The brain is our most complex organ, with an estimated 1011 neurons. With the spinal cord, it forms the central nervous system which controls our movements and our senses, holds our memories and creates our thoughts. Because of this, neurodegenerative disorders can be extremely distressing and a thorough understanding of how the nervous system develops is essential if progress is to be made in finding ways to treat them. Critically, this includes understanding how the nervous system forms, i.e., the nature of the signals that promote neural identity (neural induction) and determine correct positional information (patterning). The zebrafish (Danio rerio) has become established as a model for embryological studies due to ease of experimental manipulation. Taking advantage of this, the aims of this PhD were to contribute to unravelling the molecular mechanisms of neural induction and patterning, using a variety of embryological and molecular methods. In the first project, functional analyses of the eve1 gene identified a key factor for posterior neural development. Eve1 was found to be a critical posteriorising factor, with an additional role in posterior neural induction. An outstanding question in neural induction is the relative contribution to this process of two key developmentally important signalling pathways, Bmp and Fgf. In the second project, differential analyses of maternal versus zygotic Bmp and Fgf signalling revealed crucial maternal roles for these two pathways in neural development as neural and epidermal capacitators. The results further suggested that Fgf signalling may be the critical neural inducer. Finally, as a third project, a zebrafish ectodermal explant assay was developed using the organiser-deficient ichabod mutant. The aim was to develop a system to analyse how key molecules directly affect ectoderm and neural development, free of mesoderm and endoderm influences, as signalling from these layers can directly or indirectly influence neural development.

L’induction neurale est le processus au travers duquel les cellules ectodermiques de l’embryon deviennent neurales. De nombreuses études sur les mécanismes contrôlant ce processus on été réalisées mais du fait de sa complexité, de nombreuses questions restent sans réponse. Au cours de ce travail de thèse, je me suis intéressé à l’étude de l’induction neurale sous une perspective évolutive en étudiant ce processus chez le céphalocordé amphioxus, l’un des plus proches parents des vertébrés. J’ai pu mettre en évidence que, comme les vertébrés, l’amphioxus possède un organisateur. J’ai également confirmé une conservation du rôle des voies de signalisation BMP et FGF respectivement dans l’induction de l’épiderme et la régionalisation du tissu neural. Cependant, au contraire des vertébrés, le signal FGF ne semble pas être un acteur prépondérant de l’induction neurale. Au contraire, un rôle important de la voie de signalisation Activine/Nodal a été mis en évidence.Les résultats obtenus soutiennent d’une part la conservation de certains aspects de ce mécanisme chez tous les chordés, et suggèrent d’autre part l’implication de certains acteurs comme la voie Activine/Nodal jusque là inconnue chez les vertébrés. La position phylogénétique de l’amphioxus et la conservation globale de ce processus entre les céphalochordés et les vertébrés nous permettent de suggérer que l’ancêtre des chordés formait du tissue neurale au travers des mécanismes mis en évidence dans cet étude. Ces résultats nous permettent également de proposer de nouvelles études chez les vertébrés visant à établir un rôle putatif de la voie Activine/Nodal au cours de ce processus, rôle jusque la complètement inconnu. / Neural induction is the process through which embryonic ectodermal cells become neural. Many studies on the mechanisms controlling this process have been made, but because of its complexity, many questions remain unanswered. In this thesis, I have focused my interest on the study of neural induction in an evolutionary context studying this process in the cephalochordate amphioxus, one of the closest relatives of vertebrates. I have highlighted that amphioxus, as vertebrates, possesses an organizer. I have demonstrated a conservation of the role of BMP and FGF signals in the induction of the epidermis and the regionalization of neural tissue respectively. However, in contrast to vertebrates, FGF signal does not appear to be a major player in neural induction. Instead, an important role of Activin/Nodal signaling pathway has been demonstrated. These results support, first, the conservation of several aspects of this mechanism in all chordates, and second, they suggest the involvement of the Activin/Nodal signaling in this process, something previously unknown in vertebrates. The phylogenetic position of amphioxus and the overall conservation of this process between cephalochordates and vertebrates allow us to suggest that the ancestor of chordates formed its neural tissue through mechanisms highlighted in this study. These results also allow us to propose new studies in vertebrates for establishing a putative role of the Activin/Nodal signaling during this process, a role previously completely unknown.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20247号 / 医博第4206号 / 新制||医||1020(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 伊佐 正, 教授 鈴木 茂彦, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree Of Master of Science in Medicine, 2016. / Dental stem cells (DSCs) have been identified in teeth and their supporting tissues. They represent an exclusive source of adult stem cells, easily isolated and manipulated for tissue repair and regeneration. This research project evaluated the neurogenic potential of the dental pulp stem cells (DPSCs) and stem cells from the pulp of human exfoliated deciduous teeth (SHEDs) in a South African cohort. Sixty non-carious permanent and deciduous teeth were extracted from healthy patients aged between 18 and 30 years and 5 and 10 years, at the University of the Witwatersrand's Oral Health Clinic in Johannesburg Charlotte Maxeke Academic Hospital, South Africa. The cells, isolated from the extracted pulp tissue were cultured, counted and then phenotyped by flow cytometry analysis. The cells were further expanded in a neural induction medium and immunocytochemistry analysis for Ki-67, doublecortin (DCX) and nestin were performed. Large colonies of both DPSCs and SHEDS were harvested from the extracted pulp tissues and positively cultured. Flow cytometry analysis confirmed the presence of CD44+ and CD29+ cells as well as the known mesenchymal stem cell markers CD90 and CD105. Both DPSCs and SHEDs demonstrated successful proliferation and neural differentiation. This study confirmed that DPSCs and SHEDs are highly proliferative human adult stem cells that exhibit a neurogenic potential that may contribute in the treatment of neurological disorders. / AC2017

En els darrers anys s'ha produït un gran avenç en l'enteniment dels mecanismes implicats en la inducció de la placoda òtica. Tanmateix, poc es coneix encara de com s'estableix un domini amb competència neural i un altre no-neural i aquest ha estat l'objectiu d'aquesta tesi doctoral. Hem analitzat els mecanismes moleculars rellevants per la regionalització primarenca de la placoda òtica i hem explorat el paper de Sox3 i Lmx1b en l'establiment i manteniment d'un territori competent neural, emprant l'embrió de pollet com organisme model. Els resultats mostren que el gen Sox3, inicialment expressat en un territori extens, es regionalitza en un domini òtic i epibranquial proneural. La sobreexpressió de Sox3 a estadis preòtics, indueix la generació de precursors neuronals que expressen Sox2 i Delta1, però aquests no aconsegueixen progressar a estadis de major diferenciació. A la vegada, Sox3 és capaç de inhibir la expression de Lmx1b, un gen expressat en el domini no-neural, suggerint que el seu patró final depèn de l'activitat neurogènica de la oïda interna. Finalment, presento evidències que la senyalització mitjançada per BMP té un paper primerenc en l'establiment de l'expressió de Lmx1b en el territory òtic, però que ni l'activitat de BMP ni l'expressió de Lmx1b influencien el procés de determinació neural. En conclusió, els nostres resultats posen de relleu nova informacióour dels mecanismes moleculars que governen els primers passos de la competencia neural i regionalizació de la placoda òtica en un territori neural i un no-neural. / During the last years, a great progress has been made in understanding the mechanisms involved in otic induction but the mechanism behind otic patterning into neural and non-neural domains is still an open question and the major aim of this work was to address this question. We have analyzed the molecular mechanisms underling the early regionalization of the otic placode, and explored the role of Sox3 and Lmx1b in the establishment and maintenance of a neural competent domain in the otic placode by using the chick as a model system. The results show that Sox3 expression initially expressed in a broad domain gets regionalized in otic/epibranchial proneural domain. Overexpression of Sox3 at preotic stages can induce ectopic neuronal precursor cells expressing Sox2 and Delta1 but does not allow the ectopically developed neuronal precursor cells for further differentiation. Sox3, besides providing neural competence to the proneural domain, regulates the posterior non-neural gene Lmx1b suggesting that its final expression pattern depends on the neural activity. Finally, I present evidence that BMP signaling has an early role in inducing Lmx1b expression in the otic field but that neither BMP activity nor Lmx1b expression influence neural commitment. Taken together, our results provide new information and shed light on the molecular mechanisms that underlie the first steps of the neural competence and otic patterning in proneural and non-neural domain.

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