The tumour suppressor p53 actually exists as 9 protein isoforms. Among them, D133p53a, b and g result from the use of an alternative promoter and lack the N-terminal transactivation domain. In addition to its multiple functions maintaining cell integrity, p53 is also able to block angiogenesis, a process strongly contributing in tumour development. Here I have examined the role of p5 isoforms in the regulation of angiogenesis and tumor progression. I also focused my work on FGF-2 regulation by p53. In a first part, full length p53 (p53) and/or D133p53 isoforms were selectively knocked-down with siRNAs in human glioblastoma cells U87. Conditioned medium produced by tumour cells knocked- down for D133p53 inhibited endothelial cell - EC - migration and tubulogenesis. Furthermore, in the chicken chorioallantoïd membrane CAM, D133p53 knockdown gave rise to smaller tumours devoid of vessels, whereas, in mice, it strongly inhibited tumour growth. Interestingly, the double knockdown of p53 and D133p53 also slowed town tumour growth in mice. Taqman Low Density Array revealed distinct gene expression profiles of pro and anti-angiogenic factors regulation following D133p53 and/or p53 knockdown. In particular, D133p53 knockdown resulted in specific down-regulation of Angiogenin and hepatocyte growth factor, whereas the main angiogenic factors FGF-2 and VEGF-A were not significantly affected. Secondly we investigated the regulation of FGF-2 by p53 and its isoforms D133p53 in a human osteosarcoma cell line U2OS, at translational, transcriptional and secretion levels. It resulted in a sophisticated mode of regulation mediated by a transient IRES-dependent translation inhibition of FGF-2. Our data reveal D133p53 isoforms as activators of angiogenesis and tumour progression, through a specific modulation of the angiogenic balance. These isoforms exhibit dominant-negative effect towards p53 but also intrinsic activities, while underlining the importance of considering D133p53 expression in cancers, as well as the potential antitumoural interest of drugs targeting this p53 isoform.

Tgf-β, Wnt, and Fgf signaling pathways are required for many developmental processes. Here, I investigated the requirement of these signaling pathways during tail regeneration and limb development in the Mexican axolotl (Ambystoma mexicanum). Using small chemical inhibitors during tail regeneration, I found that the Tgf-β signaling pathway was required from 0-24 and 48-72 hours post tail amputation (hpa), the Wnt signaling pathway was required from 0-120 hpa, and the Fgf signaling pathway was required from 0-12hpa. Tgf-β1 was upregulated after amputation and thus may mediate Tgf-β signaling pathway during tail regeneration. Both Smad-mediated and non-Smad mediated Tgf-β signaling were activated as early as 1hpa. Smad-mediated Tgf-β signaling via activated pSmad2 and pSmad3, and via phosphorylated Erk and Akt. Two different Tgf-β signaling pathway inhibitors, SB505124 and Naringenin, differentially regulated pSmad2, pSmad3, p-Erk, and p-Akt, while SB505124 and Naringenin both inhibited tail regeneration; only SB505124 reduced cell proliferation. Wnt/β-Catenin signaling was increased and was enhanced by Wnt-C59. Disruption of the Wnt signaling pathway directly or indirectly activated Erk and Akt signaling. Disruption of the Fgf signaling pathway decreased p-Erk and increased p-Akt. All three signaling pathways affected cell proliferation and mitosis during tail regeneration. The Wnt pathway inhibitor Wnt-C59 prevented forelimb bud outgrowth. The critical window for Wnt signaling regulating forelimb bud outgrowth was approximately developmental stage 40-42. Wnt signaling ligand Wnt3a and tight junction protein Zo-1 were expressed in the epidermis of the forelimb bud and both were down-regulated by Wnt-C59. Moreover, both Wnt and Fgf signaling pathways affected cell proliferation and mitosis of mesodermal cells during forelimb bud outgrowth. Overall, my results show that Tgf-β, Wnt, and Fgf signaling pathways are required for axolotl tail regeneration. All three pathways affect Erk and Akt signaling and guide cell proliferation and mitosis. The Wnt signaling pathway is required for forelimb bud outgrowth, and it appears to regulate expression of Wnt3a and Zo1, and control cell proliferation and mitosis of mesodermal cells underlying the forelimb epidermis. These data enrich understanding of signaling network dynamics that underlie tissue regeneration and vertebrate limb development.

<p>Angiogenesis can be defined as the formation of new blood vessels from pre-existing ones. Angiogenesis is required for development and maintenance of our vascular system and thus of fundamental importance to our existence. The endothelial cells that line the inside of the vessels de-differentiate, migrate, proliferate and re-differentiate during angiogenesis. Angiogenesis is tightly regulated, controlled by several angiogenic factors of various classes that promote angiogenesis but also by anti-angiogenic factors that counteract the effect of the pro-angiogenic factors. We have examined three factors involved in angiogenesis regulation, Vascular endotelial growth factor (VEGFR) -3, the matrix protein laminin-1 and the collagen XVIII derived fragment endostatin. </p><p>Five tyrosine phosphorylation sites in the cytoplasmic tail of VEGFR-3 were identified by phosphopeptide mapping (PPM). The data was confirmed by PPM using point-mutated receptors generated by site-directed mutagenesis.</p><p>Laminin-1 was found to promote angiogenesis in the chicken chorioallantoic membrane assay and in a synergistic fashion together with suboptimal levels of fibroblast growth factor 2 (FGF-2) in embryoid bodies. Laminin-1 also promoted endothelial tubular morphogenesis in vitro, and upregulated the expression of the endothelial differentiation marker Jagged-1. </p><p>Endostatin was shown to affect endothelial FGF-2-induced cell survival and morphogenesis. This was a result of direct binding to endothelial cells and induction of tyrosine phosphorylation of many proteins including the adaptor protein Shb. The apoptotic and morphogenic responses induced by endostatin was shown to be dependent on Shb. Further, endostatin inhibited endothelial migration and affected molecules implicated in migration. In particular, FGF-2 induced actin reorganization, and β-catenin regulation was modulated by endostatin. </p>

Angiogenesis can be defined as the formation of new blood vessels from pre-existing ones. Angiogenesis is required for development and maintenance of our vascular system and thus of fundamental importance to our existence. The endothelial cells that line the inside of the vessels de-differentiate, migrate, proliferate and re-differentiate during angiogenesis. Angiogenesis is tightly regulated, controlled by several angiogenic factors of various classes that promote angiogenesis but also by anti-angiogenic factors that counteract the effect of the pro-angiogenic factors. We have examined three factors involved in angiogenesis regulation, Vascular endotelial growth factor (VEGFR) -3, the matrix protein laminin-1 and the collagen XVIII derived fragment endostatin. Five tyrosine phosphorylation sites in the cytoplasmic tail of VEGFR-3 were identified by phosphopeptide mapping (PPM). The data was confirmed by PPM using point-mutated receptors generated by site-directed mutagenesis. Laminin-1 was found to promote angiogenesis in the chicken chorioallantoic membrane assay and in a synergistic fashion together with suboptimal levels of fibroblast growth factor 2 (FGF-2) in embryoid bodies. Laminin-1 also promoted endothelial tubular morphogenesis in vitro, and upregulated the expression of the endothelial differentiation marker Jagged-1. Endostatin was shown to affect endothelial FGF-2-induced cell survival and morphogenesis. This was a result of direct binding to endothelial cells and induction of tyrosine phosphorylation of many proteins including the adaptor protein Shb. The apoptotic and morphogenic responses induced by endostatin was shown to be dependent on Shb. Further, endostatin inhibited endothelial migration and affected molecules implicated in migration. In particular, FGF-2 induced actin reorganization, and β-catenin regulation was modulated by endostatin.

The vertebrate nervous system is a highly complex and intriguing structure with diverse functions. To understand the functional nervous system, we first have to be aware of how it is assembled during development. In this thesis the mechanism of early diversification and regionalisation necessary for subsequent formation of part of the nervous system, namely the telencephalon and the placodes, will be addressed. We have identified signalling molecules involved in the dorsoventral patterning of the telencephalon and we propose a mechanism for the induction and differential specification of the olfactory and lens placodes. The telencephalon is regionalised along the dorsoventral axis during development. The cells situated dorsally will give rise to the cerebral cortex while the ventral and intermediate cells are mainly progenitors for the basal ganglia. The cerebral cortex is associated with higher cognitive functions whereas the basal ganglia control movements. We provide evidence that dorsal and intermediate telencephalic cells are re-specified from cells with an intrinsic ventral character. Dorsal telencephalic cells are specified at stage 10 in chick, while the intermediate cells are specified a few hours later, at stage 14. The expression of Wnt and Fibroblast growth factors (Fgfs) coincides with the time point when the dorsal cells are specified, and we provide evidence that Wnt and FGF signals act in a sequential way to specify dorsal telencephalic cells. The retinoic acid (RA) synthesising enzyme Raldh3 is expressed in proximity to the telencephalon, and our result suggests that RA is both required and sufficient to induce intermediate telencephalic cell types. Additionally, Fgf8 is expressed in the anterior neural ridge and the ventral telencephalic cells require FGF signals that oppose RA to maintain their character. The olfactory and lens placodes contribute to the special sense organs associated with olfaction and vision, respectively. Olfactory and lens placodes are specified at gastrula stage in chick, and become spatially separated at the neural fold stage. We provide evidence that Bone morphogenetic protein (BMP) signalling is required for the induction of a pool of placodal progenitor cells. Furthermore, time of exposure to BMP signals plays a key role in the differential specification of the olfactory and lens placodes, where continued exposure to BMP signals promotes lens character at the expense of olfactory placodal cells.

The development of the nervous system is a complex process. Cell divisions, cell differentiation and signalling interactions must be tightly regulated. To comprehend the mature nervous system, we have to understand its assembly during development. Two main questions were addressed in this thesis: (1) how is the caudal part of the central nervous system specified and (2) how is the early development of the olfactory placode regulated? By using tissue and whole embryo assays in the chick, we identified signalling molecules involved in these processes and propose possible mechanisms for their function. The central nervous system is regionalized along its rostrocaudal axis during development. However, the mechanisms by which cells in the caudal part of the neuraxis acquire rostrocaudal regional identity have been unresolved. We provide evidence that at gastrula stages cells in the caudal neural plate are specified as cells of caudal spinal cord character in response to Wnt and FGF signals and that cells of rostral spinal cord and caudal hindbrain character only emerge later at neurulation stages in response to retinoic acid signalling acting on previously caudalized cells. In the hindbrain and spinal cord distinct motor neuron subtypes differentiate at precise rostrocaudal positions from progenitor cells. We provide evidence that cells in the caudal neural plate have acquired sufficient positional information to differentiate into motor neurons of the correct rostrocaudal subtype. The olfactory placode gives rise to all the structures of the peripheral olfactory system, which, in the chick consists of the olfactory nerve, the sensory epithelium, where the olfactory sensory neurons (OSN) are located and the respiratory epithelium, that produces the mucus. Several studies have addressed the role of signalling cues in the specification of OSNs but much less is known about the regulation of sensory versus respiratory patterning and the events controlling early neurogenesis in the developing olfactory placode. We show that by stage 14 the olfactory placode is specified to give rise to both cells of sensory and respiratory epithelial character. Moreover, cells of respiratory epithelial character require BMP signalling, whereas cells of sensory epithelial character require FGF signalling. We suggest a mechanism in which FGF and BMP signals act in an opposing manner to regulate olfactory versus respiratory epithelial cell fate decision. BMP signalling has also been implicated in the regulation of neurogenesis in the sensory epithelium, and we show that BMP signals are required for the generation of OSNs, because in the absence of BMP signalling cells in the sensory epithelium do not mature. Independently, we also analyzed the role of Notch signalling during early olfactory development both in vitro and in vivo and provide evidence that active Notch signalling is required to prevent cells in the olfactory placode from premature differentiation.

The vertebrate nervous system is extremely complex and contains a wide diversity of cell types. The formation of a functional nervous system requires the differential specification of progenitor cells at the right time and place. The generation of many different types of neurons along the rostro-caudal axis of the CNS begins with the initial specification of a few progenitor domains. This initial coarse pattern is refined by so-called secondary organizers arising at boundaries between these domains. The Isthmic Organizer (IsO) is a secondary organizer located at the boundary between the midbrain and the hindbrain. Although the function and maintenance of the IsO are well understood, the processes underlying its initial specification have remained elusive. In the present work we provide evidence that convergent Wnt and FGF signals initiate the specification of the IsO during late gastrulation as part of the neural caudalization process. The initial step in the generation of the nervous system is the division of the embryonic ectoderm into three cell populations: neural cells giving rise to the CNS, neural plate border cells giving rise to the peripheral nervous system, and epidermal cells giving rise to the outer layer of the skin. While the choice between neural and epidermal fate has been well studied, the mechanism by which neural plate border cells are generated is less well understood. At rostral levels of the neuraxis, the neural plate border gives rise to the olfactory and lens placodes, thickenings of the surface ectoderm from which sensory organs are derived. More caudally, the neural plate border generates neural crest cells, a transient population that migrates extensively and contributes to neurons and glia of the peripheral nervous system. How the early patterning of the central and peripheral nervous systems are coordinated has remained poorly understood. Here we show that the generation of neural plate border cells is initiated at the late blastula stage and involves two phases. During the first phase, neural plate border cells are exposed to Wnt signals in the absence of BMP signals. Simultaneous exposure to Wnt and BMP signals at this early stage leads to epidermal induction. Wnt signals induce expression of Bmp4, thereby regulating the sequential exposure of cells to Wnt and BMP signals. During the second phase, at the late gastrula stage, BMP signals play an instructive role to specify neural plate border cells of either placodal or neural crest character depending on the status of Wnt signaling. At this stage, Wnt signals promote caudal character simultaneously in the neural plate border and in the neural ectoderm. Thus, the choice between epidermal and neural plate border specification is mediated by an interplay of Wnt and BMP signals that represents a novel mechanism involving temporal control of BMP activity by Wnt signals. Moreover, the early development of the central and peripheral nervous systems are coordinated by simultaneous caudalization by Wnt signals.

Angiogenesis, the formation of new blood vessels from preexisting ones through creation of new vessel branch points by sprouting or vessel splitting, is an important part of tissue growth in both physiological processes like wound healing and pathological conditions such as cancer. Growth factors like VEGF-A, FGF-2 and PDGF-BB are involved in both types of angiogenesis. Screening for genes regulated by VEGF-A stimulation in endothelial cells revealed up regulation of the endothelial cell specific glycoprotein endocan. Endocan itself did not stimulate angiogenesis. VEGF was a specific inducer since FGF-2, PDGF-BB, HGF and EGF did not alter expression. The signaling molecule PI3K was a negative regulator of endocan expression. Endocan was expressed in tumor cells and vessels, suggesting that although endocan did not directly regulate angiogenesis it can serve as a marker for angiogenic tumors. In two models of wound healing angiogenesis, the chick extra-embryonal CAM assay and the mouse cornea assay, we observed that blood vessels grew into avascular areas as functional mural cell covered loops by elongation of preexisting vessels. Loop formation was simultaneous with contraction of the avascular matrix mediated by proto/myofibroblasts. Reducing the contractibility of the stroma reduced vessel ingrowth, showing that contraction was necessary for mediating and directing growth of the vascular loops. These findings suggest a model for biomechanical regulation of vascularization that is complementary to sprouting angiogenesis which is guided by gradients of growth factors. In defining the role of growth factors, in the CAM assay, we found that FGF-2 and PDGF-BB induced vessel ingrowth, while VEGF-A, EGF and HGF did not. TGF-beta reduced the effect of FGF-2. By use of specific receptor kinase inhibitors we found an absolute requirement VEGF- and PDGF-receptor activity for vascularization while FGF- and TGF-beta-receptor function was dispensable. This suggests that functional VEGF- and PDGF-receptors are needed for vessel elongation.

Au cours du développement embryonnaire, le destin d'une cellule est spécifié par l'expression de gènes dits de lignage. Le contrôle de l'expression de ces gènes est essentiel à la cohérence du développement embryonnaire. Pour savoir comment s'effectue ce contrôle, nous avons choisi un modèle de spécification dans le cerveau postérieur des vertébrés, le rhombencéphale. Au cours de son développement, cet organe comprend sept groupes de cellules homogènes, appelés rhombomères (r) et notés de r1 à r7, qui subissent des processus de spécification distincts. La voie de spécification de r3 et r5 est la mieux connue car elle est contrôlée par un facteur unique, Krox20. En l'absence de ce facteur, les cellules de r3 et r5 ne sont pas correctement spécifiées et voient leur destin neuronal modifié. Dans ce travail de thèse, nous dévoilons les mécanismes qui permettent de contrôler le nombre de cellules exprimant le gène Krox20, donc la taille de r3 et r5. Ces mécanismes contrôlent la dynamique de transcription de Krox20, en régulant l'activité de ses éléments régulateurs. Deux éléments, B et C, sont responsables de l'initiation de l'expression de Krox20 ; un troisième, noté A, l'amplifie et la prolonge grâce à une activité autorégulatrice. Nous montrons (i) que les cellules n'acquièrent l'identité r3/r5 que si elles activent l'élément A, (ii) que l'activation de l'élément A suit un mode tout-ou-rien, selon le niveau d'initiation de Krox20. Ces conclusions ont été obtenues par l'analyse d'un modèle mathématique, contraint par des données expérimentales obtenues chez le poisson-zèbre, et suffisamment résolutif pour décrire l'activation de A à l'échelle moléculaire.

The regenerative capacity of mature mammalian CNS neurons after axonal injury is severely limited by a variety of mechanisms. Retrograde repression is the continuous inhibition of the expression of growth phenotypes by tonic signals produced by target tissues and transmitted to the neuron cell body via retrograde axonal transport. Loss of target contact through axonal injury is thought to interrupt this retrograde signal and allow the up-regulation of growth-associated proteins. Most CNS neurons, however, possess many widespread axon collaterals, such that retrograde repression is maintained by intact sustaining collaterals even if some axons are injured.<p>In this project we investigated whether or not retrograde repression plays a role in limiting the expression of GAP-43 in transcallosal neurons. Because TCNs possess local axon collaterals to nearby cortex and project distal axons to homologous areas of contralateral cortex, we hypothesized that the simultaneous interruption of retrograde repressive signals from both ipsilateral and contralateral cortex would result in an up-regulation of GAP-43 expression in at least some TCNs.<p>We found that a bilateral infusion of a function blocking antibody to FGF-2 into the parietal cortex of rats using implanted osmotic mini-pumps resulted in a significant increase in the level of expression of GAP-43 mRNA in TCNs identified by retrograde fluorescent labeling. In contrast, neither ipsilateral or contralateral antibody infusions alone increased GAP-43 expression significantly compared to controls. The level of expression of GAP-43 in TCNs did not significantly increase after stereotactic callosotomy alone, but callosotomized animals treated with an ipsilateral infusion of anti-FGF-2 had levels of increased GAP-43 expression equivalent to those seen in animals that had received bilateral antibody infusions.<p>We conclude that FGF-2 provides a retrograde repressive signal for at least some mature mammalian TCNs, and that the expression of growth-associated proteins can be up-regulated in CNS neurons by simultaneously blocking retrograde repressive signals from all existing axon collaterals. The ability to alter the gene expression of mature CNS neurons in both normal and injured states through the targeted infusion of a pharmacological agent may have potential clinical implications in the future.