Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Includes bibliographical references (leaves 112-120). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Roger Bradley. Includes bibliographical references (leaves 122-140).

Complex multicellular organisms are composed of at least 200 cell types, which contain the same DNA "black box" of genetic information. It is the precise regime according to which they express their genes, exquisitely controlled by gene regulatory circuits, that defines their cellular identity, morphology and function. We have developed an in vivo biotinylation method that uses genetically encoded components in zebrafish, termed biotagging, for genome-wide regulatory analysis of defined embryonic cell populations. By labelling selected proteins in specific cell types, biotagging eliminates background inherent to analyses of complex embryonic environments via highly stringent biochemical procedures and targeting of specific interactions without the need for cell sorting. We utilised biotagging to characterise the in vivo translational landscape on polysomes as well as the transcriptional regulatory landscape in nuclei of migratory neural crest cells, which intermix with environing tissues during their migration. Our migratory neural crest translatome presented both known and novel players of the neural crest gene regulatory network. An in depth look into the active nuclear transcriptome uncovered a complex world of non-coding regulatory RNAs that potentially specify migratory neural crest identity and present evidence of active bidirectional transcription on regions of open chromatin that include putative cis-regulatory elements. Analysis of our transcribed cis-regulatory modules functionally links these elements to known genes that are key to migratory neural crest function and its derivatives. We also identified a novel cohort of circular RNAs enriched at regions of tandem duplicated genes. Last but not least, we recovered developmentally regulated long non-coding RNAs and transcribed transposable elements. To functionally dissect the biological roles of these factors, we have built two Ac/Ds-mediated in vivo toolkits for efficient screening of putative enhancers and for CRISPR/Cas9-based transcriptional modulation. Overall, our methods and findings present a comprehensive view of the active coding and non-coding landscapes of migratory neural crest on a genome-wide scale that refine the current regulatory architecture underlying neural crest identity.

Branchial arches (BAs) are a series of transient structures that develop on the ventro-lateral surface of the head in vertebrate embryos. BAs initially appear as a series of similar segments; as development proceeds each BA will contribute to different structures. Here, it was investigated the transcriptional mechanisms that instruct the different fates of the BAs in development. Initially, each BA contains a blood vessel, known as aortic arch (AA) artery, that connects the dorsal aorta with the heart. Remodelling of the AAs is crucial to form the adult heart circulation. This process leads to regression of the anterior AAs, running though the first and second BAs (BA1 and BA2), and persistence of the AAs contained in more posterior BAs (PBA). To identify the mechanisms that control remodelling of the AAs, we compared the transcriptomes and epigenomic landscapes of different BAs. Using RNA-seq and H3K27Ac ChIP-seq, we uncovered the activation of a vascular smooth muscle cell (VSMC) differentiation transcriptional program exclusively in the PBAs (and not in BA1/BA2). In support of this finding, we show that VSMC differentiation occurs specifically in the PBAs, but not BA1-2 in mouse embryonic development. Despite the absence of VSMC differentiation in developing BA1-2, cells harvested from these tissues reveal a spontaneous tendency to differentiate towards VSMC fate when grown in vitro, and activate several VSMC-specific genes (Myocd, Acta2, Tagln, Jag1). Together, our results suggest that forming VSMCs is a key process for the persistence of AAs. We also showed that cells derived from all BAs have the potential to differentiate to VSMCs in vitro. However, only cells in the PBAs differentiate to VSMCs in vivo, resulting in the maintenance of posterior AAs. In this study, we also uncovered a novel transcriptional principle that specifies the fate of BA2. Using ChIP-seq, we found that binding of Meis transcription factors establish a ground pattern in the BAs. Hoxa2, which specifies BA2 identity, selects a subset of Meis-bound sites. Meis binding is strongly increased at these sites, which coincide with active enhancers, linked to genes highly expressed in the BA2 and regulated by Hoxa2. Thus, Hoxa2 modifies a ground state binding of Meis to instruct segment-specific transcriptional programs.

Neural crest cells arise during neurulation, a process that occurs during the third week of embryogenesis. These diverse cells then divide into various subtypes including cranial neural crest cells and cardiac neural crest cells. Each of these subtypes gives rise to a wide range of features throughout the fetus. While these cells are extremely diverse, they are also incredibly sensitive to their surrounding environment. Many maternal conditions affect neural crest cell division and migration, but maternal alcohol consumption and hyperglycemia due to gestational diabetes will be discussed in detail, with special attention paid to tissues that derive from cranial neural crest cells. While the initial mechanisms of the pathology vary for both of these conditions, what is remarkable is that they ultimately cause effects in similar ways. Both mechanisms lead to the creation of reactive oxygen species, which in turn trigger apoptotic pathways. Neural crest cell death causes a variety of congenital anomalies in fetuses, including craniofacial defects and cardiac outflow tract defects. Treatment options that have been researched in both conditions also vary, but are based on similar principles. Antioxidant therapies reduce the production of reactive oxygen species, thus reducing the severity of the anomalies affecting the fetus during development. Both maternal alcohol consumption and gestational diabetes are important public health concerns, and their management is of utmost priority in society. By decreasing the rates of women who consume alcohol during pregnancy, and managing gestational diabetes in those at highest risk, the rates of fetal congenital defects could be decreased.

La crête neurale (CN) est une structure multipotente transitoire de l'embryon de vertébrés. La CN céphalique (CNC), mais pas la CN troncale (CNT), fournit des tissus mésenchymateux (squelette, derme et tissus adipeux de la face). Cette capacité de la CNC est liée à l'absence d'expression des gènes de type Hox. Cependant, les cellules de la CNT possèdent des potentialités mésenchymateuses à l'état dormant, qui peuvent s'exprimer en culture. Les mécanismes moléculaires qui régulent les potentialités mésenchymateuses de la CN le long de l'axe antéro-postérieur restent incompris. Chez l'embryon d'oiseau, nous avons étudié l'influence des facteurs de transcription Hox et Six sur la formation du mésenchyme par la CN. D'une part, nos analyses in vivo et in vitro montrent que Six1 est présent dans des cellules mésenchymateuses de la CN et du mésoderme, suggérant un rôle dans le développement musculo-squelettique de la tête. D'autre part, nous avons testé l'hypothèse d'un rôle inhibiteur des facteurs Hox. Nos résultats montrent que l'expression ectopique de Hoxa2 dans les cellules de CNC en culture inhibe la production d'ostéoblastes, sans affecter celle des cellules nerveuses et mélanocytaires. Dans la CNT, nous avons trouvé que la différentiation osseuse, cartilagineuse et adipocytaire, est fortement réduite après la surexpression de Hoxa2, sans effet sur les autres phénotypes dérivés de la CN. Ces résultats suggèrent que les potentialités mésenchymateuses de la CN sont régulées, au moins en partie, par un mécanisme commun aux cellules de CNC et CNT, mettant en jeu une inhibition de l'activité du gène Hoxa2. / The neural crest (NC) is a transitory multipotent structure of the vertebrate embryo. The cephalic NC (CNC), not the trunk NC (TNC), gives rise to mesenchymal cell types (contributing to craniofacial skeleton, dermis and adipose tissue). This capacity of the CNC has been linked to the absence of Hox gene expression in the most rostral region of the embryo. However, TNC cells do have mesenchymal potentialities, although in a dormant state in vivo, but which can be disclosed after NC in vitro culture. The molecular mechanisms that regulate mesenchymal potentials of the NC cells along the rostral-caudal axis are still elusive. Here, we have used the avian embryo model to investigate the possible influence on NC mesenchymal fate, of Hox and Six transcription factor genes. On the one hand, in vivo and in vitro culture analyses show that Six1 gene is expressed in mesenchymal cell populations derived from both cranial NC and mesoderm, suggesting a role for Six1 in muscle-skeletal development in the head. On the other hand, we have tested the hypothesis of an inhibitory action of Hox genes on NC cell mesenchymal differentiation using NC in vitro cultures. In CNC cells, we found that ectopic expression of Hoxa2 strongly reduces the production of osteoblasts, while neural and melanocytic phenotypes are unaffected. In the cultured CNT cells, overexpression of Hoxa2 results in largely impaired differentiation into bone cells, chondrocytes and adipocytes, whereas other NC derivatives are unchanged. These results suggest that mesenchymal potentials of the CNC and TNC are controlled, at least in part, via a common mechanism that involves inhibition of Hoxa2 gene activity.

No description available.

Les capacités de cicatrisation de la gencive en font un modèle de régénération tissulaire naturelle. Ces capacités sont liées en grande partie à l’activité des fibroblastes. Composante cellulaire principale du tissu conjonctif gingival, ils sont au cœur de la régulation des réponses inflammatoires et des processus de cicatrisation. Nous avons supposé que ce tissu pouvait contenir des cellules souches, pouvant expliquer en partie, ces capacités de réparation. Au cours de cette thèse, nous avons pu mettre en évidence la présence de cellules souches mésenchymateuses aux propriétés communes avec les cellules souches adultes dérivées des crêtes neurales. Ces cellules expriment des marqueurs spécifiques des cellules souches et des crêtes neurales. Par ailleurs, elles présentent des capacités d’auto-renouvellement et de multipotence. Elles sont, en effet, capables de se différencier en adipocytes, ostéocytes et chondrocytes. Nous nous sommes plus particulièrement intéressés à la différenciation chondro/endochondrale. La culture des cellules, sous forme de sphères en suspension, a permis de mettre en évidence leurs capacités de différenciation en tissus cartilagineux et articulaires. Elles s’organisent spontanément en plusieurs types cellulaires différents, générant notamment des chondrocytes hypertrophiques et des synoviocytes selon leur localisation au sein des sphères et du milieu de culture utilisé. Le comportement de ces cellules soumises à ces conditions a permis de montrer leurs facultés à reproduire, in vitro, des processus proches de ceux retrouvés au cours du développement. Ces résultats permettent une meilleure compréhension des phénomènes de différenciation des cellules souches adultes, ouvrant ainsi de nouvelles perspectives pour des applications en thérapie cellulaire articulaire et osseuse. / The healing capacity of the gingiva makes it a model of natural tissue regeneration. These capabilities are largely related to the fibroblast activity. They are the main cellular component of the gingival connective tissue and they regulate inflammatory responses and healing process. We hypothesized that this tissue could contain stem cells, which could explain, in part, these repair capabilities. In this thesis, we were able to demonstrate the presence of mesenchymal stem cells with properties shared with the neural crest-derived adult stem cells. These cells express specific markers of stem cells and neural crest. Moreover, they do have the capacity to self-renew and multipotency. They are, indeed, able to differentiate into adipocytes, chondrocytes and osteocytes. We have particularly focused on the chondro / endochondral differentiation. When cultivated as micromasses cultures in suspension, cells were able to differentiate into cartilage and joint tissues. They organize themselves spontaneously into several different cell types, including hypertrophic chondrocytes and synoviocytes depending on their location within the micromasses and the culture medium used. The behavior of these cells under these conditions has shown their ability to replicate in vitro, close to those found during the development process. These results allow a better understanding of adult stem cells differentiation, opening new perspectives for applications in joint and bone cell therapy.

Résumé non transmis / SOX10 is a transcription factor with well-known functions in neural crest and oligodendrocyte development. Mutations in SOX10 were first associated with Waardenburg-Hirschsprung disease (WS4; deafness, pigmentation defects and intestinal aganglionosis). However, variable phenotypes that extend beyond the WS4 definition are now reported. The neurological phenotypes associated with some truncating mutations are suggested to be the result of escape from the nonsense-mediated mRNA decay pathway; but, to date, no mechanism has been suggested for missense mutations, of which approximately 20 have now been reported, and about half of which are redistributed in vitro to nuclear bodies of undetermined nature and function. Here, we reported that the paraspeckle protein p54NRB, which plays a crucial role in the regulation of gene expression during many cellular processes including differentiation, and is a member of the Drosophila behavior Human Splicing (DBHS) protein family, interacts and acts synergistically with SOX10 to regulate several target genes. Interestingly, this multifunctional protein, as well as two other members of the DBHS protein family, co-localized with SOX10 mutants in nuclear bodies, suggesting the possible paraspeckle nature of these foci or re-localization of the DBHS members to other subnuclear compartments. Remarkably, the co-transfection of wild-type and mutant SOX10 constructs led to the sequestration of wild-type SOX10 in mutant-induced foci. However, only foci forming mutants exclusively found in the nucleus altered synergistic activity between SOX10 and p54NRB. We proposed that such a dominant negative effect may contribute to or be at the origin of the progressive neurological phenotype observed in affected patients.One of the roles of p54NRB is the regulation of gene expression via nuclear retention, by binding to hyperedited IRAlu sequences this protein blocks their efficient export to the cytoplasm (Zhang and Carmichael., 2001), we then decided to get into the world of editing. Editing, is a molecular mechanism characterized by the deaminase conversion of adenosines into inosines (A-to-I). In mammals, this molecular modification, is performed by a cluster of three enzymes named Adenosine deaminases acting on RNA (ADARs 1-3) (Wagner RW et al., 1989).In order to evaluate the role of ADAR1 in NC development, we decided to conditionally invalidate the expression of this enzyme using the NC specific HtPA-Cre line. Two main crossing strategies were followed, one including the Rosa26R-LacZ marker (RADR crossing) to track the NCCs and one not (CADR crossing). Globally, the Adar1 deficient pups harvested from the CADR crossing presented with 100% mortality within the first three days after birth. The survival rate of the mutants generated using the second strategy (RADR) was higher, however, none of the mutants survived up to P30. In general, the mutants of the latest crossing, presented with pleiotropic NC phenotype: abnormal melanocyte, ENS and sciatic nerve defects were observed.

Comment les signalisations dynamiques et les mouvements morphogénétiques régionalisent et permettent la formation de tissus complexes durant l'embryogenèse est très peu compris. J’ai caractérise au cours de ma thèse, les évènements signalisants qui sont mis en place au cours de la myogenèse précoce chez l'embryon de poulet. J'ai montre que les progénitures musculaires présents dans les somites requièrent l'activation dynamique des voies de signalisation Wnt et Notch. L’activation transitoire de la signalisation Notch est requise pour adopter un destin myogénique. Le ligand de Notch Dll1 est exprime de manière mosaïque dans les cellules migrantes des crêtes neurales qui passent près du somite. Gain et perte de fonction de Dll1 dans les crêtes neurales modifient la signalisation Notch dans les somites, résultant en un délai ou une prématuré myogenèse. Nos résultats indiquent que les crêtes neural régulent la formation précoce du muscle par un mécanisme unique mené par la migration des cellules des crêtes neurales exprimant Dll1 qui déclenche l'activation transitoire de la signalisation Notch dans certains progénitures musculaires sélectionnes. Cette dynamique signalisation garantie une différentiation progressive du pool de progénitures musculaires. / How dynamic signalling and extensive tissue rearrangements interplay to generate complex patterns and shapes during embryogenesis is poorly understood. During my PhD, I have characterized the signalling events taking place during early morphogenesis of chick skeletal muscles. I observed that muscle progenitors present in somites require dynamic activation of Wnt and Notch signalling. I showed that a transient activation of NOTCH signalling is required to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. These results suggest that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.