京都大学 / 新制・課程博士 / 博士(医学) / 甲第23417号 / 医博第4762号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一特定拠点, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cancers often resurrect embryonic molecular programs to promote disease progression. In melanomas, which are tumors of the neural crest (NC) lineage, a molecular signature of the embryonic NC is often reactivated. These NC factors have been implicated in promoting pro-tumorigenic features like proliferation, migration and therapy resistance. However, the molecular mechanisms that establish and maintain NC identities in melanomas are largely unknown. Additionally, whether the presence of a NC identity has any clinical relevance for patient melanomas is also unclear. Here, using comparative genomic approaches, I have a) identified a novel role for GDF6-activated BMP signaling in reawakening a NC identity in melanomas, and b) identified a NC signature as a clinical predictor of melanoma progression. Like the genomes of many solid cancers, melanoma genomes have widespread copy number variations (CNV) harboring thousands of genes. To identify disease-promoting drivers amongst such huge numbers of genes, I used a comparative oncogenomics approach with zebrafish and human melanomas. This approach led to the identification of a recurrently amplified oncogene, GDF6, that acts via BMP signaling to invoke NC identities in melanomas. In maintaining this identity, GDF6 represses the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, allowing melanoma cells to remain undifferentiated and survive. Functional analysis in zebrafish embryos indicated a role of GDF6 in blocking melanocyte differentiation, suggesting that the developmental function of GDF6 is reiterated in melanomas. In clinical assessments, a major fraction of patient melanomas expressed high GDF6, and its expression correlated with poor patient survival. These studies provide novel insights into regulation of NC identities in melanomas and offer GDF6 and components of BMP pathway as targets for therapeutic intervention. In additional studies, I wanted to test whether a broader NC identity in melanomas had any clinical relevance. In these studies, I performed transcriptome analysis of zebrafish melanomas and derived a 15-gene NC signature. This NC gene signature positively correlated with the expression of SOX10, a known NC marker in human melanomas. Patients whose melanomas expressed this signature showed poor overall survival. These findings identify an important predictive signature in human melanomas and also illuminate the clinical importance of NC identity in this disease.

La migration cellulaire est un phénomène essentiel au développement, à l'immunité et à la cicatrisation. Pourtant, l'activation des programmes de migration en dehors des situations physiologiques peut avoir des effets néfastes. Par exemple, la migration cellulaire permet aux cellules d'une tumeur primaire d'envahir de nouveaux territoires et d'installer des tumeurs secondaires ou métastases. Lorsqu'une migration cellulaire est initiée à partir d'un tissu épithélial, ces cellules doivent acquérir des caractéristiques mésenchymateuses. Pour cela, elles diminuent leur adhérences cellule-cellule, perdent leur polarité apico-basale, réorganisent leur cytosquelette, changent d'adhérence à la matrice et modifient la composition et l'organisation de la matrice. C'est ce qu'on appelle la transition épithélio-mésenchymateuse (TEM). La famille des Métalloprotéinases Matricielle (MMP) est connue pour participer au remodelage de la matrice. Les MMPs sont au nombre de 25 et sont sécrétées ou membranaires. L'une de ces MMP membranaires est MMP14 ou MT1-MMP. Elle participe à la migration physiologique et pathologique via la dégradation de composants de la matrice. Elle dégrade également des protéines non matricielles sécrétées ou membranaires. De plus, MMP14 agit indépendamment de son activité catalytique en régulant par exemple l'activation de petites GTPases, de voies de signalisation et en contrôlant l'expression de gênes. Cependant, beaucoup d'études sur MMP14 ont été faites in vitro et ex vivo et il n'est pas clair si toutes les fonctions de MMP14 sont retrouvées in vivo. Plus spécifiquement les fonctions possibles de MMP14 dans la TEM et la migration in vivo sont encore mal définies. Nous proposons d'utiliser les crêtes neurales (CN) de l'embryon de poulet comme modèle pour étudier MMP14 au cours de la TEM et de la migration in vivo. Les CN sont des cellules embryonnaires retrouvées dans la partie dorsale du tube neural. Les CN réalisent une TEM pour quitter le tube neural avant de parcourir de longues distances et donner de nombreux types cellulaires. Les CN se séparent en deux populations, les CN céphaliques retrouvées dans la tête et les CN troncales dans le reste de l'embryon. Ces deux populations de CN réalisent des TEM différentes, avec une TEM rapide et massive pour les CN céphaliques et plus lente et en continue pour les CN troncales. Même si ces TEM sont différentes, elles présentent une diminution des jonctions cellulaires, une perte de la polarité apico-basale, un changement d'adhérence à la matrice et une réorganisation de la matrice. Une particularité des CN troncales est la localisation du noyau en position basale de l'épithélium juste avant la sortie du tube neural. Plusieurs substrats de MMP14 sont retrouvés dans la TEM et la migration des CN et une étude a montré par PCR la présence de l'ARNm de MMP14 dans les CN céphaliques de poulet. L'objectif de la thèse est d'explorer la fonction de MMP14 au cours de la TEM et de la migration des CN. Nous avons montré que MMP14 est exprimée dans les deux populations de CN au cours de la TEM et de la migration. / Cell migration is an essential event during embryonic development, immunity and wound healing. Furthermore, the activation of migration program in non-physiologic conditions can have side effects. For example, cell migration promotes invasion of primary tumor cells in new territories and the formation of secondary tumors or metastasis. When an epithelial tissue initiates migration, epithelial cells need to gain mesenchymal attributes. To this end, they decrease their cell-cell adhesions, loss their apico-basal polarity, reorder their cytoskeleton, change their matrix adhesions and modify the matrix composition and organization. This event is named epithelial-mesenchymal transition (EMT). The family of Matrix Metalloproteinase (MMP) is known to reshape the matrix. MMP family is composed of 25 members which are secreted or linked to the membrane. One of the membrane-bound MMP is MMP14 or MT1-MMP. MMP14 is known to promote physiological and pathologic cell migration by inducing degradation of numerous matrix components. MMP14 cleaves also non-matrix proteins which are secreted or membrane-bound. Moreover, MMP14 can act independently of its catalytic activity for example in the regulation of small GTPases, signaling pathway and in gene expression control. However, the vast majority of MMP14 related studies were conducted in vitro or ex vivo and it is not clear whether some of its functions occur in vivo. More specifically, MMP14's putative functions in EMT and migration are still ill-defined. We propose to use the Neural Crest (NC) of chick embryo as model to study MMP14 during in vivo EMT and migration. NC is an embryonic cell population located in the dorsal part of the neural tube. NC cells realize an EMT to leave the neural tube before performing a long-distance migration and producing a myriad of cell types as neurons, bones and cartilages of the face and pigment cells. NC cells are divided in two populations, the cephalic NC in embryo's head and the trunk NC in the posterior part. The cephalic NC perform a fast and massive EMT while the trunk NC's EMT is slower and continuous. Although the EMT are different, they conserve common characteristics with a decrease of cell junctions, a loss of the apico-basal polarity, a change of matrix adherence and a rearrangement of the matrix. One particularity of trunk NC is the epithelium basal position of the nucleus just prior their exit from the neural tube. Many MMP14's substrates are found during NC EMT and migration and a study suggested by PCR that chick cephalic NC express MMP14 mRNA. The goal of this thesis is to explore the function of MMP14 during chick NC EMT and migration. Our results show that MMP14 is expressed by the two populations of NC during EMT and migration. Moreover, MMP14 cell localization changes from apical to basal during EMT. Loss of function experiments show that MMP14 is needed for NC EMT. Our rescues with various MMP14 versions indicate that: 1/ the cytoplasmic domain is not essential, 2/ the extracellular domain is needed and 3/ the catalytic activity is not required for EMT. MMP14 is involved in the control of cell junctions by a switch between cadherin-6B and cadherin-7 but not in the remodeling of the matrix during NC EMT. We have also showed that MMP14 is necessary for the change of cell polarity during EMT. Furthermore, we have showed that MMP14 is needed for the formation of matrix adherence. In conclusion, our study shows that MMP14 is involved in NC EMT and migration and that NC are a good model to investigate MMP14 function in vivo.

Maternal consumption of ethanol during pregnancy contributes to a set of pathologies, grouped together as the fetal alcohol spectrum disorders, affecting as many as 5% of live births in the United States annually. Ethanol acts widely in the developing embryo, affecting many tissues, but causing deficits in neuronal and neural crest populations particularly. These deleterious effects cause archetypical craniofacial expression and neurological deficits, including microcephaly and neuronal dysfunction. Severity of symptoms is linked to frequency of maternal alcohol consumption as well as the maximum blood alcohol concentration reached by the mother. The teratology of ethanol has been widely researched over the last four decades, with the link between the neural crest pathology and the fetal alcohol spectrum phenotype becoming clearer. Animal model studies have managed to replicate many of the symptoms seen in humans afflicted with fetal alcohol spectrum disorders, and have allowed us to elucidate the biochemical mechanisms behind the disease. There is no singular pathway responsible for the fetal alcohol spectrum disorders: over half a dozen models of dysfunction have been identified, and ethanol’s ability to react with a series of targets means that more pathways are likely to be discovered. Current theories regarding the effects of ethanol on the neural crest have implicated apoptosis of the cephalic neural crest, mediated by G-protein coupled receptors, activation of a phospholipase C pathway, and subsequent release of intracellular calcium; perturbations of the actin cytoskeleton leading to migration dysfunction of neural crest cells in the developing neural tube; lack of functional trophic molecules, specifically Shh, likely due to dysfunction of the cholesterol biosynthetic pathway; lack of retinoic acid production; oxidative stress, production of reactive oxygen species, and iron dysregulation; and genetics, which seems to confer greater susceptibility and resistance to ethanol in certain individuals. Ultimately, a global model for ethanol’s actions on the developing fetus eludes researchers, as do any potential treatments, and more research is required to further elucidate ethanol’s teratogenic mechanism.

La neurofibromatose type1 (maladie de Von-Rechlinghausen) est une affection autosomique dominante, causée par des mutations polymorphes du gène NF1, dont la protéine, la Neurofibromine, agit comme un suppresseur de tumeur en opérant une contrôle négatif des protéines de RAS. D’un point de vue embryologique, cette maladie affecte les dérivés de la crête neurale (CN), une structure embryonnaire pluripotente, capable de générer des dérivés variés tels que des neurones, des cellules gliales, périvascullaires, squelettiques et pigmentaires. Les cellules de la CN subsistent aussi chez l’adulte, à l’état de cellule souches, pouvant être impliqués dans des processus régénératifs. Toutefois, lorsque leur programme morphogénétique est altéré, elles peuvent générer des processus tumoraux, à l’origine de tumeurs multiples dans la peau, les nerfs (tumeurs bénignes et malignes des gaines nerveuses, neurofibromes,) et le cerveau (50% des cas de tumeurs cérébrales avec un tiers de gliomes des voies optique sont cancéreuses). La compréhension des mécanismes de cette maladie est limitée par la faible corrélation qui existe entre génotypes et phénotypes, à savoir l’adéquation entre la nature hautement polymorphe des anomalies génétiques et la diversité des manifestations cliniques. L’objectif de l’étude est d’analyser les conséquences de l’invalidation du gène NF1 sur le comportement des cellules de la CN (CCN), leur prolifération, leur capacité de migration et leur potentiel de différenciation, chez un modèle expérimental. De plus, nous tentons d’élucider l’impact des modulations épigénétiques de l‘activité du NF1.Nous avons développé un système qui permet l’inactivation totale du gène NF1 dans les cellules de la CN spécifiquement en utilisant des molécules d’ARN interférent (silencing) transfectées par éléctroporation bilatérale dans les CCN, au stade précoce de la neurulation, en utilisant l’embryon de poulet comme modèle expérimental. Suite à l’invalidation du gène NF1, nous avons obtenus des déficits multi-systémiques qui consistent principalement en des altérations de la gangliogénèse céphalique, avec des phénotypes gliomateux, mais aussi des défauts périvasculaires qui affectent tant les parois adventitielles des artères branchiales, que les péricytes des capillaire faciaux et cérébraux, associés des asymétries faciales et des formations néoplasiques intra-cérébrales. Précocement, nous montrons que ces déficits peuvent être corrélés aux altérations du comportement migratoire, prolifératif et apoptotiques des cellules de la CN.Parallèlement, nous avons cherché à déterminer l’implication des régulations épigénétiques sur l’activité de NF1. Nous nous sommes focalisé sur l’activité des Histones Désacétylases (HDAC), qui contrôlent la configuration chromatinienne. Il s’avère que les transcrits de la classe I de famille des HDACs, les HDAC1, 2 et 8, normalement accumulés dans les CCN au cours de leur migration et selon un patron d’expression spatial et temporal similaire à celui de NF1, présentent des variations significatives suite au silencing de NF1. Nous avons testé l’inactivation sélective de ces gènes; Ainsi, nous montrons que l’invalidation de HDAC8 seule, permet de reproduire les altérations des phénotypes vasculaires observés chez les embryons hypomorphes pour NF1. Qui suggère un rôle prépondérant de HDAC8 dans la régulation de la vasculogenèse et de la différentiation des CCN en péricytes. Qui pourrait être par l’activation ectopique des gènes Sox9 soutenant la transdifférenciaton pathologique des péricytes en processus gliomateux ou en calcifications intracérébrales. / The neurofibromatosis-type 1 (NF1) (Von Recklinghausen disease) is an autosomal disorder, which stems from misrgulation of Neurofibromin (NF1), a gene encoding a tumour-suppressor protein which acts as a negative regulator of RAS proteins. Mutations of NF1 are causally linked to many types of tumours located in skin, nerves, but also in the brain (intra- cerebral tumours and gliomas). NF1 patients have a high risk of developing both benign and malignant tumours. The diversity of deficits and the nature of cellular lineages attribute all these tumoral manifestations to deregulation of neural crest cell (NC) derivatives. The NC is a multipotent stem cell population that contributes to a variety of cell types in vertebrate embryo, which include skeletogenic, glial, pigment cells as well as pericytes. In order to understand the pathologic process of this disease, it is essential to analyze the molecular mechanisms involved in the survival, proliferation and differentiation of NC.Our objectives are therefore to gain insights into the molecular cascade responsible for the diversity of NC derivatives at cephalic level. We opt for a drastic approach consisting in eradicating NF1 activity from NC at the beginning of their migration. In our experimental model, we can analyze developmental interactions of NC and the epigenetic regulation of the NF1 gene, at their level. Espically class1 Histone deacetylases (HDAC) family of molecules. So we have developed a system which allows complete inactivation of the NF1 gene in NC specifically using interfering RNA molecules (silencing) transfected by electroporation in the bilateral NC, during the early stage of neurulation, using the chick embryo as an experimental model.We show that HDAC8 inactivation can reproduce the alterations of vascular phenotypes observed in NF1 hypomorphic embryos. Suggesting an important role of HDAC8 in regulating vasculogenesis and differentiation of pericytes NC. That could be by ectopic activation of Sox9 gene supporting the pathological transdifférenciaton pericytes in gliomateux process or intracerebral calcifications.

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Student Number : 9502128Y - MSc dissertation - School of Anatomical Sciences - Faculty of Science / Retinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10-5M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.

The migration of the cranial neural crest is an essential part of cranio-facial development in every vertebrate embryo. The cranial neural crest (CNC) is a transient population of cells that forms the lateral border of the anterior neural plate. In the tailbud stage Xenopus embryo, the neural crest cells delaminate from the neural tube, and undergo a large-scale migration from the dorsal to ventral region of the embryo. The CNC travels along distinct pathways, and populates specific regions of the embryos face. Once the CNC ceases migrating, it differentiates into a variety of tissues that are essential for cranio-facial structure and function. Some of these tissues include bones, muscle, cartilage, and ganglia. The CNC receives a concert of signals from neighboring tissues during and after CNC migration as well as signals transmitted among CNC cells, which act together to determine the fate of each CNC cell. Therefore, the proper migration of the CNC is an essential part of cranio-facial development. What molecules are important for the process of CNC migration? As one might imagine, a milieu of different molecules and interactions are essential for this complicated embryological process to occur. The work presented in this dissertation will focus on the role of a cell adhesion molecule that is important for Xenopus CNC migration. Typically, the amount of cell adhesion decreases within tissues undergoing migration. This behavior is essential to allow fluidity within the tissue as it moves. However, cell adhesions are fundamental for cell migration to occur because the moving cells need a platform on which to mechanically propel themselves. These interactions can occur between the migrating cell and extracellular matrix molecules (ECM), or can happen between cells. The cranial neural crest utilizes both cell-ECM and cell-cell interactions during the process of migration. The amount of cell adhesion mediated by either of these mechanisms will depend on where the cell is located within the CNC. Cells located at the periphery of the CNC tissue, which is surrounded by a matrix of ECM, will have more cell-ECM interactions. Cells located deeper in the CNC tissue, where there is little ECM, will rely more on cell-cell interactions. The work presented in this thesis focuses on a cell-cell adhesion molecule that is part of the cadherin superfamily of molecules. With this in mind, these studies should be descriptive of the environment within the CNC, and to a less degree the environment between the CNC and the surrounding tissues. The work presented in this dissertation will focus on cadherin-11, which is a classical cadherin that is specifically expressed in the cranial neural crest during its migration. How does cadherin-11 function in the CNC during this process? The work presented here suggests that the main role of cadherin-11 in the CNC is to perform as a cell adhesion molecule. However, too much cell adhesion is inhibitory to migration. In this respect, many of the studies described in this work indicate that cadherin-11 mediated cell adhesion is tightly regulated during CNC migration. Here I show that cadherin-11 is extracellularly processed by ADAM metalloproteases, ADAM9 and ADAM13, which removes the adhesive domain of cadherin-11. This extracellular cleavage event occurs throughout CNC migration, and is likely the main mechanism that regulates cadherin-11 mediated cell adhesion. Cleavage of cadherin-11 by ADAMs does not seem to affect its ability to interact with cytoplasmic binding partners, â-catenin and p120-catenin. This observation supports the idea that the “purpose” of cadherin-11 cleavage is to regulate cell adhesion, and not to induce (cell autonomous) signaling events. Additionally, the secreted extracellular domain of cadherin-11 (EC1-3) retains biological activity. This fragment can bind to a number of cell surface molecules in tissue culture including full-length cadherin-11 and specific members of the ADAM family. This observation suggests that EC1-3 may interact with full-length cadherin-11 molecules in vivo, and inhibit cadherin-11 mediated cell adhesion during CNC migration. EC1-3 can rescue CNC migration in embryos that overexpress cadherin-11, further supporting this hypothesis. Many of the above observations have been published in my first-author paper entitled “Extracellular processing of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest migration” published in the journal Molecular Biology of the Cell in 2009. Some of the unpublished work in this dissertation further focuses on how EC1-3 effects CNC migration in an ex vivo environment. During these studies, the observation was made that overexpression of EC1-3 in a cranial neural crest explant produces abnormal directional movement. In these experiments, it appeared as though certain regions of the CNC explant were “attracting” other regions of the explant. The preliminary studies described in chapter IV are aimed at answering the question; does EC1-3 attract migrating CNC cells? Here, we generated a Matlab program in order to effectively quantify the amount of directional movement of CNC explants presented with a source of EC1-3. In addition to quantifying cell directionality, this program can also decipher between cells moving with random or directed motion, and measure the velocity of cell migration within certain coordinates. Therefore, this program should be useful other ex vivo studies that require the observation of these features. To conclude, the work presented in this dissertation suggests that the role of cadherin-11 during cranial neural crest migration is predominately based on the adhesive function. In order for CNC migration to proceed, the amount of cadherin-11 mediated cell-cell adhesion is tightly regulated throughout this process. These cell-cell interactions are likely important for “sheet” and “branch” migration where CNC cells maintain a lot of cell-cell cohesion.

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