoblast differentiation. This study has made a substantial contribution to the identification of novel genes and pathways involved in controlling human suture morphogenesis and craniofacial diversity. The results from this research will stimulate new areas of inquiry which will one day aid in the development of better diagnostics and therapeutics for craniosynostosis, and other craniofacial and more general skeletal abnormalities.

This body of work is concerned with the genetics of craniofacial morphology and specifically with that of the cranial sutures which form fibrous articulations between the calvarial bones. The premature fusion of these sutures, known as craniosynostosis, is a common developmental abnormality and has been extensively utilised here as a tool through which to study the genetics of suture morphogenesis and craniofacial diversity. Investigations began with a search for polymorphisms associated with normal variation in human craniofacial characteristics. Denaturing High-Performance Liquid chromatography was used to identify polymorphisms in two genes causative for craniosynostosis by analysing DNA from a large cohort of individuals from four ethnogeographic populations. A single nucleotide polymorphism in fibroblast growth factor receptor 1 was identified as being associated with variation in the cephalic index, a common measure of cranial shape. To further, and specifically, investigate the molecular processes of suture morphogenesis gene expression was compared between unfused and prematurely fusing/fused suture tissues isolated from patients with craniosynostosis. Two approaches, both utilising Affymetrix gene expression microarrays, were used to identify genes differentially expressed during premature suture fusion. The first was a novel method which utilised the observation that explant cells from both fused and unfused suture tissue, cultured in minimal medium, produce a gene expression profile characteristic of minimally differentiated osteoblastic cells. Consequently, gene expression was compared between prematurely fused suture tissues and their corresponding in vitro de-differentiated cells. In addition to those genes known to be involved in suture morphogenesis, a large number of novel genes were identified which were up-regulated in the differentiated in vivo state and are thus implicated in premature suture fusion and in vivo osteoblast differentiation. The second microarray study involved an extensive analysis of 16 suture tissues and compared gene expression between unfused (n=9) and fusing/fused sutures (n=7). Again, both known genes and a substantially large number of novel genes were identified as being differentially expressed. Some of these novel genes included retinol binding protein 4 (RBP4), glypican 3 (GPC3), C1q tumour necrosis factor 3 (C1QTNF3), and WNT inhibitory factor 1 (WIF1). The known functions of these genes are suggestive of potential roles in suture morphogenesis. Realtime quantitative RT PCR (QRT-PCR) was used to verify the differential expression patterns observed for 11 genes and Western blot analysis and confocal microscopy was used to investigate the protein expression for 3 genes of interest. RBP4 was found to be localised on the ectocranial surface of unfused sutures and in cells lining the osteogenic fronts while GPC3 was localised to suture mesenchyme of unfused sutures. A comparison between each unfused suture (coronal, sagittal, metopic, and lambdoid) demonstrated that gene expression profiles are suture-specific which, based on the identification of differentially expressed genes, suggests possible molecular bases for the differential timing of normal fusion and the response of each suture to different craniosynostosis mutations. One observation of particular interest was the presence of cartilage in unfused lambdoid sutures, suggesting a role for chondrogenesis in posterior skull sutures which have generally been thought to develop by intramembranous ossification without a cartilage precursor. Finally, the effects of common media supplements used in in vitro experiments to stimulate differentiation of calvarial suture-derived cells were investigated with respect to their ability to induce in vivo-like gene expression. The response to standard differentiation medium (ascorbic acid + β-glycerophosphate) with and without dexamethasone was measured by both mineralisation and matrix formation assays and QRT-PCR of genes identified in the above described microarray studies. Both media induced collagen matrix and bone nodule formation indicative of differentiating osteoblasts. However, the genes expression profiles induced by both media differed and neither recapitulated the levels and profiles of gene expression observed in vivo for cells isolated from both fused and unfused suture tissues. This study has implications for translating results from in vitro work to the in vivo situation. Significantly, the dedifferentiation microarray study identified differentially expressed genes whose products may be considered candidates as more appropriate osteogenic supplements that may be used during in vitro experiments to better induce in vivo-like osteoblast differentiation. This study has made a substantial contribution to the identification of novel genes and pathways involved in controlling human suture morphogenesis and craniofacial diversity. The results from this research will stimulate new areas of inquiry which will one day aid in the development of better diagnostics and therapeutics for craniosynostosis, and other craniofacial and more general skeletal abnormalities.

This thesis has involved the analysis of Eph/ephrin-A gene expression with respect to NC (neural crest) cell segmentation, identified EphA4 as a putative regulator of cell-cell communication during NC cell migration and more specifically, established that this protein plays a critical negative regulatory role in EMT.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biological Sciences, 2005

There is substantial evidence from the literature that suggests a close association between the skeletal and nervous systems at the anatomical and physiological level, and this cellular and molecular cross-interaction has been termed the neuro-osteogenic network, critical for skeletal growth, homeostasis, and repair. The Eph and ephrin molecules are the largest family of receptor tyrosine kinases. These membrane-bound molecules are contact-dependent and predominantly function through cell-to-cell interactions. Forward signalling occurs when the ephrin ligand activates signalling in the Eph expressing cell while reverse signalling can also occur where Eph receptors activate signalling through the ephrin ligand expressing cells. The Eph/ ephrin interactions have known roles independently in the skeletal system regulating bone homeostasis, and skeletal growth and repair, and within the nervous system influencing axon guidance and patterning. Therefore, the roles of Eph/ ephrin mediated cross talk between neural and bone marrow stromal cell (BMSC) populations in regulating neural differentiation, adhesion and migration were investigated. An in vitro neural differentiation assay was established for the human neuroblastoma line SH-SY5Y which showed upregulated expression of EPHRINA2, EPHRINA5 and EPRHINB1 with the progression of neuronal differentiation. Previous studies in our laboratory have shown that human BMSC highly express EPHA3, EPHA4, EPHB2 and EPHB4 at the gene and protein levels. Therefore, the roles of Eph/ ephrin interacting pairs in mediating cross -communication between BMSC and neurons, respectively, were assessed. These included the stromal expressing Eph receptors with neural expressing ephrin ligands (1) EphA3/A4 with ephrinA2 (2) EphA3/A4 or EphB2 with ephrinA5 and (3) EphB2/B4 with ephrinB1, respectively. To examine the importance of ephrin reverse signalling on neural differentiation, adhesion and migration, soluble EphA3-Fc, EphA4-Fc, EphB2-Fc, EphB4-Fc fusion molecules, were used to activate reverse signalling of the ephrinA2, ephrinA5 and ephrinB1 ligands, respectively. The gene expression of early and mature neuronal and sensory markers in the SH-SY5Y line showed a differential response following activation of the ephrinA and ephrinB ligands during neural induction. However, there was no significant difference in neuronal morphology. The activation of both ephrinA and ephrinB ligands inhibited SH-SY5Y neural adhesion, with reduced neural adhesion in response to EphA4-Fc, EphB2-Fc and EphB4-Fc for two hours, and in response to EphA4-Fc and EphB4-Fc for twenty-four hours. EphB2-Fc and EphB4-Fc stimulation did not exert an effect on neural transwell migration where these molecules were not in direct contact with the neural-like cells. There was no significant difference in neural adhesion, or neural and stromal migration, in response to blocking ephrinB1 on the neural population. To determine functional relevance of the neuro-osteogenic network, the avian embryo dorsal root ganglia (DRG) that innervate the hind limbs, were cultured in vitro in the presence of a range of Eph-Fc molecules. DRG neurite cell bodies within the Eph-Fc occupied spaces was significantly decreased for DRG pooled from the lumbar region while a strong trend of differential DRG neurite cell body number and neurite invasion was observed within the Eph-Fc occupied spaces for DRG from specific lumbar compartments. Future studies would investigate the importance of ephrinA2, ephrinA5 and ephrinB1 mediated cross-communication between the neural and BMSC populations during skeletal and neural development and fracture repair.
Thesis (M.Phil) -- University of Adelaide, Adelaide Medical School, 2021

Membrane attachment of ephrin ligands plays an important role in Eph receptor activation. Membrane anchorage is thought to provide a clustering effect to ephrins that is necessary for stimulation of Eph receptor kinase activity. The presence of soluble A-type ephrin in conditioned media of numerous cultured cancer cell lines and normal endothelial cells prompted me to question the purpose of ephrin release. In this thesis I show that ephrin A1, a potent angiogenic factor, is released from several cancer cell lines and is a substrate for tissue transglutaminase, a multifunctional enzyme with the ability to form covalent crosslinks between substrate proteins. I show that tissue transglutaminase crosslinking primes soluble ephrin A1 to promote Eph A2 activity. These results suggest a role for soluble A-type ephrins in promoting Eph receptor activity at distant sites and also indicate that ephrin A1 may be acting as a soluble angiogenic factor during tumor neovascularization.

The role that signaling molecules play during morphogenesis and their interactions is a field of intense study and the sea urchin represents a facile system to study these aspects of development in the early embryo. In many instances, the S. purpuratus genome contains relatively simple receptor-ligand signaling systems compared to vertebrate counterparts and this provides interesting opportunities to study their diversity of function during the morphogenetic events that shape the embryo. The Eph-Ephrin signaling components are an excellent example of this and they are represented by dozens of members in the vertebrate system with developmental functions that include axon guidance, cell migration and tissue segregation. In contrast, the sea urchin genome contains a single Eph receptor and a single Ephrin ligand and by interacting with different effectors of signal transduction, this simple, bipartite system can fulfill a variety of functional roles during morphogenesis. Studying the function of Eph-Ephrin signaling in the sea urchin embryo, I have revealed two distinct morphogenetic movements in which Eph-Ephrin signaling is necessary; apical constriction of ciliary band cells and pigment cell migration. In both examples, a functionally relevant Ephrin gradient establishes spatial information in the developing tissues, producing a reaction from cells expressing the Eph receptor. In the case of pigment cells, the distribution of migrating cells is affected and in the case of ciliary band cells, apical constriction occurs. The different outcomes of Eph-Ephrin signaling in these two tissues exemplifies signaling components communicating spatial information and initiating morphogenetic programs with outcomes dependent on cellular context. Furthermore, I have identified downstream components of Eph-Ephrin signaling that have necessary functions in both models, illustrating how different cellular programs can be induced by the same signaling iii iv components. My research contributes to understanding fundamental aspects of how complex 3 dimensional tissues arise from the genes and regulatory elements encoded in metazoan genomes.
Graduate

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2010
A Evolução e o Desenvolvimento (Evo-Devo) é uma area da biologia que tem por objectivo o estudo e a interpretação de conhecimentos de ambas as areas de evolução e da biologia do desenvolvimento. Tenta assim, explicar e testar as teorias evolutivas ao nivel morfologico. (David 2001) A transição de invertebados para vertebrados é um marco importante na historia evolutiva das especies. A relação filogenética no Filo Chordata é importante para compreender esta transição. O Filo é constituido de tres subfilos Vertebrata, Cephalocordata e Urochordata. As caracteristicas que os une neste Filo é a presença de uma notocorda, um tubo nervoso dorsal, tubo nervoso dorsal, fendas branquiais, um endostélio e uma cauda pós-anal, em pelo menos uma fase de sua vida. Contudo, a relação filogenética dentro do Filo Chordata foi controversa. No século XIX os urocordados foram colocados na base da filogenia do Filo, estando deste modo, os cefalocordados mais proximos dos vertebrados (Fig.1). No entanto, dados moleculares recentes levaram a reversao destas posições estipulando que os cefalocordados se separam antes, localizando-se basalmente ao grupo Urochordata-Vertebrata (Fig.1) (Delsuc 2006). O Filo Cephalochordata compreende dois generos (Branchiostoma and Epigonichtys). O anfioxo (Branchiostoma floridae - especie americana - Branchiostoma lanceolatum -especie europeia) possede uma faringe com fendas branquiais, uma cauda pos anal, um sistema circulatorio ausente de coração e um sistema excretor rudimentar (Fig.2). Contudo carecem de algumas caracteristicas de vertebrado como as células migratorias da crista neural, um endoesqueleto, um cérebro regionalizado e orgãos sensorias pares. Por apresentar um genoma não duplicado, uma copia unica para a maioria das familias multigenicas dos vertebrados, pelas suas caracteristicas morfologicas e do desenvolvimento transitorias aos vertebrados, pela disponibilidade de ferramentas de manipulação génica e vantagens de manutenção em laboratorio e do genoma de Branchiostoma floridae estar completamente sequenciado, o anfioxo é considerado como sendo um bom organismo modelo para estudar a transição de invertebrado a vertebrado. Em 1987, o primeiro receptor de efrina foi clonado. Os receptors (Eph) e ligandos (efn) de efrinas formam a maior das 14 subfamilias de receptores do tipo tirosina kinase. Estas possuem um dominio extracellular N-terminal que permite a interacção com o ligando; um dominio intracelular com funçao de kinase; um dominio SAM; e um dominio PDZ (Fig.4). Os ligandos estão subdivididos em dois grupos, as efnA que se encontram ancoradas à membrana via GPI (glycosylphosphatidylinositol) e as efn B que estão ancoradas por um dominio transmembranar (Fig.4). Os receptores EphA interagem preferencialemente com as efnA e as EphB com as efnB. Esta interacção ligando-receptor tem um papel crucial em processos celulares como a adesão, comunicação, rearranjos de citoesqueleto, divisão, migração, a activação de vias de sinalização citoplasmaticas que promovem a expressão génica. Deste modo, estão implicados em processos de desenvolvimento como a 5 migração das células da crista neural, segmentação e somitogénese, formação de sinapses e proriedades promotoras de tumores. Os receptores e ligandos de efrinas estão presents em todos os Metazoa (de esponjas a vertebrados) em numero (tab. 1), padrões de expressão e funções distintas. Varias das funções desempenhadas pelas efrinas em vertebrados são também encontradas em invertebrados. Foi sugerido que as funções das efrinas teriam evoluido de desempenhar um papel mais simples em processos cellular, ainda mantido em grupos mais basais na filogenia, para papeis mais versateis e diversificados, como os observados em cordados. Neste projecto propusemos um estudo de Evo-Devo da familia de genes das efrinas, pelo seu importante papel no desenvolvimento e pela sua presença em todas as espécies de Metazoa estudadas até a data, usando como modelo experimental o anfioxo europeu, devido à sua posiçao filogenética e caracteristicas. Com o objecctivo de melhor compreender a historia evolutiva e as funções durante o desenvolvimento deste genes os principais passos desenvolvidos foram: i) anotar e descrever os receptores e ligandos de efrinas em Branchiostoma floridae; ii) clonar e sequênciar os genes de efrina na especie europeia Branchiostoma lanceolatum ; iii) elucidar a relação filogenética da familia génica das efrinas em genomas de Metazoa disponiveis (Tab.1) ; iv) determinar os padroes de expressão, em diversos estadios de desenvolvimento, em ambos ligandos e receptores de efrina em Branchiostoma lanceolatum por hibridação in situ. A relação filogenética dos receptores de efrinas esta descrita na Fig.9 desta dissertação e foi realizada com sequências de proteinas no programa Mega4. Nesta analise pode-se verificar que as Eph de vertebrados agrupam-se monofiléticamente juntas. Os genes de cefalocordados estão basais aos de vertebrados, agrupando-se com os dos não-cordados. As Eph de urocordados formam um grupo polifilético, sugerindo a ocorrência de duplicações independentes nesta linhagem, estando CiEphE, CiEphD e CiEphG mais proximos dos vertebrados. Os receptores de efrina dos Cefalocordados parecem ter divergido mais cedo do que os receptores de efrina de urocordados CiEphE, CiEphD and CiEphG, estando de acordo com a topologia proposta por Delsuc (2006) para o Filo Chordata. Os genes de Nematostella vectensis agrupam-se com o outgrupo Ephydatia fluviatilis, sugerindo que esta especie esteve sujeita a duplicaçoes independentes. A topologia obtida para os ligandos de efrina esta representada na Fig.10. É de notar que os ligandos dos vertebrados formam dois grupos monofiléticos distintos, um que agrupa as efnA e outro as efnB. As efnA de vertebrados parecem estar filogenéticamente mais proximas das efnA de urocordados (CiefnAa, CiefnAb, CiefnAc and CiefnAd) e efnB mais proximas das efn de cefalocordados (Bfefn1, Bfefn2 e Bfefn3). Tal como observado com os receptors, os ligandos dos não-cordados formam um grupo monofilético. O estudo da expressão dos receptores (BlEph1 e BlEph2) e ligandos (Blefn1 e Blefn2) de efrina foi feita pela técnica de hibridação in situ em embriões de Branchiostoma lanceolatum durante varios estadios de desenvolvimento (morulas de 32 celulas até larvas de 60horas). No estadio de morula todos os genes expressam-se no embrião inteiro. 6 Durante a neurulação BlEph1 expressava-se na mesoderme lateral, nos somitos em formação e possivelmente no precursor da vesicular cerebral. Na neurula tardia encontrava-se expresso na notocorda e no tubo neural e na future região oral. No estadio de pré-boca expressava-se na notocorda anterior e posterior, na boca e faringe em formação. No estadio de larva este gene expressava-se na notocorda anterior e posterior, na vesicular cerebral, na boca, no endostélio, na glandula club-shaped, no diverticulum. (Fig.11) Durante a gastrulação, BlEph2 parecia expressar-se em todos os tecidos. Um padrão segmentado parecia aparecer durante a neurulação que parece seguir o padrão dos somitos em formação. No estadio de pré-boca BlEph2 expressava-se na boca em formação e na notocorda anterior. Nas larvas a expressão deste gene parecia localizar-se na boca e na região da faringe. (Fig.12) O padrão de expressão dos ligandos de efrina (Blefn1 e Blefn2) parecia estar de acordo com o padrão observado nos receptores. Em gastrulas Blefn1 expressava-se maioritariamente na mesoderme justo ao blastoporo. Durante a neurulação a expressão era mesodermica, junto ao fecho do tubo neural e também parecia expressar-se em populacões de neuronios. No estadio pré-boca parecia expressar-se na futura região oral, na notocorda posterior e a endoderme posterior. No estado de larva a expressão parecia mais restringida a zona da boca e da faringe, mas ligeiramente expresso nos restantes tecidos, exceptuando a vesicula cerebral. (Fig.13) Durante a gastrulação Blefn2 parecia expressar-se na gastrula inteira. Durante a neurulação a expressão parecia restringida a mesoendoderme, exceptuando o dominio mais posterior. Em embriões pré-boca parecia localizar-se na região da faringe em formação, na glandula de muco e na notocorda posterior. Em larvas a expressão parecia localizar-se na região da boca e da faringe e também na notocorda posterior (Fig.5F). Na literatura esta descrito que as efrinas expressam-se em cordados nas zonas dos somitos em formação, na notocorda, no tubo neural, na mesoderme paraxial e nos nervos perifericos, como demonstrado pelas imagens obtidas por hibridação in situ (Fig.11 a 14). Em vertebrados estes genes também participam na formação de varias estruturas faciais incluido a região oral e os seus padrões de inervação, padrão também observado nos receptores e ligandos de efrinas de Branchiostoma lanceolatum sugerindo a co-opção destes genes para esta função durante o desenvolvimento em Chordados (Fig.11-14). Com este trabalho foi possivel elucidar algumas das questoes sobre a expressão e a posição filogenética dos membros da familia genica das efrinas no anfioxo europeu (Branchiostoma lanceolatum). A estrutura destes genes parece ser conservada desde as esponjas aos vertebrados, mas também as funções desempenhadas durante no desenvolvimento. Entre estes incluem-seos processos a nivel cellular que, ao longo da evolução sofreram fenomenos de complexificação, na transição para cordados. Adicionado a estas observações, a topologia filogenética sugere fenomenos de co-opção para as novas funçoes de duplicações especificas de linhagem. Existem também evidências para convergência evolutiva entre espécies e de evolução paralela dentro da mesma espécie. Os cefalocordados parecem ocupar, relativamente a esta analise, uma posição entre os não-cordados e os vertebrados o que suporta a nova filogenia sugerida por Delsuc et al em 2006. No geral, pode-se sugerir que os genes da familia das efrinas diversificaram varias vezes ao longo da evolução dos 7 Metazoa e também que o ancestral dos urocordados e vertebrados apresentaria um unico receptor de efrina e dois ligandos. Contudo, dados adicionais sobre as funções, padrões de expressão génica e filogenia dos Metazoa seriam necessarios para melhor estabelecer uma relação evolutiva da familia génica das efrinas e o seu papel durante a evolução.
The Ephrin receptor and ligand gene families are implicated in several cellular processes such as cellular adhesion, communication, division, migration, and compartmentalization. These play an important role in development including, for instance, neural crest cell migration, somitogenesis, axon guidance, and have even been shown to have tumor promoting properties. They are described to be present from sponges to vertebrates in multiple copies, maintaining a conserved genomic structure. Cephalochordates, recently placed at the base of the Chordata, are considered to be the living animal that best approximates the ancestor at the transition from invertebrates to vertebrates. The phylogenetic analysis of this project shows that Eph and efn families have been independently expanded in amphioxus, vertebrate, and other Metazoan clades suggesting that each have convergently evolved complex Eph/efn complements. In the European amphioxus (Branchiostoma lanceolatum) the Eph/ephrin gene family seems to express mostly in the forming mouth apparatus, somites and notochord, as assessed by whole-mount in situ hybridization (ISH). The Eph/ephrin ISH expression pattern corresponds to some vertebrate characteristics for these genes, but they are also implied in several cellular processes similarly to invertebrates. In spite of the evidence, it is difficult to assess the exact evolutionary relationship and developmental role of these genes in amphioxus with these data, or to extrapolate to Metazoa.

Die Eph-Familie, welche die Eph-Rezeptoren und die ephrin-Liganden umfasst, repräsentiert die größte Subgruppe der Rezeptortyrosinkinasen. Sowohl die Eph-Rezeptoren als auch die ephrin-Liganden sind Membran-assoziierte Proteine, deren Interaktion einen Typ der Zellkontakt-vermittelten Kommunikation darstellt und vielfältigste zelluläre Funktionen beeinflusst. Die aktuellen Eph/ephrin-bezogenen Forschungsschwerpunkte liegen vor allem im Bereich der Entwicklungs- und Tumorbiologie, wie z.B. dem Einfluss der Eph-Familie auf Angiogenese und Axonaussprossung. Die Rolle des Eph/ephrin-Systems in der Entwicklung der Atherosklerose und bei Reparaturmechanismen wie der Reendothelialisierung ist jedoch noch weitgehend unerforscht. Die vorliegende Arbeit befasst sich anhand von in vitro Experimenten mit HUVEC und HUAEC mit dem Einfluss des Liganden ephrin-A1 und des Rezeptors EphA2 auf die endotheliale Proliferation und Migration als elementare Mechanismen der Reendothelialisierung.:INHALTSVERZEICHNIS I ABKÜRZUNGSVERZEICHNIS V ABBILDUNGSVERZEICHNIS IX TABELLENVERZEICHNIS XI 1 EINLEITUNG 1 1.1 Die Rolle des Endothels in der Atherosklerose 1 1.2 Die Migration von Endothelzellen 6 1.3 Die Eph-Familie 9 1.4 Potentielle Funktionen der Eph-Familie in der endothelialen Proliferation und Migration 14 1.5 Zielstellung der Arbeit 16 2 MATERIAL 17 2.1 Geräte 17 2.2 Zellkulturmaterial 18 2.3 Verwendete Kulturmedien 18 2.4 Verwendete Zelllinien und E. coli Stämme 19 2.5 Verwendete Kit-Systeme 19 2.6 Verwendete Oligonukleotide 20 2.7 Verwendete Plasmide 21 2.8 Konstruierte Plasmide 23 2.9 Chemikalien 23 2.10 Puffer, Gele, Lösungen 23 3 METHODEN 24 3.1 Zellbiologische Methoden 24 3.2 Proteinbiochemische Methoden 28 3.3 Molekularbiologische und gentechnische Methoden 32 3.4 Scratch Assay 44 3.5 Beurteilung der Zellproliferation mittels BrdU-Inkorporation 44 3.6 Wundheilungsassay 45 3.7 Migrationsassay 48 3.8 Baculovirale Talin-RFP- und Aktin-GFP-Expression 49 3.9 Datenanalyse und statistische Auswertung 49 4 ERGEBNISSE 50 4.1 Molekularbiologische und proteinbiochemische Charakterisierung verschiedener Eph/ephrine in proliferierenden Endothelzellen 50 4.2 Molekularbiologische Charakterisierung von ephrin-A1, EphA2 und EphA4 unter Kontaktinhibition in HUVEC 56 4.3 Einfluss eines Zellzyklusarrests auf die Expression verschiedener ephrin-Liganden und Eph-Rezeptoren in Endothelzellen 58 4.4 Hemmung von ephrin-A1 und EphA2 mittels siRNA 63 4.5 Einfluss einer siRNA-vermittelten Hemmung von ephrin-A1 und EphA2 auf die Proliferation von HUVEC 65 4.6 Überexpression von ephrin-A1 mittels eines adenoviralen Vektors 67 4.7 Einfluss einer Überexpression von ephrin-A1 auf die Proliferation von HUVEC 68 4.8 Reinitiation von Proliferation und Migration in HUVEC mittels Scratch Assay 69 4.9 Einfluss der Modulation des ephrin-A1- sowie EphA2-Gehaltes auf das Wundheilungsverhalten von HUVEC 73 4.10 Charakterisierung des Migrationsverhaltens von HUVEC unter veränderter ephrin-A1-Expression mittels live cell imaging 75 4.11 Einfluss einer veränderten ephrin-A1-Expression auf den EphA2-Phosphorylierungsstatus 78 4.12 Grenzzonenverhalten von HUVEC an ephrin-A1-beschichteten Oberflächen 80 4.13 Fluoreszenzmikroskopische Charakterisierung fokaler Adhäsionen und des Aktin-Zytoskelettes unter modulierter ephrin A1 Expression 81 5 DISKUSSION 84 5.1 Die Eph-Familie im Kontext der Reendothelialisierung 84 5.2 Einfluss des Eph/ephrin Systems auf das Proliferationsverhalten von Zellen 86 5.3 Auswirkungen einer ephrin-A1-EphA2-Wechselwirkung auf die Proliferation 90 5.4 Einfluss der Eph-Familie auf die zelluläre Migration und Wundheilung 93 5.5 Zusammenfassende Betrachtung und Ausblick 106 6 ZUSAMMENFASSUNG 109 6.1 In deutscher Sprache 109 6.2 In englischer Sprache 111 7 LITERATURVERZEICHNIS 113 8 DANKSAGUNG 133 9 ANHANG 134 10 ERKLÄRUNGEN 138 10.1 Erklärungen zur Eröffnung des Promotionsverfahrens 138 10.2 Erklärung über die Einhaltung der aktuellen gesetzlichen Vorgaben im Rahmen der Dissertation 140
The Eph-family represents the largest subgroup of tyrosine kinase receptors. Both the Eph-receptors and the ephrin-ligands are membrane-associated proteins whose interaction is a way of contact-dependent communication that influences a wide variety of cellular functions. In scientific research the Eph-family is known for its impact on developmental and tumour-biology, whereas the role of Eph-receptors and ephrin-ligands in atherosclerosis and repair mechanisms is still not well understood. The aim of the present study was to examine the influence of the ligand ephrin-A1 and its interaction with its receptor EphA2 on endothelial proliferation and migration by performing cellcultural experimentation using HUVEC and HUAEC.:INHALTSVERZEICHNIS I ABKÜRZUNGSVERZEICHNIS V ABBILDUNGSVERZEICHNIS IX TABELLENVERZEICHNIS XI 1 EINLEITUNG 1 1.1 Die Rolle des Endothels in der Atherosklerose 1 1.2 Die Migration von Endothelzellen 6 1.3 Die Eph-Familie 9 1.4 Potentielle Funktionen der Eph-Familie in der endothelialen Proliferation und Migration 14 1.5 Zielstellung der Arbeit 16 2 MATERIAL 17 2.1 Geräte 17 2.2 Zellkulturmaterial 18 2.3 Verwendete Kulturmedien 18 2.4 Verwendete Zelllinien und E. coli Stämme 19 2.5 Verwendete Kit-Systeme 19 2.6 Verwendete Oligonukleotide 20 2.7 Verwendete Plasmide 21 2.8 Konstruierte Plasmide 23 2.9 Chemikalien 23 2.10 Puffer, Gele, Lösungen 23 3 METHODEN 24 3.1 Zellbiologische Methoden 24 3.2 Proteinbiochemische Methoden 28 3.3 Molekularbiologische und gentechnische Methoden 32 3.4 Scratch Assay 44 3.5 Beurteilung der Zellproliferation mittels BrdU-Inkorporation 44 3.6 Wundheilungsassay 45 3.7 Migrationsassay 48 3.8 Baculovirale Talin-RFP- und Aktin-GFP-Expression 49 3.9 Datenanalyse und statistische Auswertung 49 4 ERGEBNISSE 50 4.1 Molekularbiologische und proteinbiochemische Charakterisierung verschiedener Eph/ephrine in proliferierenden Endothelzellen 50 4.2 Molekularbiologische Charakterisierung von ephrin-A1, EphA2 und EphA4 unter Kontaktinhibition in HUVEC 56 4.3 Einfluss eines Zellzyklusarrests auf die Expression verschiedener ephrin-Liganden und Eph-Rezeptoren in Endothelzellen 58 4.4 Hemmung von ephrin-A1 und EphA2 mittels siRNA 63 4.5 Einfluss einer siRNA-vermittelten Hemmung von ephrin-A1 und EphA2 auf die Proliferation von HUVEC 65 4.6 Überexpression von ephrin-A1 mittels eines adenoviralen Vektors 67 4.7 Einfluss einer Überexpression von ephrin-A1 auf die Proliferation von HUVEC 68 4.8 Reinitiation von Proliferation und Migration in HUVEC mittels Scratch Assay 69 4.9 Einfluss der Modulation des ephrin-A1- sowie EphA2-Gehaltes auf das Wundheilungsverhalten von HUVEC 73 4.10 Charakterisierung des Migrationsverhaltens von HUVEC unter veränderter ephrin-A1-Expression mittels live cell imaging 75 4.11 Einfluss einer veränderten ephrin-A1-Expression auf den EphA2-Phosphorylierungsstatus 78 4.12 Grenzzonenverhalten von HUVEC an ephrin-A1-beschichteten Oberflächen 80 4.13 Fluoreszenzmikroskopische Charakterisierung fokaler Adhäsionen und des Aktin-Zytoskelettes unter modulierter ephrin A1 Expression 81 5 DISKUSSION 84 5.1 Die Eph-Familie im Kontext der Reendothelialisierung 84 5.2 Einfluss des Eph/ephrin Systems auf das Proliferationsverhalten von Zellen 86 5.3 Auswirkungen einer ephrin-A1-EphA2-Wechselwirkung auf die Proliferation 90 5.4 Einfluss der Eph-Familie auf die zelluläre Migration und Wundheilung 93 5.5 Zusammenfassende Betrachtung und Ausblick 106 6 ZUSAMMENFASSUNG 109 6.1 In deutscher Sprache 109 6.2 In englischer Sprache 111 7 LITERATURVERZEICHNIS 113 8 DANKSAGUNG 133 9 ANHANG 134 10 ERKLÄRUNGEN 138 10.1 Erklärungen zur Eröffnung des Promotionsverfahrens 138 10.2 Erklärung über die Einhaltung der aktuellen gesetzlichen Vorgaben im Rahmen der Dissertation 140

Eph receptors and their ephrin ligands play important roles in guiding mouse and Xenopus cranial neural crest (CNC) cells to their destinations. My objective was to determine if Ephs and ephrins also regulate avian CNC pathfinding. By double labeling for Eph or ephrin RNA and a neural crest marker protein, I was able to clearly distinguish neural crest from ectoderm and head mesenchyme and show that avian CNC cells express EphA3, 4, and 7 and EphB 1 and 3 and migrate along pathways bordered by non-neural crest cells expressing ephrin-B 1. Surprisingly, avian CNC cells also express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Consistent with these findings, explanted avian CNC cells are labeled by both ephrin-B I and EphB2 Fc fusion proteins. Given the choice between growing out onto substrate-bound fibronectin (FN) or FN plus clustered Fc protein in the stripe assay, these cells show no preference for either condition. Conversely, given the choice between FN or FN plus clustered ephrin-B1 or EphB2 Fc fusion protein, the cells strongly localize to stripes containing only FN. This response is mitigated in the presence of soluble ephrin-B1/Fc or EphB2/Fc, but not in the presence of soluble Fc alone. These findings show that avian CNC cells have a mutually exclusive distribution with non-neural crest cells expressing ephrin-B 1 and EphB2 RNA in situ and are repelled from ephrin-B1 and EphB2 protein in vitro, suggesting that their migration is guided by both forward signaling through a variety of Eph receptors as stimulated by ephrin-B1 and reverse signaling through ephrin-B2 as stimulated by EphB2. I further explore the phylogeny of Ephs and ephrins and show that these genes diversified at different times in evolutionary history, such that the ancestral chordate likely had a single receptor for two different ligands.