How do embryos develop with such poise from a single zygote to multiple cells with different identities, and yet survive? At the four-cell stage of the C. elegans embryo, only the blastomere EMS adopts the endo-mesoderm identity. This fate requires SKN-1, the master regulator of endoderm and mesoderm differentiation. However, in the absence of the RNA binding protein POS-1, EMS fails to fulfill its fate despite the presence of SKN-1. pos-1(-) embryos die gutless. Conversely, the RNA binding protein MEX-5 prevents ectoderm blastomeres from adopting the endo-mesoderm identity by repressing SKN-1. mex-5(-) embryos die with excess muscle at the expense of skin and neurons. Through forward and reverse genetics, I found that genes gld-3/Bicaudal C, cytoplasmic adenylase gld-2, cye-1/Cyclin E, glp-1/Notch and the novel gene neg-1 are suppressors that restore gut development despite the absence of pos-1. Both POS-1 and MEX-5 bind the 3’UTR of neg-1 mRNA and its poly(A) tail requires GLD-3/2 for elongation. Moreover, neg-1 requires MEX-5 for its expression in anterior ectoderm blastomeres and is repressed in EMS by POS-1. Most neg-1(-) embryos die with defects in anterior ectoderm development where the mesoderm transcription factor pha-4 becomes ectopically expressed. This lethality is reduced by the concomitant loss of med- 1, a key mesoderm-promoting transcription factor. Thus the endo-mesoderm identity of EMS is determined by the presence of SKN- 1 and the POS-1 repression of neg-1, whose expression is promoted by MEX-5. Together they promote the anterior ectoderm identity by repressing mesoderm differentiation. Such checks and balances ensure the vital plurality of cellular identity without the lethal tyranny of a single fate.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

The central theme of development, how cells are organized into functional structures and assembled into whole organisms, is addressed by developmental biology. One important feature of embryonic development is pattern formation, which is the generation of a particular arrangement of cells in three-dimensional space at a given point of time. Central to this work is the model system of the zebrafish, Danio rerio. The aim of the first part of this study was to try to understand how a distinct part of the embryonic brain called midbrain-hindbrain boundary (MHB), a region that acts as an organizer for the adjacent brain regions, is established in vertebrates. spiel-ohne-grenzen (spg) is one mutant which interferes with MHB development. Here, I addressed the role of pou2 in brain development by molecular, phenotypical and functional analysis. By genetic complementation and mapping I could elucidate the molecular nature of this mutant and found that the pou2 gene encoding the POU domain transcription factor is affected in spg mutant embryos. By chromosomal syntenic conservation, phylogenetic sequence comparison, and expression and functional data I imply that pou2 is the orthologue of the mammalian Oct4 (Pou5F1) gene. I find by detailed expression and transplantation analysis that pou2 is cell autonomously required within the neuroectoderm to activate genes of the MHB and hindbrain primordium, like pax2.1, wnt1, gbx2 or krox20. By gain-of-function experiments I demonstrate that pou2 synergizes with Fgf8 signaling in order to activate particularly the hindbrain primordium. Since pou2 is already provided to the embryo by the mother, I generated embryos which lack maternal and zygotic pou2 function (MZspg) to reveal a possible earlier than neuroectodermal role of pou2. In the second part of this work I demonstrate that pou2 is a key factor controlling endoderm differentiation. By expression and gain-of-function analysis I suggest a cell autonomous function for Pou2 in the first step of endodermal differentiation. By gain-of-function experiments involving the gene encoding the HMG transcription factor Casanova (Cas) I show that both Cas and Pou2 are necessary to activate expression of the endodermal differentiation marker sox17 in a mutually dependent way, and that the ability of Cas to ectopically induce sox17 strictly requires Pou2. I conclude that both maternal and zygotic pou2 function is necessary for commitment of endodermal progenitor cells to differentiate into endodermal precursor cells.

In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarity signaling from its posterior sister cell, P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (A/P) axis and to specify the endoderm fate of its posterior daughter cell, E. Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or various mutant combinations of the Wnt pathway components disrupt EMS polarity completely. Here we describe the identification of a pathway that is defined by two tyrosine kinase related proteins, SRC-1 and MES-1, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC-1, a C. elegans homolog of c-Src, functions downstream of MES-1 to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells. In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK-3 regulates C. elegans germline development, a function of GSK-3 that is not associated with Wnt signaling. It is required for the differentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans.

Dans la première partie de cette thèse effectuée en collaboration avec le groupe expérimental de C. Chazaud (Clermont Université), nous avons étudié théoriquement un processus de différenciation cellulaire intervenant avant l’implantation de l’embryon dans l’utérus. Il s’agit de la spécification des cellules de la masse cellulaire interne (MCI) en épiblaste (EPI) et en endoderme primitif (EPr), processus dans lequel les facteurs de transcription Nanog et Gata6 jouent un rôle essentiel. En effet, en absence de Nanog, les cellules de la MCI acquièrent toutes une identité EPr, tandis qu’en absence de Gata6, elles se différencient toutes en EPI. De plus, la voie de signalisation Fgf/Erk active l’expression de Gata6 et inhibe celle de Nanog. Enfin, Nanog active la sécrétion dans le milieu extracellulaire de Fgf4, une molécule qui active la voie de signalisation Fgf/Erk en se liant au FgfR2. Nous avons développé un modèle mathématique pour ce réseau de régulations, fondé sur des équations différentielles ordinaires décrivant l’évolution temporelle des niveaux de protéines Nanog, Gata6, Fgf4 et Fgfr2 et de l’activité de la voie Fgf-Erk. Nous avons validé ce modèle en montrant qu’il récapitule les résultats expérimentaux obtenus in vivo, dans les embryons wild-type et dans les mutants Nanog-/- et Gata6-/-. De plus, l’analyse des résultats du modèle permet de proposer un nouveau mécanisme pour l’émergence d’une population mixte de cellules EPI et EPr au sein de la MCI. Ce mécanisme repose sur le fait que le système décrit par notre modèle peut présenter trois états stationnaires stables, dont les niveaux d’expression de Nanog et Gata6 correspondent à l’EPI, l’EPr et la MCI non-différenciée, respectivement. De plus, le modèle a été utilisé afin d’interpréter des résultats expérimentaux récents et contre-intuitifs, concernant les embryons hétérozygotes Gata6+/-. Enfin, nous avons établi des prédictions théoriques, dont certaines ont été ultérieurement vérifiées en laboratoire. <p>Dans la seconde partie de la thèse, effectuée dans le laboratoire d’O. Pourquié (Université de Strasbourg), nous avons étudié un processus de différenciation in vitro, par une approche expérimentale. Il s’agit de la différenciation des cellules souches embryonnaires (ES) en cellules de mésoderme paraxial, un tissu dont dérivent –au cours du développement embryonnaire– les cellules formant notamment les vertèbres, les côtes, la peau et les muscles squelettiques du dos.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Au cours de gastrulation chez la Drosophile, deux vagues successives de constriction ont lieux au niveau des cellules ventrales menant à l'invagination du mésoderme. La première vague de constriction est stochastique et entraine la constriction de 40% des cellules mesodermales réparties aléatoirement et est contrôlée par le facteur de transcription Snail. La seconde vague de constriction arrive immédiatement après et implique également la constriction des 60% manquant de cellules mésodermales. Cette seconde vague est contrôlée par le facteur de transcription Twist et requière la présence de la protéine sécrétée Fog. L'invagination complète du mésoderme riquière la redistribution de la protéine moteur Myosine II au niveau de l'apex des cellules en cours de constriction. Il a été montré que la mutation de Snail mène à une perte des deux phases de constriction, mais qu'une indentation sur les cellules du mésoderme permet de rétablir la seconde phase de constriction Twist dépendante. Nous avons cherché à étudier les interaction entre les deux phases de constriction, la protéine sécrétée Fog et le moteur moléculaire Myosine II à l'aide d'une simulation numérique. Nous avons également chercher à étudier la corélation entre l'invagination globale du mésoderme et la phosphorylation de la Bêta-Cathenine qui est impliquée dans l'activation de Twist. Nous avons étudier l'invagination de l'endoderme postérieur qui présente de nombreuses similitude avec l'invagination de l'endoderme et leurs interactions. Enfin également à l'aide d'une simulation numérique, nous avons testé l'hypothèse de l'apparition d'une invagination dans un organisme primitif mécano-sensible ( la gastræ d'HAECKEL ) au contact avec le plancher océanique. / During Drosophila gastrulation, two waves of constriction occur in the apical ventral cells, leading to mesoderm invagination. The first constriction wave is a stochastic process mediated by the constriction of 40% of randomly positioned mesodermal cells and is controlled by the transcription factor Snail.The second constriction wave immediately follows and involves the other 60% of the mesodermal cells. The second wave is controlled by the transcription factor Twist and requires the secreted protein Fog. It is known that Snail mutation lead to the loss of the two constriction phases but a mechanical poking on the mesoderm cells can rescue de second phase of Twist dependent constriction. The interactions between the two constriction phases, la secreted protein Fog and the molecular motor Myosin II with a numerical simulation. The posterior endoderm invagination that presents similarities with mesoderm invagination have been study, as well as the interaction between them. Finally with an other numerical simulation, the hypothesis of an induced invagination on a primitive mechanosensible organism ( the HAECKEL grastrae ) on the contact with the oceanic floor has been tested.

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.