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L'acide rétinoïque (AR), métabolite actif de la vitamine A, est une molécule qui a une fonction pléiotropique au cours du développement embryonnaire dont l'excès ou le déficit entraine diverses pathologies cardiaques. Les mutants perte de fonction pour l'enzyme de synthèse de l'AR, la rétinaldéhyde déshydrogénase 2 Raldh2), possèdent de multiples défauts cardiaques. Ces embryons meurent vers le neuvième jour de développement mais cette létalité peut être sauvée en ajoutant de l'AR dans la nourriturre des femelles en gestation. Les mutants Raldh2-/- sauvés ont un Tronc Artériel Commun (TAC) qui est un vaisseau unique au pôle artériel du coeur à la place de l'AO et du TP. Le but de ma thèse a été de comprendre les mécanismes moléculaires et cellulaires conduisant à ce TAC. Ce TAC est à dominance aortique et les connexions des artères coronaires au TAC sont malpositionnées. Nos résultats montrent que la réduction de la longueur de la VE des mutants Raldh2-/- sauvés est associée à une hypoplasie du mésoderme pharyngé. Les mutants Raldh2-/- sauvés possèdent une altération de l'expression de gènes essentiels pour l'élongation de la VE, la rotation de la VE est aussi affectée . Les cellules de la crête neurale cardiaque (CCNC) sont nécessaires pour la septation de la VE et leur contribution à la VE n'est pas altérée dans les mutants Raldh2-/- sauvés. En revanche, elles sont anormalement localisées à proximité du myocarde et anormalement orientées perpendiculairement au futur plan de septation. Cette délocalisation des CCNC est corrélée à une augmentation du processus de transition endothélio-mésenchymateux. L'AR est donc une molécule essentielle pour la morphogenèse de la VE. / Retinoic acid (RA), the active metabolite of vitamin A, is a molecule with pleiotropic functions during development. An excess or deficiency of RA during development leads to various cardiac pathologies. Loss-of-function of retinaldehyde dehydrogenase 2 (Raldh2), the enzyme that synthesises RA, results in various cardiac defects. Mutant embryos die at around the ninth day of embryonic development (E9), but this lethality can be rescued by supplementing the gestating female's diet with RA. RA-rescued Raldh2-/- displayed a persistent truncus arteriosus (PTA), a unique artery at the arterial pole of the heart, rather than separate aorta and pulmonary trunk. The aim of my thesis was to understand the molecular and cellular mechanisms leading to PTA. This PTA is predominantly an aortic phenotype, associated with malpositioned of the coronary artery connections. Our results show that the reduced length of RA-rescued Raldh2-/- OFTs was linked to pharyngeal mesoderm hypoplasia. RA-rescued Raldh2-/- mutants expressed altered levels of several genes that are essential for OFT elongation and OFT rotation was also affected. Cardiac neural crest (CNC) cells play an essential role in OFT septation and their contribution to the OFT was not altered in RA-rescued Raldh2-/- mutants. However, CNC cells display abnormal characteristics, and are found in close proximity to the myocardium, oriented perpendicularly to the plane of septation. This mis-location of the CNC cells was associated with increased endothelial-to-mesenchymal transition (EMT). Accordingly, an increased contribution of endocardial EMT to valve mesenchyme. In conclusion, RA is essential for OFT morphogenesis.

Les cellules de la crête neurale (CN) sont une population de cellules multipotentes que pendant le développement embryonnaire vont migrer et se différencier vers divers lignages comme mélanocytes, muscle lisse, neurones périphériques et entériques, glie ainsi que tissus mésenchymateux cranio-faciaux y compris ceux de la dent. Dans le contexte de l’étude de modèles pour l’ingénierie tissulaire de la dent, nous avons établi une nouvelle lignée de cellules souches embryonnaires (ES) à partir de blastocystes issus de croisements entre un souris Wnt1-Cre et souris rapportrices fluorescentes, les Rosa26 mT/mT. Dans ce system, les cellules qui acquièrent l’identité CN et expriment le gène Wnt1 vont devenir fluorescentes grâce à l’activation de la protéine Tomato, ce qui permet de suivre 1) leur différenciation in vitro 2) isolement et 3) devenir lorsqu’elles sont utilisées dans de modèles in vivo. En parallèle, nous avons mis au point un nouveau protocole simplifié de différenciation (monocouche et milieu défini), vers un phénotype CN. Finalement nous avons tenté de développer un protocole d’induction d’une compétence odontogénique. Notre étude montre que la lignée Wnt1 Cre/Tomato 1) présentent toutes les caractéristiques d’une lignée ES classique i.e. expression de marqueurs de pluripotence, caryotype normal, capacité à se différencier in vitro et in vivo en tissus dérivés des 3 feuillets embryonnaires 2) acquièrent une identité CN, après induction in vitro avec notre protocole de différenciation. 3) Par l’intermédiaire de réassociations tissulaires in vitro, nous avons montré que ces cellules sont capables d’interagir avec un épithélium oral pour former des tissus squelettiques oro-faciaux. Ce nouvel outil cellulaire devrait aider à la compréhension des signaux impliqués dans le dialogue ectomésenchymateux qui sous-tend la formation des tissus durs de la face mais aussi plus généralement permettre suivir le devenir de cellules CN dans des modèles d’ingénierie tissulaire. / Neural crest cells are multipotent progenitor cells that, during embryogenic development, migrate and differentiate into diverse lineages such as melanocytes, smooth muscle, peripheral and enteric neurons, glial cells as well as craniofacial mesenchymatic components, including teeth. In the context of the development of an odontogenic model for tissue engineering, we have generated a new cell line of embryonic stem cells (ES) obtained from blastocysts from crossing Wnt1-CRE mice with fluorescent reporter Rosa26 mT/mT mice. In this Cre/Lox system the cells that have acquired a CN identity and thus expressing Wnt1, will become and remain fluorescent due to the activation of Tomato expression. We have generated a simplified protocol in a monolayer cell culture in defined serum-free medium in order to differentiate the cells into CN cells, named ES-CN cells. Second, we investigated the signals necessary for the odontogenic specification of these ES-CN cells. Our study provides evidence that the Wnt1-CRE/Tomato cell line 1) is a competent ES cell line with the expression of pluripotent markers, a stable karyotype and the ability to differentiate in vitro and in vivo into all the three embryonic germ layers, 2) acquires in vitro a CN identity after induction with our protocol, 3) expresses odontogenic markers in hypoxic culture conditions and 4) is able to interact with an oral epithelium in order to form orofacial skeletal tissues via the tissue reassociation in vitro. This novel cell model should facilitate the understanding of the mechanisms implicated in the ectomesenchymatic interaction, at the base for formation of orofacial skeletal tissues, and will provide the possibility to follow the fate of ES-CN cells tissue engineering models of wounded orofacial structures in general.

by Cheung Chui Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 96-107 (2nd gp.)). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.i / ABSTRACT (CHINESE) --- p.iii / ACKNOWLEDGEMENTS --- p.v / TABLE OF CONTENT --- p.vii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION / Chapter 1.1 --- Early development of the central nervous system --- p.1 / Chapter 1.2 --- Neural crest cells and Cardiac neural crest cells --- p.1 / Chapter 1.3 --- Role of neural crest cells in cardiovascular development --- p.4 / Chapter 1.4 --- Methods in tracing neural crest cells --- p.7 / Chapter 1.5 --- Neural crest-related defects --- p.14 / Chapter 1.6 --- Animal models for studying neural crest defects --- p.16 / Chapter 1.7 --- Recent studies on the migration of cardiac neural crest cells in mammals --- p.19 / Chapter 1.8 --- Objectives of the present study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Pregnant mice --- p.26 / Pregnant Splotch mice (Sp2H) --- p.26 / Preparation of the handling medium --- p.27 / Preparation of the culture medium --- p.27 / Gas mixtures for embryo culture --- p.30 / Preparation of wheat germ agglutinin-gold conjugates (WGA-Au) --- p.30 / Preparation of the fixative --- p.30 / DNA solution for genotyping of Splotch embryos --- p.31 / Primers used in PCR for genotyping of Splotch embryos --- p.31 / PCR reagent system --- p.32 / 10XTBE --- p.32 / Chapter 2.2 --- Methods --- p.33 / Isolation of embryos from pregnant mice --- p.33 / In situ labelling of exogenous dye --- p.34 / Orthotopical grafting of neural crest fragment --- p.36 / Whole embryo culture --- p.37 / Morphological examination of cultured embryos --- p.38 / Histological examination of cultured embryos --- p.38 / Examination of labelled cells in sectioned embryos --- p.39 / Genotyping of Splotch embryos by PCR --- p.40 / Gel electrophoresis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3. 1 --- Initial migration of cardiac neural crest cells in normal ICR mouse embryos --- p.43 / Gross morphological examination of cultured embryos --- p.43 / Distribution of WGA-Au labelled cells in ICR normal mouse embryos --- p.45 / Chapter 3.2 --- Initial migration of cardiac neural crest cells in Splotch embryos --- p.50 / Genotyping --- p.50 / Morphological examination of Splotch mutant embryos --- p.50 / Morphological examination of in vivo Splotch embryos --- p.53 / Distribution of WGA-Au labelled cells in Splotch Embryos --- p.54 / Chapter 3.3 --- Transplantation of neural crest fragments in Splotch embryos --- p.60 / Morphological features of Splotch embryos after orthotopic grafting --- p.60 / Histological examination of Splotch embryos after grafting --- p.61 / Distribution of WGA-Au labelled cells in Splotch embryos after grafting --- p.62 / Chapter CHPATER FOUR --- DISCUSSION / Chapter 4.1 --- Development of embryos in vitro --- p.65 / Chapter 4.2 --- Methodology --- p.70 / In situ labelling of WGA-Au in embryos --- p.70 / Counting of labelled cells in Sploch embryos --- p.72 / Transplantation of neural crest fragments --- p.72 / Chapter 4.3 --- Initial migration of cardiac neural crest cells --- p.74 / Distribution of cardiac neural crest cellsin normal mouse embryos --- p.74 / Differences in the distribution of labelled neural crest cells In different genotypes of Splotch embryos --- p.78 / Distribution of cardiac neural crest cells in Splotch embryos After transplanting of neural crest fragments --- p.83 / Chapter 4.4 --- Factors in extracellular matrix affecting the migration of neural crest cells --- p.88 / Chapter CHAPTER FIVE --- CONCLUSION --- p.91 / REFERENCES --- p.96 / FIGURES AND LEGEND / TABLES / GRAPHS

Greenhill et al. (2011) developed a gene regulatory network of the main genes and interactions known to play a role in melanocyte biology, and generated a mathematical model to describe the behaviour of this complex network using semi quantitative data (ISH expression data). In this project we sought to collect expression data from four genes of the melanocyte GRN (sox10, kit, mitfa and dct) to develop a quantitative model that is able to describe the data more accurately. Moreover, we intended to identify more genes that are part of the melanocyte development process to be incorporated to the GRN. We analysed microarray data that compared differentially expressed genes between sox10 mutant and wild type embryos and validated five genes with a key role in melanocyte biology as downregulated in mitfa mutant embryos, which are downstream of mitfa in the GRN. We suggest that kit plays the role of factor Y in the Greenhill et al. (2011) GRN: Mitfa drives kit expression, and kit expression is transiently driven by Sox10 at early stages of development. As part of the feedback loop, kit seems to drive and maintain mitfa expression, however this needs to be validated. Finally we developed an experimental set up to obtain an estimate of gene expression per melanocyte from sox10, kit, mitfa and dct, using both qPCR and ISH cell count measurements. With this estimate we performed a parameter optimisation procedure, and found a set of parameters for the mathematical model that predicted the experimental data very accurately. The new model suggests that low expression values of sox10 are sufficient to drive mitfa expression in high levels. It also predicts that high expression of sox9b is needed to achieve the high expression levels of dct seen in the data, although these predictions need to be experimentally tested. This study represents the first attempt to obtain fine-scale gene expression data from melanocytes for the development of a quantitative mathematical model in zebrafish.

The multisystemic tumors characteristic of the monogenic neoplastic diseases, tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), share common signaling aberrations upon the loss of heterozygosity in either the TSC1 or TSC2 genes. However, their physical manifestations are vastly different and can generally be classified as being either neurological (TSC) or mesenchymal (TSC & LAM; referred to herein as LAM for simplicity) in origin. In this study, I present a comprehensive stem cell model of LAM utilizing multiple TSC2 knockout (TSC2-/-) pluripotent stem cell lines differentiated to the putative cell of origin for mesenchymal tumors, neural crest cells (NCCs). TSC2-/- NCCs faithfully recapitulate LAM phenotypes and temporal RNA-seq analysis of neural and neural crest differentiation was performed to model disease pathogenesis. Analysis revealed immediate activation of stress response signaling resulting in protein aggregation and lysosome and autophagosome accumulation upon neuralization in TSC2-/- cells. This resulted in acute and lasting effects specific to neural progenitor cells (NPCs), that are transient and ameliorated in NCCs. These lineage-specific effects resulted in selective sensitization of NPCs to cell death via proteasome inhibition, suggesting a potential therapeutic avenue for neurological TSC, but not LAM. Thus, a genome-wide CRISPR knockout screen was performed in TSC2-/- NCCs. Analysis of synthetic lethal genes reveals pathways previously targeted for LAM, but provides gene-level resolution to the vulnerable nodes within these pathways. Importantly, 18 novel gene targets were identified that display synthetic lethality to TSC2-/- cells with high specificity. 3 genes within this list were targetable using commercially available small molecule inhibitors, one of which, FGFR1, shows highly selective lethal targeting of TSC2-/- NCCs. Importantly, this model system, paired with the expansive resource of transcriptomic and synthetic lethal data, serves as a foundation for the development of next generation treatment strategies for LAM, and potentially the entire spectrum of TSC manifestations.

Neural crest is a vertebrate-specific population of embryonic precursor cells thought to have been essential in vertebrate evolution. During development, a group of naïve ectoderm cells are induced to become neural crest and then undergo series of developmental events to give rise to diverse derivatives. Failure of these events often leads to malfunction of neural crest derived tissues and organs. This thesis focuses on the genetic regulation of two events during neural crest development, induction and differentiation. Neural crest induction refers to the specification of ectoderm cells to the neural crest lineage. It is believed that combinatorial activity of transcription factors governs neural crest induction, but the function of specific transcription factors in this process are not yet clear. The AP-2 family of transcription factors is implicated in control of neural crest development, but whether there is a cell autonomous role of AP-2 transcription factors in neural crest induction has remained uncertain. Here I show that in zebrafish, two AP-2 family members, Tfap2a and Tfap2c, are required redundantly for neural crest induction, and that this requirement is cell autonomous. Failure of neural crest induction in the zebrafish embryos that are devoid of Tfap2a and Tfap2c is not caused by defects in cell survival or cell proliferation, but rather appears to result from a failure neural crest cell fate specification. Simultaneous knockdown of Tfap2a and Tfap2c is one of the only known genetic manipulations that result in failure of neural crest induction. Thus the Tfap2a/c double knockdown embryos will be useful for further studies on the emergence of neural crest during both development and evolution. The second section of my thesis concerns differentiation of neural crest derived zebrafish melanophores. This study reveals that Tfap2a and another AP-2 family member, Tfap2e, redundantly and autonomously regulate melanophore differentiation. This is the first report on the function of Tfap2e in any animal. Given that the expression of AP-2 transcription factors is tightly associated with the metastasis potential of human melanoma, my study reinforces the view that cancer cells co-opt regulatory pathways employed in embryonic development.

Les cellules de la crête neurale migrent extensivement et forment le système nerveux autonome comprenant les ganglions parasympathiques, sympathiques et entériques, qui maintiennent l'homéostasie. Dans cette étude, j'explore les migrations, interactions neuronales et dépendances moléculaires lors de la formation des circuits nerveux autonomes. Je démontre que les précurseurs des ganglions parasympathiques dérivent des précurseurs des cellules de Schwann (SCPs) qui envahissent les nerfs préganglionaires jusqu'à leur destination, proche des organes cibles (Espinosa-Medina et al., 2014). D'autre part, je montre un parallélisme entre le mécanisme de migration des précurseurs parasympathiques et celui d'une population de précurseurs du système nerveux ¿sophagien, qui migrent le long le nerve vague. Enfin, je propose un réexamen du système nerveux sacré, qui régule les fonctions urinaire, digestive et reproductrice et qui est considéré comme parasympathique depuis plus d'un siècle, sans argument moléculaire. Je présente une signature moléculaire pour distinguer les neurones parasympathiques crâniens et les neurones sympathiques thoraco-lombaires et démontre que le système nerveux sacré est en fait sympathique. En conséquence, le système nerveux autonome est composé de trois divisions contrastées par leur origine embryonnaire aussi que leur anatomie adulte: une parasympathique d'origine et de connectivité exclusivement crânienne, une sympathique spinale, allant de l'étage cervical au sacré (Espinosa-Medina et al., 2016) et une division entérique que son origine aussi bien que sa connectivité placent à l'interface des systèmes sympathique et parasympathique. / Neural crest cells migrate extensively to form the autonomic nervous system including sympathetic, parasympathetic and enteric ganglia essential for regulating bodily homeostasis. In the present work, I explore the migratory mechanisms and neuronal interactions during autonomic circuit assembly, as well as their molecular dependencies. I show that parasympathetic ganglia derive from Schwann cell precursors (SCPs) and migrate along their preganglionic nerves to locate close to their target tissues (Espinosa-Medina et al., 2014). In line with this work, I show that vagal-associated SCPs give rise to part of the oesophageal nervous system, whereas cervical sympathetic-like crest cells colonize all the gastrointestinal tract, demonstrating a dual origin and different migration mechanisms for enteric neurons. Finally, I revise the identity of the sacral autonomic outflow, whose allocation to the parasympathetic nervous system has been accepted for a century. Sacral autonomic neurons control rectal, bladder, and genital functions and analysis of their cellular phenotype was lacking. Here I present a differential molecular signature for cranial parasympathetic versus thoraco-lumbar sympathetic neurons and show that, in this light, the sacral autonomic outflow is sympathetic. Accordingly, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively (Espinosa-Medina et al., 2016). Interestingly the enteric nervous system, which receives input from both sympathetic and parasympathetic nerves, shares with each system aspects of its ontogeny.

In recent years, mobile devices have played a significant role in daily life. As a result, Mobile Applications have become very popular. An increasing number of people are using mobile applications to handle an increasing number of personal tasks, as well as for amusement. To date, there are thousands of mobile applications available for a variety of purposes. However, there are very few specifically designed for local church use. Mobile applications are potentially more convenient and easier to operate than normal website for parishioners. Designing and implementing a mobile application for Crest Community Church, in Riverside, based on Android Developer Tools (ADT) and Android Software Development Kit (Android SDK) is this project’s primary objective. The objective of this project is to provide a communications platform where members of Crest Community Church can share news, discuss coming church events, promote and enhance the connectivity of this worship community, and provide mobile access to information not available on their present systems. A distinct feature of this project allows for communication between mobile application and webserver by creating a HTTP URL Connection. As a result, church members can publish news and events in a discussion forum as well as a dialog with other church members in a near real time format. The Crest users can also indicate that posted prayer request has been prayed for. In addition, this new application displays general church information, and provides the address and driving direction for potential visitors. Church members can also check the church’s calendar of events, special meetings and information about the Passion Center for Children ministry. This project has the ability to store and track pertinent information about each user, the content of all discussions, and retains member’s key contact data. This project successfully implemented a church mobile application. It demonstrated that a tailored mobile application can enhance the sense of community via internet for this congregation. It feather shows that mobile applications can greatly assist churches in community with members as well as visitors. The application also expanded the internet footprint of Crest Community Church, and offers other churches an avenue to improve their connectivity for the future.