Mok Wing Fai Simon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 91-97). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Table of content --- p.v / List of Figures --- p.viii / List of Tables --- p.x / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Origin of the Neural Crest Cells / Chapter 1.1.1 --- Formation of the Neural Tube --- p.1 / Chapter 1.1.2 --- The Neural Crest cells and the Vagal Neural Crest Cells --- p.2 / Chapter 1.1.3 --- The migration profiles of Neural Crest Cells Originated from the Axial level other than Vagal Neural Crest --- p.4 / Chapter 1.1.4 --- Development of the Gastrointestinal Tract and the Enteric Nervous System --- p.5 / Chapter CHAPTER TWO: --- MIGRATION OF NEURAL CREST CELLS IN NORMAL ICR AND DOM MUTANT MOUSE EMBRYOS / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Pregnant mice --- p.39 / Chapter 2.2.2 --- The Handling Medium --- p.39 / Chapter 2.2.3 --- The Culture Medium --- p.40 / Chapter 2.2.4 --- Preparation of Wheat Germ Agglutinin-Gold Conjugates (WGA-Au) --- p.42 / Chapter 2.2.5 --- "Preparation of 1,´1ة-dioctadecyl-´3ة 3,3 '3,3 226}0ة-tetramethyl indocarbocyanine perchlorate (Di-I) " --- p.43 / Chapter 2.2.6 --- Preparation of Carnoýةs Solution --- p.43 / Chapter 2.2.7 --- Preparation of Paraformaldehyde --- p.43 / Chapter 2.2.8 --- Pregnont Dominant Megacolon (Dom) Mice --- p.44 / Chapter 2.2.9 --- DNA Extraction for Genotyping of Dom Embryos --- p.45 / Chapter 2.2.10 --- Primers Used in PCR for Genotyping of Dom Embryos --- p.45 / Chapter 2.2.11 --- PCR Reagent System --- p.46 / Chapter 2.2.12 --- 10XTBE --- p.46 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of Embryos from Pregnant Mice --- p.47 / Chapter 2.3.2 --- In situ labeling of exogenous dye --- p.48 / Chapter 2.3.3 --- Whole Embryo Culture --- p.49 / Chapter 2.3.4 --- Morphological Examination of Cultured Embryos --- p.49 / Chapter 2.3.5 --- Histological Examination of Cultured embryos --- p.50 / Chapter 2.3.6 --- Genotyping of Dom F1 Generation --- p.51 / Chapter 2.3.7 --- Genotyping of Dom Embryos by PCR --- p.52 / Chapter 2.3.8 --- Gel Electrophoresis --- p.52 / Chapter 2.3.9 --- Counting of WGA-Au Labelled Cells --- p.53 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Genotyping --- p.54 / Chapter 2.4.2 --- Examination on Gross morphology of Control and Experimental Embryos --- p.54 / Chapter 2.4.3 --- Morphological Examination of DOM Mutant Embryo after culture --- p.57 / Chapter 2.4.4 --- Initial Stage of Vagal and Trunk Neural Crest Cells Migration in Mouse Embryos --- p.62 / Chapter 2.4.5 --- Initial Stage of Vagal and Trunk Neural Crest Cells Migration in DOM Embryos --- p.64 / Chapter 2.4.6 --- Distribution of Labelled Cells in ICR Embryos after WGA-Au Labelling --- p.65 / Chapter 2.4.7 --- Distribution of WGA-Au Labelled Cells in DOM Embryos --- p.69 / Chapter CHAPTER THREE: --- DISCUSSION / Chapter 3.1 --- Development of embryos in vitro --- p.78 / Chapter 3.2 --- Comparison of the Two Exogenous Dyes --- p.80 / Chapter 3.3 --- Migration Pathway of the Vagal and Trunk Neural Crest Cells --- p.81 / Chapter 3.4 --- Counting of Labelled Cells in DOM Embryos --- p.83 / Chapter 3.5 --- Initial Stage of Vagal and Trunk Neural Crest Cells Migration of Different Genotypes of the DOM Embryos --- p.84 / Chapter 3.6 --- Differences in Distribution of WGA-Au Labelled Cells in Different Genotypes of DOM Embryos --- p.85 / Chapter CHAPTER FOUR: --- CONCLUSION --- p.88 / REFERENCES --- p.91 / "FIGURES, LEGEND TABLE AND APPENDIX"

L’ectoderme embryonnaire devient spécifié en ectoderme non-neural, plaque neurale et bordure neurale à la fin de la gastrulation. Les cellules de bordure neurale sont les progéniteurs de la crête neurale et des placodes. La crête neurale est une population transitoire de cellules multipotentes, qui se forme au cours de la neurulation. Quand les bourrelets neuraux s’élèvent pour former le tube neural, les cellules de la crête neurale subissent une transition épithélio-mésenchymateuse, migrent dans l'ensemble du corps pour atteindre leur destination finale et se différencier. La crête neurale donne naissance à de multiples dérivés tels que les neurones et les cellules gliales du système nerveux périphérique, le cartilage et les os du visage, ou encore les mélanocytes. Des régulations complexes, impliquant de nombreuses signalisations et la transcription de gènes-clé, orchestrent ces événements. Cependant, les premières étapes menant à la formation de la crête neurale et à la spécification précoce de la bordure neurale sont encore peu comprises. Nous avons analysé le transcriptome de la crête neurale d'embryon de l'amphibien Xenopus laevis, à la recherche de nouveaux régulateurs des premières étapes de la formation de la crête neurale. Nous avons constaté que le régulateur de la glycolyse PFKFB4, est exprimé dans l’ectoderme dorsal de la jeune gastrula et dans les cellules de la crête neurale. Ici, nous démontrons que PFKFB4 régule la spécification de l’ectoderme via la voie de signalisation Akt, indépendamment de la glycolyse, démontrant ainsi la première fonction non-glycolytique des enzymes PFKFB. En outre, cette régulation est essentielle pour permettre aux progéniteurs de l'ectoderme d’être spécifiés en plaque neurale, crête neurale, placodes ou ectoderme non neural, mettant en évidence un nouveau point de contrôle de développement. De plus, nous démontrons que PFKFB4 régule des étapes ultérieures de la formation de la crête neurale. Notre travail met en évidence que les régulateurs du métabolisme cellulaire possèdent des fonctions non-métaboliques pour contrôler des étapes de développement au cours du développement embryonnaire. / Embryonic ectoderm becomes specified into non-neural ectoderm, neural plate and neural border at the end of gastrulation. Neural border cells are the progenitors of the neural crest and placodes. The neural crest is a transient population of multipotent cells, which forms during neurulation. As the neural border elevates to form the neural tube, neural crest cells undergo an epithelial to mesenchymal transition, migrate extensively into the whole body to reach their final destinations and differentiate. Neural crest gives rise to multiple derivatives such as neurons and glia, facial cartilage, bones, melanocytes and sympatho-adrenal cells. A complex interplay of signaling and transcriptional regulations orchestrates these early patterning events. However, the first steps leading to NC formation and early specification at the NB are less understood. We analysed the NC transcriptome of frog embryos, to look for novel regulators of the early steps of NC formation. We found that the well-known glycolysis regulator PFKFB4, is expressed in early gastrula dorsal ectoderm, and in neurula neural crest cells. Here, we demonstrate that PFKFB4 regulates ectoderm specification via Akt signaling independently of glycolysis, thus demonstrating the first non-glycolytic function of PFKFB enzymes. Moreover, this regulation is essential to allow ectoderm embryonic progenitors to be patterned into neural plate, neural crest, placodes and definitive ectoderm, highlighting a novel developmental checkpoint. Moreover, we also demonstrate that PFKFB4 regulates later steps of neural crest formation. Our work highlights that regulators of cell metabolism accumulate non-metabolic related functions to control developmental steps during embryonic development.

Eph receptors and their ephrin ligands play important roles in guiding mouse and Xenopus cranial neural crest (CNC) cells to their destinations. My objective was to determine if Ephs and ephrins also regulate avian CNC pathfinding. By double labeling for Eph or ephrin RNA and a neural crest marker protein, I was able to clearly distinguish neural crest from ectoderm and head mesenchyme and show that avian CNC cells express EphA3, 4, and 7 and EphB 1 and 3 and migrate along pathways bordered by non-neural crest cells expressing ephrin-B 1. Surprisingly, avian CNC cells also express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Consistent with these findings, explanted avian CNC cells are labeled by both ephrin-B I and EphB2 Fc fusion proteins. Given the choice between growing out onto substrate-bound fibronectin (FN) or FN plus clustered Fc protein in the stripe assay, these cells show no preference for either condition. Conversely, given the choice between FN or FN plus clustered ephrin-B1 or EphB2 Fc fusion protein, the cells strongly localize to stripes containing only FN. This response is mitigated in the presence of soluble ephrin-B1/Fc or EphB2/Fc, but not in the presence of soluble Fc alone. These findings show that avian CNC cells have a mutually exclusive distribution with non-neural crest cells expressing ephrin-B 1 and EphB2 RNA in situ and are repelled from ephrin-B1 and EphB2 protein in vitro, suggesting that their migration is guided by both forward signaling through a variety of Eph receptors as stimulated by ephrin-B1 and reverse signaling through ephrin-B2 as stimulated by EphB2. I further explore the phylogeny of Ephs and ephrins and show that these genes diversified at different times in evolutionary history, such that the ancestral chordate likely had a single receptor for two different ligands.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: References located at the end of each chapter.

Eph receptors and their ephrin ligands play important roles in guiding mouse and Xenopus cranial neural crest (CNC) cells to their destinations. My objective was to determine if Ephs and ephrins also regulate avian CNC pathfinding. By double labeling for Eph or ephrin RNA and a neural crest marker protein, I was able to clearly distinguish neural crest from ectoderm and head mesenchyme and show that avian CNC cells express EphA3, 4, and 7 and EphB 1 and 3 and migrate along pathways bordered by non-neural crest cells expressing ephrin-B 1. Surprisingly, avian CNC cells also express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Consistent with these findings, explanted avian CNC cells are labeled by both ephrin-B I and EphB2 Fc fusion proteins. Given the choice between growing out onto substrate-bound fibronectin (FN) or FN plus clustered Fc protein in the stripe assay, these cells show no preference for either condition. Conversely, given the choice between FN or FN plus clustered ephrin-B1 or EphB2 Fc fusion protein, the cells strongly localize to stripes containing only FN. This response is mitigated in the presence of soluble ephrin-B1/Fc or EphB2/Fc, but not in the presence of soluble Fc alone. These findings show that avian CNC cells have a mutually exclusive distribution with non-neural crest cells expressing ephrin-B 1 and EphB2 RNA in situ and are repelled from ephrin-B1 and EphB2 protein in vitro, suggesting that their migration is guided by both forward signaling through a variety of Eph receptors as stimulated by ephrin-B1 and reverse signaling through ephrin-B2 as stimulated by EphB2. I further explore the phylogeny of Ephs and ephrins and show that these genes diversified at different times in evolutionary history, such that the ancestral chordate likely had a single receptor for two different ligands.

Le système nerveux entérique (SNE) provient des cellules de crête neurale entériques (CCNEs) qui migrent au sein de l'intestin embryonnaire, prolifèrent et se différencient en cellules gliales et neurones formant des ganglions interconnectés. Mon projet de thèse vise à comprendre comment les propriétés biochimiques et mécaniques de l'intestin embryonnaire influencent la colonisation et la différenciation des ccnes. L'absence d'endothéline-3 (EDN3), un facteur biochimique exprimé dans la paroi intestinale, est une des causes de la maladie de hirschsprung, caracterisée par une aganglionose du côlon distal. Nous montrons pour la première fois que l'EDN3 stimule l'adhésivité des CCNEs en augmentant leurs adhérences focales dépendantes des intégrines beta1 ainsi que la dynamique de leurs protrusions membranaires. De plus, nous avons mis en évidence l'existence d'une interaction génétique entre Edn3 et Itgb1 gouvernant le développement du SNE. Par ailleurs, les propriétés mécaniques du microenvironnement influençant la migration et la différenciation cellulaire , nous avons analysé par des approches biophysiques les propriétés élastiques de l'intestin embryonnaire et leurs impacts sur les comportements des ccnes. Nous avons montré que l'intestin embryonnaire se rigidifie au cours de son developpement et que la migration en 3D des CCNEs est inhibée lorsque la rigidité de l'environnement dépasse un certain seuil. Enfin, nous avons démarré l'analyse de l'effet de l'élasticité sur la différenciation des progéniteurs entériques. L'ensemble de nos résultats permettent de mieux comprendre les mécanismes contrôlant le développement du SNE. / The enteric nervous system (ENS) is derived from enteric neural crest cells (ENCC) that migrate along the length of the intestine through the gut mesenchyme. During this process, ENCC proliferate and differentiate into glial cells and neurons, which aggregate into ganglia. The aim of my thesis is to study how biochemical and mechanical properties of the gut tissue influence ENCC colonization and fate during embryogenesis. The absence of endothelin-3 (EDN3), a small peptide trapped in the embryonic gut mesenchyme, is one of the causes leading to hirschsprung disease, characterized by an aganglionosis of the distal colon. We highlighted for the first time that EDN3 increases ENCC adhesion properties throught 1-integrins focal adhesions and modulates their protrusion dynamics. Moreover, we evidenced a genetic interaction between Edn3 and Itgb1 during ENS development. Also, it is now well established that mechanical properties of the microenvironment influence fundamental mechanisms such as cell migration and cell fate determination. Thus, we analysed whether the mechanical properties of the ENCC’s environment influence their behaviours. Using biophysical approaches, we evidenced a physiological stiffening of the embryonic gut during its development and showed that ENCC migration in 3D is inhibited above a certain rigidity threshold. Finally, we begun to analyse the influence of the elastic properties of the environment onto enteric progenitor cells differenciation, taking advantage of the neurosphere culture system. All together, our results contribute to the understanding of the molecular and cellular mechanisms driving physiological and pathological ENS ontogenesis.

La morphogenèse crâniofaciale chez les vertébrés est un phénomène important, strictement régulé dans l’espace et dans le temps. Elle est basée sur une série complexe d'événements moléculaires et morphogénétiques qui implique un réseau interactionnel de gènes et de facteurs de transcriptions, tels les homéoboîtes. La crête neurale (CN) est au cœur de ce processus. Cette dernière fournit la principale source du mésenchyme crâniofacial. Cette population de cellules embryonnaires transitoires va subir une transition épithélio-mésenchymateuse et migrer en plusieurs vagues vers des sites prédéfinis puis se différencier en divers types cellulaires. La CN est à l’origine de plusieurs structures : une grande partie du squelette facial dont le maxillaire, la mandibule, l’os alvéolaire qui entoure les dents ainsi qu’une partie des tissus conjonctifs crâniofaciaux. Les cellules issues des CN sont pluripotentes et offrent un espoir en régénération osseuse et cartilagineuse. Ces caractéristiques ont généré un intérêt particulier des chercheurs pour les utiliser en thérapie cellulaire afin de réparer les défauts osseux des mâchoires. Parmi les tissus crâniofaciaux, nous avons choisi d’étudier plus avant la gencive et les cellules gingivales car leur accès est le plus facile et leurs capacités de différenciation autorisent l’observation d’autres phénotypes cellulaires.La gencive est un tissu kératinisé qui entoure les dents et recouvre l’os alvéolaire. Ce tissu est composé principalement de fibroblastes gingivaux (GFs). Parmi ces cellules, se trouvent des cellules souches gingivales (GSCs) caractérisées par leur auto-renouvellement et leur multipotence. Les GSCs sont facilement recueillies chez les patients adultes, elles montrent une plasticité importante et une activité immunomodulatrice qui en font un outil de choix pour la thérapie cellulaire. De plus, la biopsie se fait sans douleur et n’entraîne ni cicatrice ni problème fonctionnel.La première partie de mon travail de doctorat avait pour objectif d’évaluer le rôle de Msx1 dans la morphogenèse crâniofaciale et par la suite d’analyser l’os alvéolaire après une extraction dentaire afin d’analyser les mécanismes associés à ce processus et l’impact de Msx1 sur la cicatrisation osseuse.La deuxième partie de mon travail est axé sur la gencive et avait pour objectif de mettre en évidence l’origine embryologique des cellules souches orales, dont les GSCs, et de déterminer si elles proviennent des crêtes neurales, du mésoderme ou d’une mosaïque des deux. Enfin, pour appliquer nos connaissances sur l’origine embryologique des cellules souches gingivales, nous avons étudié le profil immunitaire des cellules dérivées des CN. Pour cela, nous avons déterminé la capacité phagocytaire des cellules souches gingivales murines dérivées des CN et comparé à des cellules de CN d’autres espèces vertébrées. / Craniofacial morphogenesis in vertebrates is an important phenomenon, strictly regulated in space and in time. It is based on a complex series of molecular and morphogenetic events involving an interactional network of genes and transcription factors, such as the homeobox. Neural crest (NC) is at the heart of this process. The latter provides the main source of craniofacial mesenchyme. This transient population of embryonic cells will undergo epithelial-mesenchymal transition and migrate in waves to predefined sites and to differentiate into various cell types. NC is the source of several structures: a large part of the facial skeleton including the maxillary, mandibular alveolar bone around the teeth as well as connective tissue in craniofacial portion. Cells from NC are pluripotent and offer hope for bone and cartilage regeneration. These characteristics have generated particular interest to researchers for use in cell therapy to repair bone defects of the jaw. Among the craniofacial tissues, we decided to further investigate the gums and gingival cells because their access is easier and differentiation capabilities allow observation of other cellular phenotypes.The gum is a keratinized tissue around the teeth and covers the alveolar bone. This tissue is composed mainly of populations of gingival fibroblasts (GFs). Among these populations, there are gingival stem cells (GSCs) characterized by their self-renewal and pluripotency. The GSCs are easily collected in adult patients, they show significant plasticity and immunomodulatory activity that make it a tool of choice for cell therapy. In addition, the biopsy is painless and involves neither scar nor functional problem.The first part of my PhD work was to evaluate the Msx1 role in craniofacial morphogenesis and subsequently analyse the alveolar bone after tooth extraction to analyse the mechanisms involved in this process and the impact of Msx1 on bone healing.The second part of my work focuses on the gingiva and was intended to highlight the embryological origin of oral stem cells, including GSCs and determine if they come from the neural crest, mesodermal or mosaic two. Finally, to apply our knowledge of the embryological origin of gum stem cells, we studied the immune profile derived NC cells. For this, we determined the phagocytic capacity gingival murine stem cells derived from CN and compared to cells of CN other vertebrate species.

Differentiation of the head ectoderm is crucial for the evolutionary diversification of vertebrates. Expression of the genes responsible for this process is orchestrated troughout complex gene regulatory networks that are induced and modulated by Wnt, FGF and BMP signaling pathways. In addition, Wnt/β-catenin signaling, in combination with expression of the Wnt antagonists from the rostral-most part of the head ectoderm, represent a key source of information for the regionalization of the tissue along the antero-posterior axis. This allows the differentiation of the anterior ectoderm that gives rise to the anterior neural fold (ANF) and anterior part of the presumptive placodal region (PPR), and more posterior ectoderm where higher levels of active Wnt/β-catenin signaling promote differentiation into the neural crest (NC) and posterior PPR. Although the requirement of Wnt/β-catenin signalling for ANF, PPR and NC development has been intensively studied in non-mammalian vertebrate model organisms, we lack a clear picture about the situation in mammals. Furthermore, current knowledge in mammals has been gathered via experiments on the level of β-catenin and very little is known about the individual roles of the Tcf/Lef transcription factors. Thereby, we decided to manipulate the Tcf7l1, member of the...

Meis transcription factors belong to the group of TALE (three amino acids loop extension) homeodomain proteins. Meis2 proteins have a potential role in regulation of neural crest cells development and in differentiation of their derivates. Zebrafish genome has two paralogues of meis2 gene, meis2a and meis2b. CRISPR/Cas9 technology was used to prepare mutant lines of both paralogues, meis2a and meis2b, for the purpose of study of function of Meis2 transcription factors. Specific morpholinos that reduce the expression of meis2a and meis2b were used as controls. Craniofacial and cardiac development in mutant fish was analyzed in the meis2a line by RNA in situ hybridization, histological cartilage staining, and computed tomography. While we observed impaired craniofacial and cardiac development after injection of specific Morpholinos, we did not detect similar changes in the meis2a KO line. Our genetic approach has not clearly shown that the meis2a paralogue itself plays an important role in craniofacial development and cardiac development. For more detailed analysis, further experiments on fish lines with combined meis2a and meis2b knock-outs are needed. Key words Mutagenesis CRISPR, Danio rerio, neural crest cells, Meis2, transcription factor

Despite the fact that some aspects of single studies differ, there's a generally accepted view that the whole cartilaginous viscerocranium of vertebrates is neural crest derived. By the series of isotopic transplantation experiments of presumptive neural crest on the model organism Ambystoma mexicanum I partly specify this oppinion and prove that the most ventro-caudal cartilage, the second basibranchial, is of a different origin. Furher I mention the level of the presumptive neural crest where the single parts of cartilaginous viscerocranium arise from. Moreover there is one element, the first basibranchial, which has double origin. I discuss also some other neural crest derivatives such as head and outer gills mesenchyme, the trabeculae cranii, part of the cartilaginous otic capsule or the connective tissue in the head. I have performed 179 transplantations between transgenic and normal axolotl embryos. My final analysis is composed of 65 embryos of stage 40 - 42 and 7 larvae of lenght of 15 - 17 mm.