At embryonic day 3.5 (E3.5), inner cell mass (ICM) cells co-express the transcription factors NANOG and GATA6. Between E3.5 and E4.5, cells of the ICM differentiate into epiblast (Epi) and primitive endoderm (PrE). These two lineages are distinguished by the differential expression of the previously coexpressed transcription factors; Epi cells express NANOG while PrE cells express GATA6. FGF/ERK signalling is responsible for Epi and PrE differentiation but it does not explain the initial co-expression of both factors and how the mutually exclusive expression arises. β-catenin is the downstream effector of Wnt signalling, and it is also found in the membrane forming a complex with E-cadherin. Depending on it subcellular location, β-catenin has been associated with pluripotency and differentiation of mESCs, whose origin is the mouse embryo. My hypothesis was that changes in both cellular pools of β-catenin are involved in ICM differentiation. To characterize Wnt/β-catenin role during preimplantation development, I applied quantitative immunofluorescence analysis (QIF) together with chemical and classical genetics in in vitro and in vivo models. I found that high membrane β-catenin levels are associated with Epi cells from E4.0 stage, while nuclear β-catenin levels are higher in co-expressing cells at E3.5 and PrE precursors at E4.0. My results indicate that increases in nuclear β-catenin levels allow the ICM cells to be specified earlier, determined by an earlier appearance of mutually exclusive expression of GATA6 and NANOG in vitro and ex vivo. Moreover, increased β-catenin levels promote specification towards PrE fate, observed by the presence of higher percentages of PrE cells. Conversely, a decrease in β-catenin levels result in slower ICM specification into Epi and PrE. Finally, modulation of FGF/ERK signalling in mouse embryos, which is the main pathway in this cell fate choice, led to changes in β-catenin subcellular location and levels. Altogether, my results are consistent with a role for Wnt/β-catenin signalling facilitating PrE fate acquisition concomitantly with FGF/ERK signalling.

No description available.

Dans ce travail, nous nous sommes intéressés à caractériser la polarité et la croissance du myocarde au cours du développement de la souris. Travaux antérieurs ont montré que la croissance du myocarde est orientée. Cependant, les processus qui sous-tendent cette croissance orientée restent mal connus. Dans d’autres systèmes, la polarité des cellules régule plusieurs comportements cellulaires. Suivant cette hypothèse, nous montrons que la polarité et la division des cellules du myocarde sont coordonnée dans le cœur embryonnaire. Notre objectif suivant était d’obtenir des indications fonctionnelles sur les signaux de croissance du myocarde. Dans le cœur, la voie de la Polarité Cellulaire Planaire (PCP) ainsi que la voie Hippo sont requises pour sa morphogenèse. Chez la mouche, la cadhérine atypique Fat module les voies PCP et Hippo. Le rôle de Fat4, l’orthologue chez les mammifères, comme régulateur de la voie Hippo est controversé. En utilisant les mutants Fat4, nous montrons que Fat4 module la croissance du myocarde plutôt que sa polarité. A la naissance, les mutants Fat4 ont des parois ventriculaires plus épaisses, associées à une prolifération accrue des cardiomyocytes. Nous montrons aussi que Fat4 module l’activité de Yap1, l’effecteur principal de la voie Hippo dans le cœur. Néanmoins, contrairement à la mouche, nous apportons ici la preuve que Fat4 module Yap1 d’une manière non-canonique, en impliquant l’adaptator Amotl1. Ainsi, nous proposons un modèle dans lequel, lorsque Fat4 est supprimé, Amotl1 rentre dans le noyau avec Yap1 pour promouvoir la prolifération des cardiomyocytes. Nos résultats ouvrent des perspectives importantes pour la réparation du cœur blessé. / For this work, we focussed on characterizing myocardial polarity and growth during mouse development. Previous work, showed that the growth of the myocardium is oriented. However, which processes underly such oriented growth has remained unclear. In other systems, cell polarity regulates several types of oriented cell behaviour. Following this hypothesis, we show that myocardial cell polarity and division are coordinated in the embryonic heart. Next, we aimed to gain functional insights into the signals required for the growth of the myocardium. In the heart, both Planar Cell Polarity (PCP) and Hippo signalling are required for proper morphogenesis. In the fruit fly, the atypical cadherin Fat modulates both PCP and the Hippo pathways. The mammalian ortholog Fat4 regulates PCP in the kidneys and in the inner ear. However, its role as an upstream regulator of the Hippo pathway is controversial. Using Fat4 mutants, we show that Fat4 modulates myocardial growth rather than polarity. At birth, Fat4 mutants have thicker myocardial walls and this is associated with increased cardiomyocyte proliferation. Mechanistically, we show that Fat4 modulates the activity of Yap1, the main downstream effector of the Hippo pathway in the heart. However, contrary to the fruit fly, we provide novel evidence that Fat4 modulates Yap1 in a non-canonical way, that implies the adaptor protein Amotl1. Thus, we propose a model where, upon suppression of Fat4, Amotl1 translocates into the nucleus with Yap1 to promote myocardial cell proliferation. Our findings open important perspectives for the repair of the injuried heart, as Fat4 and its effector Amotl1 might play important roles in this context.

A 3,5 jours de développement (E3.5), l'embryon de souris est composé d'une couche externe de trophectoderme (TE) entourant la cavité blastocélique et la masse cellulaire interne (MCI). La MCI comprend une population hétérogène de précurseurs d'épiblaste (Epi) et d'endoderme primitif (EPr) dans une configuration "poivre et sel". A E4.5, ces cellules ségrégent, les cellules d'EPr migrant vers la surface de la MCI pour former un épithélium. A E4.75 cet épithélium donne naissance à 2 tissus distincts : un épithélium d'endoderme viscéral (EP) et à l'endoderme pariétal (EP) qui migre le long du TE. Une transition épithélium-mésenchyme est impliquée dans la formation de l'EP. Je m'intéresse au rôle de Dkk1, un inhibiteur de la voie Wnt canonique et activateur de la voie Wnt/PCP, et Noggin, un inhibiteur des BMP, dans la différenciation de l'endoderme extraembryonnaire. J'ai montré que Dkk1 est un marqueur de l'EPr qui devient apicalement polarisé à E4.5. Son expression est ensuite restreinte aux cellules de l'EP. J'ai aussi montré que Noggin est exprimé dès la préimplantation puis dans l'EP et au niveau de la charnière EV-Ep. Par des expériences de perte et de gain de fonction des voies Wnt et BMP et en utilisant les souris mutantes j'ai analysé le rôle de ces deux facteurs dans la différenciation de l'endoderme extraembryonnaire. / At 3.5 days of development (E3.5), the mouse embryo consists of an outer layer of trophectoderm (TE) surrounding the blastocelic cavity and the inner cell mass (ICM). The ICM is composed of intermingled populations of epiblast (Epi) and primitive endoderm (PrE) precursors, that sort to form two distinct tissues. At E4.75 this epithelium differentiates into visceral endoderm (VE) and parietal endoderm (PE) that migrates along TE. An epithelium-mesenchyme transition (EMT) is involved in PE formation while the VE is maintained as an epithelium. My work focuses on the role of Dkk1, a Wnt canonical pathway inhibitor and Wnt/PCP pathway activator, and Noggin, a BMP pathway inhibitor, in extraembryonic endoderm differentiation. I have shown that Dkk1 is a marker of PrE precursors and is apically polarised at E4.5. Afterwards, its expression is restricted to PE. Noggin is expressed during preimplantation and then in PE and EV-EP hinge. By gain and loss of fonction experiments of Wnt and BMP pathways and by using mutant mice, I studied the role of these two factors in extraembryonic endoderm differentiation.

The preimplantation stages of mammalian development are dedicated to the differentiation of two extraembryonic epithelia, the trophectoderm (TE) and the primitive endoderm (PrE), and their segregation from the pluripotent embryonic lineage, the epiblast. The TE and PrE are responsible for implantation into the uterus and for producing the tissues that will support and pattern the epiblast as it develops into the foetus. PrE and epiblast are formed in a two step process that involves random cell fate specification, mediated by fibroblast growth factor (FGF) signalling, and cell sorting through several mechanisms. In the present work I have addressed aspects of both steps of this process. Chimaera assays showed that epiblast precursors transplanted onto a recipient embryo rarely differentiate into PrE, while PrE precursors are able to switch their identity and become epiblast. Transient stimulation or inhibition of the FGF4-ERK pathway in the chimaeras can modify the behaviour of these cells and restore the plasticity of epiblast precursors. This work shows that epiblast precursors are refractory to differentiation signals, thus ensuring the preservation of the embryonic lineage. I have also found that atypical Protein Kinase C (aPKC) is a marker of PrE cells and that pharmacological inhibition of aPKC impairs the segregation of PrE and epiblast precursors. Furthermore, it affects the survival of PrE cells and can alter the subcellular localisation of the PrE transcription factor GATA4. These data indicate aPKC plays a central role for the sorting of the PrE and epiblast populations and links cell position within the embryo to PrE maturation and survival. Lastly, I have found that aPKC can directly phosphorylate GATA4 in vitro. Knockdown of GATA4 affects cell position within the embryo, whereas aPKC knockdown reduces the number of GATA4-positive cells. These results indicate GATA4 plays an important role in cell sorting during preimplantation development and suggest phosphorylation by aPKC could determine its presence in the nuclei of PrE cells. My work, in the light of the current knowledge, supports a model where the earliest cell fate decisions during mammalian development depend on cellular interactions and not on inherited cell fate determinants. This robust mode of development underlies the plasticity of the preimplantation embryo and ensures the formation of the first mammalian cell lineages, critical for any further progression in mammalian development.

TAD-Grenzen sind genomische Regionen mit Isolatorpotenzial, die zwischen benachbarten Chromatindomänen liegen und deren Unterbrechung zu einer pathologischen Genexpression führen kann. Die meisten TAD-Grenzen werden durch das CTCF gebunden, ein Architekturprotein, das Chromatinschleifen bevorzugt zwischen distalen Paaren von CTCF-Bindungsstellen (CBS) mit einer konvergenten Motivausrichtung bildet. An TAD-Grenzen sind die CBS häufig geclustert, wobei die Motive eine divergente Ausrichtung aufweisen und Chromatinschleifen in Richtung der stromaufwärts und stromabwärts gelegenen Regionen projizieren. Wie die CTCF-Besetzung die Isolierung an TAD-Grenzen moduliert, ist immer noch nicht ganz klar. Hier habe ich die regulatorische Logik von CTCF-geclusterten TAD-Grenzen untersucht, indem ich genomweite Analysen und in vivo-Mausexperimente an der Epha4-Pax3-TAD-Grenze kombiniert habe. Analysen einzelner Deletionen zeigten einen deutlichen hierarchischen Beitrag von CBS zur Grenzfunktion. Im Gegensatz dazu zeigten kombinierte CBS-Deletionen ein gewisses Maß an funktioneller Redundanz und Kooperativität zwischen den Stellen. Diese Analysen zeigten auch, dass die abweichende Konfiguration der CBS, die immer wieder an TAD-Grenzen zu finden ist, für eine robuste Isolierung nicht unbedingt erforderlich ist. Genomweite Analysen haben gezeigt, dass es eine Untergruppe von CBS gibt, die unabhängig von der konvergenten Ausrichtung Chromatinschleifen bilden, wofür ich einen Mechanismus der "Schleifeninterferenz" vorschlage. Weitere Vergleiche ergaben, dass das Niveau der Genexpression von den Abständen zwischen Enhancer und Promoter im linearen Genom abhängen könnte. Durch die Quantifizierung der Isolierung der Grenzen, der Pax3-Fehlexpression und der Schwere der Gliedmaßenfehlbildungen konnte ich schließlich zeigen, dass die TAD-Grenzen die Genexpression und den Phänotyp quantitativ beeinflussen. / TAD boundaries are genomic regions with insulator potential located between adjacent chromatin domains, which disruption can cause pathological gene expression. Most TAD boundaries are bound by the CTCF, an architectural protein that forms chromatin loops preferentially between distal pairs of CTCF binding sites (CBSs) with a convergent motif orientation. At TAD boundaries, CBSs are frequently clustered, with motifs displaying a divergent orientation and projecting chromatin loops towards up and downstream regions. How CTCF occupancy modulates insulation at TAD boundaries still remains elusive. Here, I dissected the regulatory logic of CTCF-clustered TAD boundaries by combining genome-wide analysis and in vivo mouse experiments at the Epha4-Pax3 TAD boundary. Analyses of individual deletions revealed a distinct hierarchical contribution of CBS to boundary function. In contrast, combined CBSs deletions revealed a certain degree of functional redundancy and cooperativity between sites. These analyses also demonstrated that the divergent configuration of CBSs, recurrently found at TAD boundaries, is not strictly required for robust insulation. Genome-wide analysis highlighted the existence of a subset of CBSs that establish chromatin loops independently of the convergent orientation bias, for which I propose a mechanism of “loop interference”. This mechanism suggests that CBS forming a robust convergent loop can simultaneously form a non-convergent loop, by stalling Cohesin complexes extruded from both sides. Further comparisons revealed that gene expression levels might depend on enhancer-promoter distances in the linear genome. Finally, by quantifying boundary insulation, Pax3 misexpression and the severity of limb malformation, I demonstrate that TAD boundaries are quantitative modulators of gene expression and phenotypes. Overall, I highlight that TAD boundary composition and strength constitute a fundamental regulatory layer in developmental processes and disease.

No description available.

Der Transkriptionsfaktor Pitx3 wird nur in postmitotischen mDA Neuronen des Mittelhirns exprimiert und zeigt eine vollständige Koexpression mit TH im VTA und der SN. Bei einem Verlust an Pitx3-Expression (aphakia-Maus) zeigen die mDA Neurone des VTA nahezu keine Veränderung, während sich jedoch die Neuronen der SN nicht entwickeln. Die Gründe hierfür sind bisher unklar.  In der vorliegenden Arbeit sollte analysiert werden, welche Rolle Pitx3 bei der Neurogenese der mDA spielt, indem Pitx3 im murinen Mittelhirn spezifisch überexprimiert wurde. Dies sind die ersten Ergebnisse einer in vivo Pitx3-Überexpressionsstudie in der Maus und die Ergebnisse könne wie folgt zusammengefast werden:  • Die Expression von TH-, DAT- und Nurr1-positiven Zellen im medialen Bereich des dopaminergen Areals, aus dem das VTA hervorgeht, ist bei allen Mutanten (Shh-Cre+/Pitx3OE, Foxa2-Cre+/Pitx, Wnt1-Cre+/Pitx3OE und En1-Cre+/Pitx3OE) signifikant erhöht. Dabei war dieser Phänotyp besonders bei den  Wnt1-Cre+/Pitx3OE Mutanten ausgebildet.  • An E12.5 ist die Expression von Lmx1a und Lmx1b  bei allen Mutanten in der VZ signifikant erhöht.  • Bei den Wnt1-Cre+/Pitx3OE Mutanten zeigte sich an E12.5 eine starke ektopische Wnt1-Expression in den Bereichen der VZ und SVZ. Ebenso konnte eine gesteigerte Proliferation (BrdU, Ki67) bei gleichzeitiger leichter Reduktion der Neurogenese (Ngn2) beobachtet werden. In den Bereichen der ektopischen Wnt1-Expression konnte weiterhin eine Inhibierung der Shh-Expression an E12.5 festgestellt werden.  • An E12.5 konnte bei den Wnt1-Cre+/Lmx1aOE und Wnt1-Cre+/Lmx1bOE Mutanten eine ektopische Wnt1-Expression in den Bereichen festgestellt werden, in denen Lmx1a/b konditional überexprimiert wurde. Hierbei zeigte sich eine starke Reduktion der TH- und Pitx3-Expression, die aber wahrscheinlich durch eine Störung des IsO hervorgerufen sein könnte.  Im Allgemeinen kann also gesagt werden, dass Pitx3 nicht nur an der Differenzierung, sondern auch an Prozessen der Koordination von Proliferation und Spezifikation – im Sinne von Aufrechterhaltung des mDA Bereiches bzw. der Abgrenzung alternativer neuronaler Zellschicksale  beteiligt sein könnte. Dabei beeinflusst Pitx3 wahrscheinlich indirekt den kanonischen Wnt-Signalweg, indem es eine Rückkopplungsschleife mit Lmx1a und/oder Lmx1b, oder einem Zwischenfaktor bildet.

Philosophiae Doctor - PhD / The use of ontologies in the mapping of gene expression events provides an effective and comparable method to determine the expression profile of an entire genome across a large collection of experiments derived from different expression sources. In this dissertation I describe the development of the developmental human and mouse eVOC ontologies and demonstrate the ontologies by identifying genes showing a bias for developmental brain expression in human and mouse, identifying transcription factor complexes, and exploring the mouse orthologs of human cancer/testis genes.Model organisms represent an important resource for understanding the fundamental aspects of mammalian biology. Mapping of biological phenomena between model organisms is complex and if it is to be meaningful, a simplified representation can be a powerful means for comparison. The implementation of the ontologies has been illustrated here in two ways.Firstly, the ontologies have been used to illustrate methods to determine clusters of genes showing tissue-restricted expression in humans. The identification of tissue restricted genes within an organism serves as an indication of the finetuning in the regulation of gene expression in a given tissue. Secondly, due to the differences in human and mouse gene expression on a temporal and spatial level, the ontologies were used to identify mouse orthologs of human cancer/testis genes showing cancer/testis characteristics. With the use of model systems such as mouse in the development of gene-targeted drugs in the treatment of disease, it is important to establish that the expression characteristics and profiles of a drug target in the model system is representative of the characteristics of the target in the system for which it is intended.