Type 1 diabetes develops through the progressive destruction of the insulin-producing beta-cells. Regeneration or replacement of beta-cells is therefore needed to restore normal glucose homeostasis. Presently, normoglycemia can be achieved by the transplantation of whole pancreas or isolated islets of Langerhans. Islet transplantation can be performed through a simple laparoscopic procedure, but the long-term graft survival is low due to poor revascularization and early cell death. This thesis examined the possibility of using different auxiliary cells (Schwann cells, endothelial progenitor cells, and neural crest stem cells) to improve the engraftment and function of endogenous and transplanted islets. Co-transplantation of Schwann cells with islets improved islet graft function early after transplantation, and caused an increased islet mass at one month posttransplantation. However, the vascular densities of these grafts were decreased, which also related to an impaired graft function. Islet grafts containing endothelial progenitor cells had a superior vascular density, with functional chimeric blood vessels and substantially higher blood perfusion and oxygen tension than control transplants. By culturing and transplanting islets together with neural crest stem cells it was found that islets exposed to these cells had a higher beta-cell proliferation compared with control islets. At one month posttransplantation, the grafts with neural crest stem cells also had a superior vascular- and neural density. The potential of intracardially injected neural crest stem cells to home to the pancreas and ameliorate hyperglycemia in diabetic mice was investigated. During a three-week period after such cell treatment blood glucose concentrations decreased, but were not fully normalized. Neural crest stem cells were present in more than 10% of the pancreatic islets at two days postinjection, at which time the beta-cell proliferation was markedly increased when compared with islets of saline-treated diabetic animals. Three weeks later, a doubled beta-cell mass was observed in animals receiving neural crest stem cells. In summary, islets can easily be transplanted together with different auxiliary cells. Some of these cells provide the possibility of improving vascular- and neural engraftment, as well as beta-cell growth and survival. Systemic administration of neural crest stem cells holds the potential of regenerating the endogenous beta-cells.

La gencive correspond à un modèle de régénération naturelle grâce notamment à sa capacité de cicatrisation « ad integrum ». Ce phénomène est permis par sa composition en fibroblastes gingivaux. Ces cellules, composante cellulaire principale du tissu conjonctif gingival, sont au cœur de la régulation des réponses inflammatoires et de la cicatrisation. Ce tissu contient, comme d’autres tissus mésenchymateux, des cellules souches ; qui expliquent en partie ces capacités de régénération. De plus, comme le tissu gingival est abondant et facilement accessible, l’utilisation de ces cellules souches pourraient être d’un intérêt prometteur en thérapie cellulaire ou pour de la modélisation in vitro. Au cours de cette thèse, nous avons pu montrer que les Cellules Souches dérivées de la Gencive Humaine (CSGH) possèdent des propriétés communes avec les cellules souches adultes dérivées des crêtes neurales. Ces cellules peuvent être qualifiées de « souche » par leur capacité d’auto-renouvèlement, d’adhésion au plastique et de multipotence. Premièrement, nous avons montré que la méthode ainsi que les produits de culture utilisés pour l’isolation des fibroblastes gingivaux in vitro à partir de biopsies de gencive avait une influence sur les cellules obtenues. Dans un second temps, une analyse clonale in vitro de populations de fibroblastes gingivaux a permis de montrer que les fibroblastes gingivaux sont composés de sous-populations qui expriment des marqueurs spécifiques des cellules souches et des crêtes neurales. Outre leur origine embryologique, l’étude de leur multipotence a aussi été caractérisée après expansion et en fonction des additifs utilisés. Pour finir, deux exemples d’utilisation de ces cellules comme modèle d’étude de la biocompatibilité de biomatériaux in vitro ont été développés; imitant la muqueuse buccale ainsi que les réactions dentaires (réparatrices et réactionnaire). / Gingiva is a natural regeneration model thanks to its "ad integrum" healing capability. Gingival fibroblasts are the main actors of this property. These cells, the main cellular component of the gingival connective tissue, regulate the inflammatory responses and healing process. This tissue contains, like many others, mesenchymal stem cells; which also partly explain these regenerative abilities. Moreover, as the gingiva is abundant and easily accessible, the use of these stem cells may interest cell therapy or in vitro model tissues responses. In this work, we demonstrated that Stem Cells Derived from Human Gingiva (SCHG) have common properties with neural crest adult stem cells. These cells can be called "stem cells" for their ability to self-renew, adhere to plastic and to differentiate. First, we have shown that the method and the culture products used for isolation of gingival fibroblasts from gingival biopsy had an influence on the obtained cells. Secondly, an analysis of in vitro clonal populations of gingival fibroblasts has shown that gingival fibroblasts are composed of subpopulations that express specific markers of stem cells and neural crests. In addition to their embryological origin, the study of their multipotency was also characterized after expansion and depending on the used additives. Finally, two examples of using these cells and dental pulp stem cells as a model to study the in vitro biocompatibility of biomaterials have been developed, mimicking oral mucosa or dentin reactions (reparative or reactional).

Les cellules de la crête neurale se caractérisent par leur capacité de migration dansl’embryon et la variété des types cellulaires qu’elles sont capables de générer (mélanocytes,système nerveux entérique (SNE) et périphérique). Chez l’homme, plusieurs maladiescongénitales affectant des organes et tissus divers, ont pour origine une anomalie demigration, prolifération, survie ou différenciation de ces cellules. Au laboratoire, nousétudions deux d’entre elles, le syndrome de Waardenburg-Hirschsprung (WS4- anomalie depigmentation, surdité et maladie de Hirschsprung (HSCR : anomalie entérique)) et lesyndrome de Mowat et Wilson (MWS – retard mental sévère, dysmorphie faciale avec ousans HSCR). A l’heure actuelle, quatre gènes ont été impliqués : l’endothéline 3 (EDN3) etson récepteur à sept domaines transmembranaires EDNRB et les deux facteurs de transcriptionZEB2 et SOX10. Au cours de ma thèse, nous avons montré que des délétions de SOX10 sontégalement responsables de 15% des cas de WS2 (défauts de pigmentation et surdité sansHSCR), élargissant le spectre des phénotypes liés à une mutation au sein de ce gène(Bondurand, Dastot-Le Moal, Stanchina et al. Am. J Hum. Genet, 2007).Parallèlement à ces études génétiques, nous avons souhaité mieux définir la fonction et lesinteractions entre les différents gènes impliqués dans le WS4 (SOX10, EDN3 et EDNRB).Pour cela, nous avons croisé les modèles murins invalidés pour ces gènes, et comparé lephénotype des simples et doubles mutants. A travers cette analyse phénotypique, nous avonsdémontré qu’une interaction entre ces molécules est nécessaire au développement normal duSNE et des mélanocytes dérivés de la crête neurale. En effet, par rapport aux simples mutants,les doubles mutants Sox10;Edn3 et Sox10;Ednrb présentent une augmentation de ladépigmentation, et une forte aggravation du phénotype entérique. Le suivi du devenir descellules formant le SNE au cours du développement nous a permis de montrer quel’aggravation du phénotype entérique est due à une diminution du pool de cellulesprogénitrices par apoptose (Stanchina et al. 2006).Dans la continuité des travaux déjà réalisés, nous avons voulu améliorer notrecompréhension du rôle joué par le gène du MWS : ZEB2, et étudier ses interactions avec lesgènes du WS4. Dans un premier temps, nous avons analysé l’effet de l’expression constitutiveou l’inhibition de ce facteur sur la survie, prolifération et différenciation des cellulesprogénitrices du SNE à l’aide d’un système de culture de progéniteurs entériques disponibleau laboratoire. Nos résultats suggèrent un effet répresseur de ZEB2 sur la différenciationneuronale. Ce facteur pourrait donc être nécessaire au maintien du pool de cellulesprogénitrices dans un état indifférencié. Nous avons ensuite étudié les interactions entre ZEB2et les gènes du WS4. Nous avons croisé les souris portant une invalidation du gène ZEB2 avecles souris invalidées pour SOX10 ou portant une mutation de EDN3 ou EDNRB, et démontréqu’une interaction entre ZEB2 et SOX10 est nécessaire au développement normal du SNE. Eneffet, par rapport aux simples mutants, les doubles mutants présentent une forte aggravationdu phénotype entérique, due à une diminution de la prolifération des cellules progénitrices et àune augmentation de la différenciation neuronale. L’analyse phénotype des mutantsZeb2;Edn3 et Zeb2;Ednrb suggère également l’existence d’une interaction entre ces troismolécules, mais l’origine du défaut entérique reste inexplorée.Ces études nous ont permis de mieux appréhender les réseaux moléculaires mis en place aucours du développement du SNE, de comprendre l’origine des anomalies entériques observéeschez les patients, améliorant leur prise en charge. / To understand in more details the molecular and cellular bases of hereditary diseases resulting from defects of neural crest (NC) development, we study several neurocristopathies, in particular Waardenburg syndrome (WS – pigmentary abnormalities and hearing loss), and Mowat-Wilson syndrome (MWS, severe mental retardation, facial dysmorphy, with or without HSCR (congenital megacolon)). To date, about ten causative genes have been identified, among which are the seven transmembrane domain receptor EDNRB and its ligand endothelin 3 (EDN3), the two transcription factors SOX10 and ZEB2.We contributed to the research efforts engaged to unravel these disorders. In particular, we identified the first mutations of SOX10 in patients presenting with WS4 (association of WS with HSCR disease) and WS2 (Bondurand, Dastot-Le Moal, Stanchina et al. Am. J Hum. Genet, 2007), and participated to functional studies describing its role during enteric nervous system (ENS) development. More recently, we identified the gene ZEB2 as responsible for MWS. The goal of my thesis was to understand the function of these genes and their interaction during the development of NC and ENS in particular. For this purpose, we combined an in vitro approach (isolation of ENS progenitors) to in vivo experiments (phenotype analysis of simple and double mutant mice). We demonstrated that an interaction between SOX10, EDN3 and EDNRB is necessary for the normal development of the ENS and melanocytes (Stanchina et al. 2006), and then focused our efforts in understanding the function of ZEB2 during the development of the ENS as well as its interactions with WS4 genes. Preliminary results suggest that ZEB2 inhibition accelerates neuronal differentiation in vitro. In the same time, generation of Zeb2;Sox10, Zeb2;Edn3 and Zeb2;Ednrb have been realized. Through phenotype analysis of Sox10;Zeb2 double mutants, we showed that a coordinated and balanced interaction between these two genes is required for normal ENS development. Indeed, double mutants present with more severe ENS defects due to decreased proliferation of enteric progenitors and increased neuronal differentiation from E11.5 onwards. These data revealed that crosstalks between these two transcription factors are crucial for proper ENS development. Analysis of Zeb2;Edn3 and Zeb2;Ednrb double mutant suggest also an interaction between these genes. Future experiments will help us to confirm these results and to determine the cellular and molecular origin of these interactions. These studies will enable us to better apprehend the molecular bases of these diseases, and to understand the origin of the enteric anomalies observed in patients. This knowledge may also help to develop new therapeutic strategies

Skeletal (cartilaginous, bony, and dental) tissues undoubtedly exemplify the key innovation of vertebrates. Among all recent vertebrates, the most numerous and successful lineage is represented by the Ray-finned fishes that, accordingly, exhibit amazing variety of skeletal architectures and phenotypic adaptations. In order to depict fundamental principles of fish cranial skeletogenesis the developmental formation of skeletal architectures was described, compared and analyzed using members of early branching fish lineages, that exemplify very different strategies of skeletogenesis. While the Senegal bichirs and the Tropical gars are heavily armored forms with massive exoskeleton and hyperossified dental structures covering the whole oropharyngeal region, the European sterlets, on the contrary, possess mostly cartilaginous skeleton and reduce their dental structures during early development. Whole analysis is underpinned by the Northen pike, teleostean species with lightened skeletal architecture with comparable number of cranial elements. The present study represents the first complex comparative analysis of their skeletogenesis and odontogenesis. This allowed to define developmental strategies founding different lineage-specific skeletal architecture of vertebrates. Comparative description of...

Le mélanome uvéal constitue le cancer intraoculaire le plus fréquent chez l’adulte. Il s’agit d’un cancer très agressif puisque plus de 50% des patients développent des métastases principalement localisées au niveau du foie. Dans le but d’identifier des gènes pronostiques de développement métastatique, nous avons comparé le transcriptome de 28 tumeurs de mélanome uvéal issues de patients ayant développé des métastases dans les trois années qui ont suivi l’énucléation et 29 tumeurs issues de patients n’ayant pas développé de métastases ou ayant développé des métastases après 36 mois. Le gène PTP4A3/PRL-3 (protein tyrosine phosphatase type IV member 3/Protein of Regenerating Liver-3) a été identifié comme prédictif de l’apparition de métastases. Il code une phosphatase et sa surexpression dans des cellules de mélanome uvéal augmente leur migration in vitro et leur invasivité in vivo. Les évènements protéolytiques à la surface des cellules sont essentiels pour la migration et l’invasivité durant plusieurs processus physiologiques ou pathologiques tels que le développement de métastases. Ces évènements sont assurés par les métalloprotéases (MMPs) qui sont responsables de la dégradation et du remodelage de la matrice extracellulaire.Dans la première partie de cette thèse, nous avons observé que la métalloprotéase transmembranaire MT1-MMP est enrichie à la surface des cellules de mélanome uvéal OCM-1, des cellules MP41 issues de xénogreffes de tumeurs de mélanome uvéal humaines ou dans des tumeurs primaires de mélanome uvéal, surexprimant PTP4A3. Nous avons aussi observé que cette accumulation de MT1-MMP à la surface des cellules de mélanome uvéal est accompagnée d’une accumulation de la sécrétion de MMP2 dans le milieu extracellulaire des cellules exprimant PTP4A3. De plus, nous avons montré que PTP4A3 et MT1-MMP s’associent physiquement et que le trafic vésiculaire de MT1-MMP est accéléré dans les cellules exprimant PTP4A3 mais pas dans celles exprimant le mutant catalytique inactif PTP4A3(C104S). Enfin, nous avons démontré que l’inhibition de l’expression de MT1-MMP dans les cellules exprimant PTP4A3 diminue leur migration in vitro et leur invasivité in vivo. Pour conclure, nos résultats indiquent que PTP4A3 agit en amont de MT1-MMP à travers une accélération de son trafic vésiculaire et son accumulation à la surface des cellules afin de promouvoir la migration et l’invasivité cellulaires.Dans la deuxième partie de cette thèse, nous nous sommes intéressés au rôle de PTP4A3 pendant le développement embryonnaire. Les mélanocytes, incluant ceux de l’uvée, dérivent de la crête neurale pendant le développement embryonnaire. Nous avons alors supposé que la fonction de PTP4A3 pendant la progression métastatique pourrait refléter un rôle de la phosphatase dans la migration des cellules de crête neurale pendant le développement embryonnaire. Dans cette partie de la thèse, nous avons montré que PTP4A3 joue un rôle important dans la migration des cellules de crête neurale céphalique pendant le développement de l’embryon de Xenopus laevis. La perte de fonction de PTP4A3 provoque une réduction du territoire de la crête neurale, alors que le gain de fonction de cette phosphatase élargit les faisceaux de migration des cellules de crête neurale céphalique. De plus, des expériences d’isogreffes montrent que les explants de crête neurale dépourvus de l’expression de PTP4A3, sont incapables de migrer dans les embryons greffés. Plus encore, l’inhibition pharmacologique de PTP4A3 dans des cellules de crête neurale en culture diminue de façon significative leur vitesse de migration in vitro. Les résultats de cette étude démontrent que PTP4A3 est requise pour la migration des cellules de crête neurale céphalique in vivo pendant le développement embryonnaire de Xenopus laevis. Donc, les effets pro-migratoire et -invasif reliés à l’expression de la protéine PTP4A3 peuvent refléter son rôle durant la migration des cellules de crête neurale. / Uveal melanoma (UM) is the most common intraocular malignancy in adults and is an aggressive tumor since about 50% of patients will develop metastases mostly in the liver. In order to identify metastasis prognostic genes, we compared 28 uveal melanoma tumors from patients who developed metastases within three years after enucleation to 29 tumors from patients who did not develop metastases or who developed metastases after 36 months. The PTP4A3/PRL-3 gene (protein tyrosine phosphatase type IV member of Regenerating Liver 3/Protein-3) was identified as a strong predictor of metastasis occurence. PTP4A3 encodes a dual specificity phosphatase and its expression in UM cells increases their in vitro migration and in vivo invasiveness. Proteolytic events at the cell surface are essential for cell migration and invasiveness during many physiological and pathological processes such as tumor metastasis. MMPs are responsible for the degradation and turnover of the extracellular matrix (ECM). In the first part of this thesis, We found that the membrane anchored MT1-MMP is enriched at the cell surface of OCM-1, xenograft MP41 or primary human uveal melanoma tumors expressing PTP4A3. We also found that membrane accumulation of MT1-MMP in presence of PTP4A3 in OCM-1 cells is accompanied by enhanced secretion of MMP2 in the extracellular medium. Moreover, we demonstrated that PTP4A3 and MT1-MMP physically associate and that the vesicular trafficking of MT1-MMP is accelerated in presence of active PTP4A3 but not in presence of the mutant PTP4A3(C104S). Furthermore, we found that inhibition of MT1-MMP expression in PTP4A3 expressing uveal melanoma cells impairs their migration in vitro and invasiveness in vivo. Collectively, our results indicate that PTP4A3 acts upstream of MT1-MMP through acceleration of its vesicular trafficking and accumulation at the cell surface to enhance cell migration and invasiveness of uveal melanoma cells. In the second part of this thesis, we investigated the role of PTP4A3 during embryonic development. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.Therefore, the pro-invasive and migratory effects related to the expression of PTP4A3 protein may reflect its role during neural crest migration. Thus, understanding the mechanism of action of PTP4A3 during NC migration may provide insight into PTP4A3 related migratory and invasive phenotypes in human uveal melanoma pathology.

The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...

Migrating to Mars is the next exploration of human space missions and adapting to the extreme environment for a long period of time needs to be studied in-prior. Studies on cells in space may promote advancements in our understanding of the human body. The aim of this study is to optimize the protocol for the preparation of EndoCβH1 cells alone in the space chamber and co-culturing EndoCβH1 cells and BCs for microgravity experiments. We analyzed the cell survival and biocompatibility of β- cells with different parameters and the best concentration was used for the co-culture test. A 1:1 ratio of β-cells and BCs were cultured in the space chamber for viability and survival test. Co-culture experiment showed a significant increase in β-cell viability in the space chamber relative to in vitro tests. The effects of mitochondrial health of the cell inside the chamber was evaluated using the mitochondrial membrane test using the fluorescent probe 5,5,6,6’- tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). We determined that the β-cell fitness inside the chamber was two times higher than the 24-well plate. To assess the viability of β-cells in a bioprinted gelatin scaffold we used the Live-dead fluorescence microscopy assay. Scaffolds were stained with Calcein AM (CAM) and propidium iodide (PI) and checked for survival. The result of scaffold staining showed that β- cells in gelatin and BCs in the media had more living cells compared to β-cells without BCs. Thus, space flight (SF) exposure to this culture system can be a platform for further studies in the treatment of diabetes.

Le suppresseur de tumeur et sérine/thréonine kinase LKB1 est un régulateur clé de la polarité cellulaire et du métabolisme énergétique en partie grâce à l'activation de sa kinase substrat AMPK. Cette protéine est un senseur métabolique pour adapter les apports énergétiques aux besoins nutritionnels des cellules confrontées à un stress. Pour cela, AMPK phosphoryle divers substrats qui activent les réactions cataboliques et inhibent les processus anaboliques dont la kinase mTOR.Au cours de ma thèse, via l’utilisation de modèles murins d’inactivation conditionnelle, j'ai découvert que Lkb1 est crucial pour la formation des cellules de crête neurale (CCN). Ces cellules multipotentes, originaires du tube neural, donnent naissance à divers dérivés, comme les cellules des os et cartilage de la face, les cellules pigmentées de la peau et les cellules gliales et neurales des nerfs périphériques et du système nerveux entérique. J'ai démontré que Lkb1 est essentiel pour la formation de la tête des vertébrés et pour la différenciation et le maintien des dérivés des CCN dans le système nerveux périphérique. J'ai également mis en évidence l’acétylation de LKB1 sur la lysine 48 par l'acétyltransférase GCN5 et son rôle dans l'ontogenèse des CCN céphaliques et la formation de la tête. De plus, j'ai découvert que Lkb1 contrôle la différenciation des cellules gliales en réprimant un programme de biosynthèse d’acides aminés couplé à la transamination du pyruvate en alanine, en amont de la voie de signalisation mTOR.Les phénotypes dus à la perte de Lkb1 dans les CCN récapitulent les caractéristiques cliniques de maladies humaines appelées neurocristopathies. L’activation anormale du suppresseur de tumeur p53 est également associée à certaines neurocristopathies et l’ablation de p53 sauve le phénotype pathologique. Ainsi, j'ai montré que Lkb1 dans les cellules gliales contrôle p53 en limitant les dommages à l’ADN. Lkb1 est aussi essentiel pour maintenir l’homéostasie lysosomale et le recyclage des protéines et ainsi empêcher la formation de granules nommés lipofuscine, chargés en protéines et lipides oxydés. De façon intéressante, les voies mTOR et LKB1/AMPK sont activées à la surface des lysosomes de façon dépendante des niveaux d’acides aminés. Des données récentes de la littérature suggèrent que les lysosomes constitueraient une plateforme de signalisation pour contrôler la protéolyse et le devenir cellulaire. Ainsi, nos données proposent que les signalisations Lkb1 et p53 pourraient réguler l'homéostasie lysosomale et en conséquence le vieillissement cellulaire.De façon intéressante, les cellules de Sertoli, des cellules somatiques épithéliales, localisées dans les tubes séminifères des testicules, et qui régissent la maturation des cellules germinales et l'homéostasie testiculaire, partagent des fonctions métaboliques similaires avec les cellules gliales. En effet, ces cellules sécrètent le lactate et l'alanine qui alimentent les mitochondries des cellules voisines (cellules germinales ou neurones respectivement) contrôlant ainsi leur survie et leur maturation. Au cours de ma thèse, nous avons observé que Lkb1 est requis pour l'homéostasie testiculaire et la spermatogenèse en régulant la polarité des cellules de Sertoli et leur métabolisme énergétique par le cycle pyruvate-alanine. Ces résultats suggèrent une conservation des régulations métaboliques par Lkb1 dans divers tissus.Dans leur ensemble, mes travaux de thèse ont apporté une meilleure connaissance des mécanismes sous-jacents des régulations métaboliques lors du devenir cellulaire. Ces résultats fournissent de nouvelles perspectives sur le développement des CCN et élargissent notre compréhension du contrôle métabolique exercé par LKB1. Enfin, mes projets de doctorat ont mis en évidence l'existence d'une communication entre les voie de signalisation Lkb1 et p53 et suggèrent l’importance de cette communication dans les pathologies humaines dues à des défauts des CCN. / The tumor suppressor LKB1 codes for a serine/threonine kinase. It acts as a key regulator of cell polarity and energy metabolism partly through the activation of the AMP-activated protein kinase (AMPK), a sensor that adapts energy supply to the nutrient demands of cells facing situations of metabolic stress. To achieve metabolic adaptations, AMPK phosphorylates numerous substrates which inhibit anabolic processes while activating catabolic reactions. In particular, AMPK inhibits the mammalian target of rapamycin (mTOR).During my PhD, based on genetically engineered mouse models, I uncovered that Lkb1 signaling is essential for neural crest cells (NCC) formation. NCC are multipotent cells that originate from the neural tube and give rise to various derivatives including bones and cartilage of the face, pigmented cells in the skin and glial and neural cells in peripheral nerves and the enteric nervous system. I demonstrated that Lkb1 is essential for vertebrate head formation and for the differentiation and maintenance of NCC-derivatives in the peripheral nervous system. I also emphasized that LKB1 is acetylated on lysine 48 by the acetyltransferase GCN5 and that this acetylation could regulates cranial NCC ontogeny and head formation. Furthermore, I discovered that Lkb1 controls NCC-derived glial differentiation through metabolic regulations involving amino acid biosynthesis coupled to pyruvate-alanine cycling upstream of mTOR signaling.Phenotypes due to Lkb1 loss in NCC recapitulate clinical features of human disorders called neurocristopathies and therefore suggest that aberrant Lkb1 metabolic signaling underlies the etiology of these pathologies. Abnormal activation of the tumor suppressor p53 has been described in some NCC disorders and p53 inactivation in neurocristopathy mouse models rescues the pathological phenotype. By using a NCC line that can be cultivated as progenitors or differentiated in glial cells in vitro, I demonstrated that Lkb1 expression in NCC-derivatives controls p53 activation by limiting oxidative DNA damage and prevents the formation of lysosomes filled with oxidized proteins and lipids called lipofuscin granules. Interestingly, activation of mTOR and LKB1/AMPK pathways is governed by amino acid sensors and takes place at the lysosome surface. Lysosomes have been proposed as a signaling hub controlling proteolysis and aging. Thus Lkb1 and p53 signaling could converge especially through lysosome homeostasis thereby potentially impacting cellular aging.Strikingly, Sertoli cells, that are epithelial somatic cells, located in seminiferous tubules in testes, and which govern germ cells maturation and whole testis homeostasis, share similar metabolic functions with glial cells. For example, they secrete lactate and alanine to fuel mitochondria of neighboring cells (germ cells or neurons respectively) to control their survival and maturation. During my PhD, we highlighted that Lkb1 is essential for testis homeostasis and spermatogenesis by regulating Sertoli cell polarity and, as observed in glial cells, energy metabolism through pyruvate-alanine cycling. These data suggest that this particular Lkb1 metabolic regulation is conserved in tissues with similar function.Taken together, these studies reveal the underlying molecular mechanisms that coordinately regulate energy metabolism and cell fate. They provide new insights into NCC development and expand our understanding of the role of LKB1 as an energy metabolic regulator. Finally, my PhD projects uncover the existence of a crosstalk between Lkb1 and p53 and underline its importance in NCC disorders.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and occurs in ~1 of every 700 live births. Individuals with DS present craniofacial abnormalities, specifically an undersized, dysmorphic mandible which may lead to difficulty with eating, breathing, and speech. Using the Ts65Dn DS mouse model, which mirrors these phenotypes and contains three copies of ~50% Hsa21 homologues, our lab has traced the mandibular deficit to a neural crest cell (NCC) deficiency in the first pharyngeal arch (PA1 or mandibular precursor) at embryonic day 9.5 (E9.5). At E9.5, the PA1 is reduced in size and contains fewer cells due to fewer NCC populating the PA1 from the neural tube (NT) as well as reduced cellular proliferation in the PA1. We hypothesize that both the deficits in NCC migration and proliferation may cause the reduction in size of the PA1. To identify potential genetic mechanisms responsible for trisomic PA1 deficits, we generated RNA-sequence (RNA-seq) data from euploid and trisomic E9.25 NT and E9.5 PA1 (time points occurring before and after observed deficits) using a next-generation sequencing platform. Analysis of RNA-seq data revealed differential trisomic expression of 53 genes from E9.25 NT and 364 genes from E9.5 PA1, five of which are present in three copies in Ts65Dn. We also further analyzed the data to find that fewer alternative splicing events occur in trisomic tissues compared to euploid tissues and in PA1 tissue compared to NT tissue. In a subsequent study, to test gene-specific treatments to rescue PA1 deficits, we targeted Dyrk1A, an overexpressed DS candidate gene implicated in many DS phenotypes and predicted to cause the NCC and PA1 deficiencies. We hypothesize that treatment of pregnant Ts65Dn mothers with Epigallocatechin gallate (EGCG), a known Dyrk1A inhibitor, will correct NCC deficits and rescue the undersized PA1 in trisomic E9.5 embryos. To test our hypothesis, we treated pregnant Ts65Dn mothers with EGCG from either gestational day 7 (G7) to G8 or G0 to G9.5. Our study found an increase in PA1 volume and NCC number in trisomic E9.5 embryos after treatment on G7 and G8, but observed no significant improvements in NCC deficits following G0-G9.5 treatment. We also observed a developmental delay of embryos from trisomic mothers treated with EGCG from G0-G9.5. Together, these data show that timing and sufficient dosage of EGCG treatment is most effective during the developmental window the few days before NCC deficits arise, during G7 and G8, and may be ineffective or harmful when administered at earlier developmental time points. Together, the findings of both studies offer a better understanding of potential mechanisms altered by trisomy as well as preclinical evidence for EGCG as a potential prenatal therapy for craniofacial disorders linked to DS.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the most common aneuploidy to affect humans and occurs in approximately 1 of 750 live births. Individuals with DS present with a wide range of clinical phenotypes. Common craniofacial phenotypic expressions include a small mandible, protruding tongue, and a flattened nasal bridge. These traits may affect the feeding, breathing, and swallowing of individuals with DS. Because some complications may go unnoticed for longer periods of time, we hypothesize that significant cardiac and GI defects may be indicative of feeding and airway difficulties. In order to better understand the secondary phenotypes resulting from DS, we have implemented a retrospective chart review of 137 infants between zero and six months of age who were evaluated through the Down Syndrome Program at Riley Hospital for Children from August 2005 to August 2008. Data regarding cardiac, gastrointestinal, endocrine, airway, auditory, and feeding abnormalities have been collected and incedences and comorbidites of these traits has been examined. Comprehensive results indicate cardiac abnormalities occur in 80% of infants, 60% experience gastrointestinal complications, feeding difficulties occur in 46%, and airway complications occur in 38% of infants. Infants with DS were found to be breastfed less over time, with an increase in tube feeds. Notably, we have found all infants with videofluoroscopic evaluations had some type of dysphagia. The presence of gastrointestinal abnormalities closely correlate with the need for tube feeds, and the comorbidity between GI anomalies and muscle tone appear to indicate the likelihood of feeding difficulties and need for altered feeding strategies. Comorbidities between feeding difficulties were nearly significant with cardiac defects and significant with GI abnormalities. Identification of such associations will help healthcare providers determine the best course of treatment and recommended feeding methodology for infants with DS. In order to utilize an in vitro model to study the craniofacial dysmorphologies seen in individuals with DS, cranial neural crest cells (NC) have been cultured. With these, we have begun to investigate the mechanisms behind a smaller trisomic mandibular precursor as compared to the euploid. With this in vitro model, we will be able to test proliferation, migration, and senescence of NC in a culture system.