Bone Morphogenetic Protein (BMP) signalling is an evolutionary conserved pathway, which functions to regulate numerous developmental processes such as cell fate determination and cellular proliferation. In Drosophila melanogaster, the ortholog of the vertebrate BMP2/4 is Decapentaplegic (Dpp). The most extensively characterised role of Dpp signalling in Drosophila is embryonic Dorsal-Ventral patterning. In this developmental environment, the Dpp morphogen acts as a step gradient to specify different concentration thresholds for target gene activation. The resulting nested domains of target gene expression in the embryo cooperate to induce the formation and subsequent maintenance of a simple extra-embryonic tissue, the amnioserosa. The amnioserosa tissue acts as an ideal model tissue to study Dpp-regulated differentiation. This study aims to identify and validate new targets of Dpp signalling, which are required for determining cell fate and differentiation of the amnioserosa tissue during embryogenesis. Additionally, this study aims to identify new regulators of the core signalling pathway. The work presented here was performed using a two tiered approach to understand in more detail the processes that regulate BMP-responsive transcription and the downstream effects. Firstly, RNA-Sequencing was performed on embryos with ectopic Dpp signalling in the early embryo. BMP-responsive target genes were idenitifed as differentially expressed when compared to control embryos. Expression studies have validated novel Dpp target genes and the list of genes that are regulated by BMP signalling has now been expanded. It can be invoked that these genes are involved in specification and/or mainentance of the amnioserosa tissue. Furthermore, I have uncovered a putative multi-tiered mechanism that exists between the Dpp and EGF signalling pathways to thus ensure correct cell fate specification and fine tuning of the Dpp signal in the Drosophila embryo. To further investigate how BMP signalling mediates such transcriptional regulation, a genome-wide RNAi screen was designed and performed to identify novel regulators of BMP transcription. Analysis of the screen data has identified a putative link between BMP-regulated transcription and transcriptional effectors of the Hippo signalling pathway, Scalloped and Yorkie. The data presented here suggests a co-regulatory requirement of these transcription factors to mediate Smad-dependent transcription.

Η Οστεοαρθρίτιδα ή αλλιώς η εκφυλιστική νόσος των αρθρώσεων δεν είναι μια μόνο νόσος αλλά μάλλον το τελικό αποτέλεσμα διαφόρων παθήσεων των αρθρώσεων. Σε μεγαλύτερη ή μικρότερη έκταση χαρακτηρίζεται πάντοτε από εκφύλιση του αρθρικού χόνδρου και ταυτόχρονη ανάπτυξη νέου οστού , χόνδρου και συνδετικού ιστού . Η εν λόγω ανάπτυξη έχει ως αποτέλεσμα την αναδιαμόρφωση του περιγράμματος της άρθρωσης . Οι φλεγμονώδεις αλλοιώσεις στην αρθρική μεμβράνη είναι συνήθως μικρές και δευτεροπαθείς . Πρόκειται για μια νόσο η οποία εμφανίζεται στο 80% των ατόμων ηλικίας πάνω από 65 έτη , ως ακτινολογικό εύρημα , ενώ στο 25% αυτών θα παρουσιάζει συμπτώματα . Διάφοροι παράγοντες επηρεάζουν την πορεία της νόσου . Εκτιμάται ότι σε λίγα χρόνια η επίπτωση της νόσου θα είναι τόσο μεγάλη έτσι ώστε δε θα επαρκούν οι ορθοπαιδικοί να χειρουργούν τους ασθενείς !!! Λόγω αυτών επιχειρείται η κατανόηση των μοριακών παθογενετικών μηχανισμών οι οποίοι λαμβάνουν χώρα στην οστεοαρθρίτιδα (ΟΑ) . Ο χόνδρος της άρθρωσης είναι αυτός που εκφυλίζεται με αποτέλεσμα την παραμόρφωση της άρθρωσης . Για την διατήρηση της ακεραιότητας του αρθρικού χόνδρου υπεύθυνη είναι μια λεπτή ισορροπία μεταξύ αναβολικών και καταβολικών διεργασιών που αφορούν τόσο στα χονδροκύτταρα όσο στην εξωκυττάρια θεμέλια ουσία . Η εκφύλιση του εκτελείται από αυξημένη δραστηριότητα των μεταλλοπρωτεασών του στρώματος (Αγκρεκανάσες) καθώς και των υπολοίπων πρωτεολυτικών ενζύμων τα οποία επάγονται από κυτοκίνες ( Il-1 , TNF-a) ή/και προϊόντα αποικοδόμησης της θεμέλιας ουσίας .Αν η εκφύλιση και η καταστροφή του χόνδρου είναι μεγαλύτερη από την παραγωγή νέας θεμέλιας ουσίας από τα χονδροκύτταρα τότε οδηγούμαστε στην ΟΑ . Νέα δεδομένα και απόψεις έρχονται συνεχώς στην επιφάνεια τα οποία υποστηρίζουν την συμμετοχή τόσο του αρθρικού υμένα όσο και του υποχόνδριου οστού στην ανάπτυξη της οστεοαρθρίτιδας . Ο κύριος στόχος αυτής της βιβλιογραφικής εργασίας είναι η λεπτομερής περιγραφή όλων των μορίων και των μονοπατιών που οδηγούν στην ενεργοποίηση των αγγρεκανασών , συνεπώς στην ανάπτυξη της οστεοαρθρίτιδας , την παράθεση των στοιχείων που εμπλέκουν , εκτός του αρθρικού χόνδρου , τον αρθρικό υμένα και το υποχόνδριο οστό στην παθογένεια της , καθώς επίσης τα διάφορα cross talks τα οποία υπάρχουν ανάμεσα στην αναβολική (σύνθεση) και καταβολική (καταστροφή) πορεία του χόνδρου , παρέχοντας έτσι μια καινούρια βάση για μελλοντική ερεύνα και ανακάλυψη νέων φαρμάκων τόσο προληπτικών όσο και θεραπευτικών της οστεοαρθρίτιδας . / -

No / Bone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

The mangrove killifish, Kryptolebias marmoratus, has the potential of becoming a strong model organism for a range of biological disciplines thanks to its ability to self-fertilise, a process only known to occur in invertebrate animals until its discovery. Selfing, a natural occurrence in this species, has lead to the formation of clonal lineages composed of highly homozygous individuals. The aim of this thesis was to further establish K. marmoratus in the field of developmental biology by providing an information infrastructure to help advance research on this peculiar animal and further promote its place in the pantheon of model organisms. To do so, I first set out to standardise K. marmoratus embryology by providing defined developmental stages with clear visual representations of key embryonic structures. This staging series is an essential tool that will ensure repeatability and consistency within and across different laboratories. Secondly, I examined several techniques for embryonic manipulation and for imaging that can be used in an array of experimental designs. Using these techniques I demonstrated microinjection of embryos by monitoring the yolk syncytial layer and its nuclei, and time-lapse analyses of the yolk surface during embryonic development. Finally, I applied the knowledge gained from my first two studies and examined Bmp signalling in K. marmoratus embryos and its influence on body patterning. By inhibiting this pathway, I found a new phenotype characterised by an extremely short and split body axis. These data highlighted the importance of studying known signalling pathways in unknown organisms as species-specific differences may improve our understanding of fundamental developmental processes. This thesis demonstrates that with its easily obtainable and manipulated embryos, K. marmoratus can be used for embryological research in the same light as other model organisms such as zebrafish or medaka. The rising amount of information on mangrove killifish will help further take advantage of this unique and intriguing species, and supports the use of this hermaphroditic vertebrate as a strong comparative model in developmental biology.

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (β-catenin/Lef1) markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways.

TGFβ signalling via Smad transcription factors is essential for axis patterning and subsequent cell fate specification during mammalian embryogenesis. However, the cellular and molecular mechanisms have been difficult to characterise in vivo due to early embryonic lethality of mouse mutants and redundant functional activities. Here I show that combined deletion of closely related Smad2 and Smad3 in mouse embryonic stem cells impairs induction of lineage specific gene expression during differentiation, while extra-embryonic gene expression is up-regulated. Preliminary data suggest that the underlying mechanism of this differentiation defect reflects the inability of Smad2/3^-/- cells to establish lineage priming. Collectively, these findings identify novel downstream target genes controlled by Smad2/3 and an absolute requirement for Smad2/3 during embryonic differentiation. TGFβ signalling via Smad1 and Smad4 is essential for induction of the transcription factor Blimp1 required for primordial germ cell specification. The direct upstream regulators of Blimp1 are unknown, but T-box factors have recently been suggested to play a role. In a second project, I performed tissue- specific ablation of the T-box transcription factor Eomes as well as components of the TGFβ signalling pathway in either the visceral endoderm or the epiblast to examine tissue-specific functions for Blimp1 induction. I show that Eomes and Smad2 functions in the visceral endoderm as well as Eomes function in the epiblast are dispensable for Blimp1 induction, but rather are required to restrict Blimp1 induction to posterior epiblast cells. In contrast, epiblast-specific Smad4 or Smad1 mutants fail to robustly induce Blimp1 in the epiblast. My preliminary analysis suggests that competence to induce primordial germ cell fate is dependent on the interplay of Smad2/Eomes functions in the visceral endoderm and Smad1/4 functions in the epiblast. Collectively, this thesis provides insight into the transition from pluripotency to cell fate specification in the mammalian embryo that is impossible to obtain from human embryos in vivo.

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (¿-catenin/Lef1 markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways. / University of Bradford, NIH and BBSRC.

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

Chez les vertébrés, une première ébauche du système nerveux central à partir du neurectoderme est obtenue par la neurulation. Ce processus mène à la formation du tube neural (TN) à partir de la plaque neurale. La neurulation est coordonnée avec l’induction d’une population de cellules multipotentes aux bordures latérales de la plaque neurale: les cellules de la crête neurale (CCNs). Le gène Pax3 encode un facteur de transcription qui est essentiel pour la formation du TN et des CCNs. Une petite région régulatrice d’environ ~250pb dans le promoteur proximal de Pax3, appelée NCE2, est suffisante pour récapituler l’induction de Pax3 ainsi que sa restriction aux bordures latérales de la plaque neurale. Le NCE2 de Pax3 est connu pour intégrer des signaux instructifs antéropostérieur (AP) provenant de la voie Wnt, via les protéines CDX (CDX 1, 2, 4), pouvant induire l'expression de Pax3 dans la plaque neurale postérieure (PNP). Nous avons démontré ici que, en plus des signaux AP, le NCE2 de Pax3 intègre des signaux instructifs dorsoventraux (DV) provenant de la voie BMP, via ses effecteurs SMAD1/5. Nos résultats indiquent que les protéines SMAD1/5 pourraient être le cofacteur manquant dans le contrôle CDX-dépendant de l’expression de Pax3 et que ce serait ces protéines qui permettraient de conférer le patron d’expression restreint de Pax3 aux bordures latérales de la PNP. Pour étayer cette affirmation, nous fournissons de nouvelles preuves que l’activité de BMP-SMAD1/5 sur l’expression de Pax3 est médiée par les CDX. Comme des défauts affectant la formation du TN et des CCNs sont à la base de plusieurs syndromes génétiques et malformations congénitales chez l’humain, nos résultats offrent ainsi une meilleure compréhension des mécanismes moléculaires sous-tendant ces pathologies. / In vertebrates, a first draft of the central nervous system from the neurectoderm is obtained by neurulation. This process leads to the formation of the neural tube (NT) from the neural plate. Neurulation is coordinated with the induction of a population of multipotent cells at the neural plate border: neural crest cells (NCCs). The Pax3 gene encodes a transcription factor that is essential for the formation of the NT and NCCs. A small regulatory region of ~250bp in the proximal promoter of Pax3, called NCE2, is sufficient to recapitulate the induction of Pax3 and its restriction to the lateral borders of the neural plate. The Pax3NCE2 is known to incorporate anterior-posterior (AP) instructive cues from the Wnt pathway, via CDX proteins (CDX1, 2, 4), which can induce the expression of Pax3 in the posterior neural plate (PNP). We have demonstrated that, in addition to the AP cues, Pax3NCE2 integrates instructive dorsal-ventral (DV) cues from the BMP pathway, via SMAD1/5 proteins. Our results indicate that SMAD1/5 proteins could be the missing co-factor in the CDX-dependent expression of Pax3 that restrict Pax3 expression to the lateral borders of the PNP. To support this assertion, we provide further evidence that the activity of BMP-SMAD1/5 on the expression of Pax3 is mediated by CDX proteins. As defects affecting the formation of the NT and NCCs are the basis of many genetic syndromes and birth defects in humans, our results provide a better understanding of the molecular mechanisms underlying these pathologies.