To some extent formation of embryoid bodies (EBs) from embryonic stem cells (ES) mimics the events during embryonal development and can be a useful model for studying lineage commitment. However, due to morphological and structural features, the effects of direct supplementation of a molecule of interest (MOl) on EBs in cell culture to study differentiation are often limited to the superficial layers. The use of biodegradable microparticles (MPs) to deliver the MOl directly within EBs might be a promising approach for controlling ES differentiation. This study aimed to develop a 3-D model combining cells and MPs loaded with osteo-inductive molecules including simvastatin and BMP2 for ES cell-derived osteogenesis within an EB model. The steps towards achieving this aim involved; i) establishing a reliable approach for producing human and / or mouse ES cell derived EBs; ii) evaluating the osteo-inductive potential for simvastatin iii) establishing a 3-D construct of ES cell derived EBs containing simvastatin loaded MPs followed by evaluation for osteogenesis through studying different markers during formation of /osteoblast- like cells/ bone-like tissue. It was found that mouse and human ES cells could readily form EBs in a controllable manner. Simvastatin pro and! or active drug were found to induce osteogenesis of ES cells in a 2-D environment over a concentration range of 0.1 and 1 ~M. A controlled simvastin release model from MPs loaded with either the prodrug or active drug was fabricated using an emulsion method. The MPs with average diameter of 12-13 urn were designed to be distributed within EBs and provide release of the MOl over a period of three weeks. A reliable method for creating 3-D constructs of ES cell derived EBs containing MPs was tested under static or with centrifugation as well as mass suspension with avidin coated- lyophilised MPs. In this study, improved incorporation of MPs within hES cells derived EBs has been presented by coating MPs with hES cell lysate before EB formation by ~ •.......................-- centrifugation method. EBs containing MPs loaded with osteo-inductive molecules (simvastatin prodrug, active drug or BMP-2) provided a robust and reproducible 3D model for osteogenesis and the lineage commitment of HUES- 7 cells into bone cells. Results were confirmed by positive expression of major osteogenic markers at the gene level (Runx-2, Osterix and osteocalcin) and protein level (Runx-2 and osteocalcin). Extracellular matrix (ECM) formation was confirmed by Picro-sirius red staining for collagen formation and matrix mineralisation by alizarin red staining. In conclusion, the model presented in this study mimics, to some extent, the spatiotemporal presentation of locally released growth factors of early development and can be beneficial for tissue engineering purposes through achieving better presentation of the molecule of interest within the multi-cellular EB model.

The anterior visceral endoderm (AVE) is a specialised subpopulation of the visceral endoderm (VE), a single layer simple epithelium that surrounds the extra-embryonic ectoderm and epiblast of the egg cylinder stage embryo. Initially induced at the distal tip of the egg cylinder, AVE cells undergo a stereotypic migration towards the prospective anterior, stopping at the interface between the underlying epiblast and extra-embryonic ectoderm (ExE). Previous research has shown that membrane enrichment of Dvl2 is present in the VE overlying the epiblast (Epi-VE). In this thesis I confirm the presence of planar cell polarity (pep) signalling in this region by assaying the subcellular localisation of additional core pep proteins Vangl2 and Daaml. I show that null embryos of the Nodal antagonist Lefty1 exhibit ectopic membrane enrichment of Dvl2 and a previously unreported AVE over-migration phenotype. Furthermore, using pharmacological inhibition of Nodal signalling I show that the TGF~ protein Nodal modulates pep signalling in the YE. Utilising DIe and confocal microscopy I perform detailed time-lapse analyses of the VE to quantify the dynamic cell behaviour and topology. Using this assay I show that wild-type embryos exhibit dynamic cell movement, which is regionally restricted to the Epi-VE. Analysis of Leftyl-/- and ROSA26lyn-Celsr-l mutants, both of which exhibit disrupted pep signalling and AVE over-migration phenotypes, indicates that normal VE dynamics and topology are disrupted. The results of this quantitation indicate that these mutants exhibit increased cell migration and neighbour exchange across the YE. These data show that regional restriction of movement is lost and results in the AVE over-migration phenotypes observed. Together these results show that regionally restricted pep signalling in the VE acts to modulate cell behaviour and topology, which in turn determines the regional restriction and normal end-point of AVE migration.

Cell migration is essential for embryonic development, it occurs in adult organisms during processes like wound healing and its misregulation contributes to pathological conditions such as metastasis. Despite this, most studies of cell migration have been undertaken in vitro. Ena/VASP proteins, believed to be actin anti-capping proteins, have been studied extensively in fibroblasts in vitro, and using Drosophila macrophages (hemocytes) within the developing embryo, the role of the Drosophila homologue of Mena, Ena, is investigated in vivo. Consistent with data from fibroblasts in vitro, Ena localised to regions of actin dynamics within migratory hemocytes, where this protein stimulated lamellipodial dynamics and positively regulated filopodial number and length. However, whilst overexpression of Ena/VASP proteins in fibroblasts reduced migration speeds, Ena overexpression in hemocytes dramatically increased migration speeds in three different assays. This positive regulation of migration speed closely resembled the increased motility of breast cancer cells that overexpress Mena and evidence presented here, suggests that this key difference may be explained by spatial constraints that are imposed upon cells within three dimensional environments. Indeed, such constraints prevented ruffling, a more detrimental form of retraction, in hemocytes in vivo. Furthermore, fibroblasts overexpressing Mena in vitro form membrane ruffles more frequently. Therefore Ena/VASP proteins drive migration by enhancing lamellipodial protrusion, but in certain environments these protrusions are lost as ruffles slowing migration. The method by which Ena regulates lamellipodial protrusion and migration speeds was then investigated: Ena increased Fascin-mediated actin bundling and the number of Fascin rich-actin bundles that coalesced. Analysis of individual actin bundles revealed that coalescence increased protrusion rate and that both protrusion rate and coalescence, increased cell migration speeds. This suggests that Ena facilitates an increase in cell migration by promoting the coalescence of Fascin bundles, and positions Ena as a key regulator of migration speeds in vivo.

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Οι έρευνες που αρχικά στόχευαν στην ανίχνευση του μηχανισμού δράσης της Δ9-τετραϋδροκανναβινόλης, του κύριου ψυχοδιεγερτικού συστατικού που εξάγεται από την ινδική κάνναβη, οδήγησαν τελικά στην ανακάλυψη ενός νέου νευρορυθμιστικού συστήματος, του ενδογενούς κανναβινοειδούς συστήματος. Το ενδοκανναβινοειδές σύστημα περιλαμβάνει μια σειρά από ενδογενείς, λιπιδικής φύσεως προσδέτες (κυρίως την ανανδαμίδη και την 2-αραχιδονοϋλγλυκερόλη), μεμβρανικούς υποδοχείς (CB1-brain type, CB2-immune type, αλλά και άλλους) που ανήκουν στην υπεροικογένεια των συζευγμένων με G πρωτεΐνες υποδοχέων, καθώς και τα ένζυμα εκείνα που είναι απαραίτητα για τη βιοσύνθεση και αποικοδόμησή τους. Στον ενήλικο εγκέφαλο τα ενδοκανναβινοειδή λειτουργούν ως παλίνδρομα σηματοδοτικά μόρια: απελευθερώνονται από μετασυναπτικά κύτταρα, ενώνονται με υποδοχείς που εντοπίζονται στους προσυναπτικούς νευρώνες και ρυθμίζουν την απελευθέρωση διαφόρων νευροδιαβιβαστών (κυρίως του ανασταλτικού γ-αμινοβουτυρικού οξέος, αλλά και του διεγερτικού γλουταμινικού οξέος). Οι κύριες επιδράσεις της τετραϋδροκανναβινόλης οφείλονται στη διέγερση αυτών των υποδοχέων και είναι ευφορία, διαταραχή πρόσφατης μνήμης και άλλων νοητικών λειτουργιών, ακινησία, αναλγησία, αύξηση της όρεξης. Στον αναπτυσσόμενο οργανισμό, η εντόπιση νωρίς κατά την εμβρυϊκή ζωή λειτουργικών CB1 υποδοχέων σε θέσεις στις οποίες δεν εκφράζονται στην ενήλικο ζωή (άτυπες θέσεις) αποτέλεσε μία από τις πρώτες ενδείξεις, οι οποίες οδήγησαν στην υπόθεση ότι το ενδοκανναβινοειδές σύστημα παίζει κάποιον/ους ρόλο/ους στην φυσιολογική εμβρυϊκή ανάπτυξη. Το ενδιαφέρον σχετικά με τη σπουδαιότητα της ύπαρξης φυσιολογικής σηματοδότησης μέσω του ενδοκανναβινοειδούς συστήματος προκειμένου να επιτευχθεί όχι μόνο φυσιολογική εμβρυογένεση, αλλά και μεταγεννητική ανάπτυξη, επικεντρώθηκε στο κεντρικό νευρικό σύστημα. Τα πειράματα που έχουν πραγματοποιηθεί μέχρι τώρα έχουν οδηγήσει στο συμπέρασμα ότι τα κανναβινοειδή συμμετέχουν σε όλες τις διαδικασίες της φυσιολογικής εμβρυϊκής ανάπτυξης του νευρικού συστήματος: πολλαπλασιασμό και δέσμευση των προγονικών νευρικών κυττάρων προς συγκεκριμένη κυτταρική σειρά, διαφοροποίηση των δεσμευμένων νευροβλαστών ή σπογγιοβλαστών, μετανάστευση των μεταμιτωτικών νευρώνων, μορφολογική και λειτουργική ωρίμανση των νευριτών, δημιουργία λειτουργικών συνάψεων, απόπτωση και μυελινοποίησηση των νευραξόνων. Σκοπός της εργασίας αυτής είναι η ανασκόπηση της διεθνούς βιβλιογραφίας σχετικά με τον φυσιολογικό ρόλο των κανναβινοειδών στην ανάπτυξη του εμβρύου και ιδιαίτερα στην ανάπτυξη του κεντρικού νευρικού συστήματος. Μετά από μία συνοπτική περιγραφή της φυσιολογικής εμβρυογένεσης του κεντρικού νευρικού συστήματος, ακολουθεί η ανάλυση του ενδογενούς κανναβινοειδούς συστήματος, καθώς και των σηματοδοτικών μονοπατιών που η ενεργοποίηση των υποδοχέων του διεγείρει. Στη συνέχεια αναπτύσσονται διεξοδικά όλες οι απόψεις περί συμμετοχής του συστήματος αυτού στην φυσιολογική εμβρυογένεση. Είναι γνωστό ότι η τετραϋδροκανναβινόλη περνά τον αιματοπλακουντιακό φραγμό, φτάνει μέχρι τον εγκέφαλο του εμβρύου σε σημαντικές συγκεντρώσεις και μπορεί επομένως να διαταράξει τη φυσιολογική σηματοδότηση από τους CB1 υποδοχείς, η οποία φαίνεται να είναι απαραίτητη για την δημιουργία του φυσιολογικού νευρικού συστήματος. Η κατανόηση των μηχανισμών μέσω των οποίων τα κανναβινοειδή συμμετέχουν στη φυσιολογική εμβρυογένεση μπορεί ερμηνεύσει τα νοητικά και κοινωνικά ελλείμματα που παρουσιάζουν απόγονοι γυναικών που κατά τη διάρκεια της κυήσεως έκαναν χρήση κάνναβης, κάποια από τα οποία διαρκούν μέχρι την ενήλικο ζωή. / Recent evidence suggests that the endogenous cannabinoid system emerges and is operative early during brain development and that CB1 receptors are located in areas that do not contain or have a small density of these receptors in adult brain. This was the first hint which led to research about the potential role of the endocannabinoid system during brain development. It is now well accepted that endocannabinoids, acting as epigenetic factors, regulate neural progenitor proliferation, commitment into neuroblasts or spongioblasts, as well as differentiation of commited cells, control of neurite outgrowth, neurotransmitter maturation and establishment of synaptic contacts. There is also a great interest in the role of cannabinoids as neuroprotective agents in the developing, as well as the adult brain, though their role as a general endogenous protection system is yet to be established. In utero exposure to tetrahydrocannabinol (THC) induces behavioral and cognitive deficits enduring into adulthood. Improving our knowledge on the molecular mechanisms of cannabinoid actions during brain development could lead to a better understanding of these deficits.

During embryonic development, precursor cells commit to specific cell fates in response to environmental cues through the establishment of lineage-specific gene expression programmes. Transcription factors are important downstream effectors of signalling pathways that initiate and maintain cell fate decisions. The haematopoietic transcription factor SCL (TAL-1) is an essential regulator of embryonic blood development. However, the exact stage at which SCL is required, its mechanisms of action, and its genomic targets are poorly understood. Characterising, jiow SCL functions - , during haematopoietic development will provide insights into how stern cells are specified. Using the embryonic stem cell/embryoid body (ES/EB) system to model early mouse development, we describe a critical role for SCL in mesoderm patterning. SCL is first expressed in PDGFRa+ FLK1+ mesoderm populations which contain lateral, paraxial and cardiac precursors. Through loss- and gain-of-function studies, we show that SCL drives lateral mesoderm specification and activates the haematopoietic programme in a direct DNA-binding independent manner, while actively repressing alternative mesodermal fates, specifically cardiac development, in a DNA-binding dependent manner. At a molecular level, we have identified direct genomic targets of SCL in Flk-1 + mesoderm populations. These include haematopoietic and cardiac transcription factors, cardiac-specific structural proteins, signalling proteins and general transcriptional repressors; thereby strengthening the dual function of SCL in mesoderm patterning. Finally, we have shown that the cardiac transcription factor GATA4 acts in a reciprocal manner, specifying cardiac precursors while repressing a lateral mesoderm fate. Collectively, this implicates SCL as a critical transcriptional regulator of cell fate decisions in early mesodermal precursors, employing distinct molecular mechanisms to impose a blood programme. Moreover, and extending earlier reports, we document the existence of an antagonistic cross-talk between haematopoietic and cardiac lineages during mesoderm patterning. In conclusion, this work offers a cellular and molecular platform to begin to dissect the network of genetic interactions involved in these developmental processes.