No description available.

Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

Mucociliary epithelia are found in the human airways and act as the first line of defence against inhaled foreign agents. Mucus traps potentially damaging particles and the cilia transport the mucus away from the airways to remove the threat. Modelling mucociliary epithelia for research purposes is challenging. This is because the airways are enclosed and are thus difficult to study directly. Instead, tissue is extracted or in vitro techniques are employed. Whilst these systems are useful, there is a need for accessible in vivo models to complement them. In this thesis I assess a new model system for studying mucociliary epithelia. This system is the larval epidermis of the amphibian, Xenopus tropicalis. Its epidermis comprises multi-ciliated cells that beat in a polarised direction reminiscent of those found in the human airways. It is also proposed to have a number of other cell types including mucus-secreting cells, but very little is known about them. The epidermis is open and accessible to manipulation meaning that it has great potential to be used in the study of mucociliary epithelia in live, native conditions. Such a system would be a valuable addition to the current models employed. However, the epidermis has not been thoroughly characterized before so its utility as a model system remains speculative.To develop and evaluate this new model, I fully characterize the epidermis, showing that it has five distinguishable cell types. This includes a population of cells called ionocytes that are shown to be essential for the health and function of the epidermis. I also test for the presence of mucins, the structural component of mucus, secreted from the epidermis in order to evaluate the proposal that mucus-secreting cells are present in the epidermis. A mucin-like protein called otogelin is identified. After characterizing the epidermal cell types, I compare them to the human mucociliary epithelium and consider potential applications and future perspectives for this model.

Mutation within the human ALS2 gene, which encodes the protein Alsin, causes a number of recessive motor neuron diseases. The ALS2 gene encodes a 180kDa protein, which has been shown to localize to early endosomes. The Alsin protein comprises three predicted guanine exchange factor (GEF) domains, the best characterised of which is the VPS9 domain for Rab5 GTPase, which is involved in the endocytosis membrane trafficking pathway, particularly in the docking and fusion of early endosomes. Furthermore, Alsin contains a Rho-GEF domain which specifically interacts with Rac-1 GTPase in the PI3K/AKT signal transduction pathway. This pathway has been implicated in numerous biological processes, including control of protein translation, via the mTOR branch of the pathway. To date, most work on the human ALS2 disease phenotypes has focused on the role of alsin in membrane trafficking, and neglected alsin’s potential role in signalling via its Rho-GEF domain. The focus of this project was to study the role of alsin in signalling during early Xenopus development, a period rich in well-characterised cell-cell signalling. I have shown that alsin is maternally loaded and zygotically expressed in the early Xenopus embryo. In cell culture, alsin is localised to early endosomes. Knockdown of alsin protein through the use of mopholinos (MO), resulted in a gastrulation defect, in particular, failure to close the blastopore caused by disrupted mesoderm induction and convergent extension movements. An animal cap assay was used to study mesoderm induction in the presence of als2-MO and activin protein, a potent mesoderm inducer. These animal caps extended normally, indicating proper mesoderm induction. By contrast, als2-MO animal caps failed to extend when co-injected with activin mRNA suggesting that alsin is important for the production and/or secretion of the activin ligand in the source cell. Subsequently it was determined that knockdown of alsin reduced the precursor protein levels of TGF-β family members activin and Xnr-2. These results suggest a novel role for alsin in mRNA stability, translational regulation or post-translational control of specific mesoderm-inducer mRNAs.

No description available.

L’adénosine extracellulaire appartient à la voie de signalisation purinergique et réguledivers processus physiologiques à travers l’activation de ses récepteurs spécifiques (adora).La disponibilité de cette purine dans l’espace extracellulaire est régulée par plusieurs ectoenzymesassurant sa production ou sa dégradation, mais également par des transporteurs denucléosides permettant son passage à travers la membrane. Chez l’adulte, le rôle del’adénosine est assez bien connu. Cependant, l’implication de cette purine au cours del’embryogenèse reste très peu étudiée. Pourtant, un excès d’adénosine dans les phasesprécoces du développement est létal chez la souris et l’oursin, démontrant l’importance de larégulation des concentrations de cette molécule de signalisation lors de l’embryogénèse. Lebut de ma thèse est de comprendre le rôle de l’adénosine au cours de l’embryogenèse enutilisant l’amphibien xénope. En effet, ce modèle a permis de mettre en évidence in vivol’implication de l’ADP au cours du développement de l’oeil chez les vertébrés. La premièrepartie de ce projet a permis de caractériser les acteurs de la voie de signalisation del’adénosine chez le xénope afin d’établir la première carte comparative de leur profild’expression embryonnaire. Cette partie a également permis de mettre en avant laphosphatase alcaline alpl pour son profil d’expression particulier, dans le rein et la rétine. Laseconde partie s’est focalisée sur l’étude fonctionnelle de cette enzyme. Les expériences deperte de fonction montrent son implication lors de la formation de ces deux tissus. / Extracellular adenosine belongs to the purinergic signalling pathway and regulatesvarious physiological processes through activation of specific receptors named adora. Theextracellular concentration of adenosine is regulated by several ecto-enzymes involved eitherin its generation or in its degradation but also by nucleoside transporters enabling its exitoutside or entry inside the cell. In adults, the functions of adenosine are quite well known,however, the its involvement during embryogenesis remains poorly studied. An excess ofadenosine in early phases of development is lethal in mouse and sea urchins, demonstratingthe importance of the extracellular adenosine level regulation during embryogenesis. The aimof my phD is to understand the role of adenosine during embryogenesis using Xenopus as avertebrate model. Indeed, the first in vivo evidence of the implication of the purinergic signallingpathway during vertebrate development, and in particular of ADP during eye formation hasbeen demonstrated using this model. The first part of this project was to characterize all theadenosine signalling pathway actors in Xenopus in order to generate the first comprehensiveand comparative embryonic expression map of these genes. This work allowed me to selectthe alkaline phosphatase alpl for functional studies based on its specific expression profile, inthe retina and kidney. These functional studies, mostly carried out by knockdown experiments,constituted the second part of this phD and showed the implication of this enzyme during theeye and kidney development.

African clawed frogs (Xenopus) represent an ideal model organism for study of regeneration mechanisms. In frogs, complete regeneration occurs in the tadpole stage. In later stages the regeneration capacity is lost. The Laboratory of Developmental biology was successful in establishment of cell culture called Xenopus tropicalis immature Sertoli cells (XtiSCs) derived from X. tropicalis testes. These cells are common progenitors of Sertoli cells and peritubular myoid cells. XtiSCs show similar characteristics as mesenchymal stem cells. MSCs hold interest of scientists for their immunomodulatory properties and multipotent differential and regeneration potential. In this thesis, we studied regeneration and migration potential of XtiSCs after X. tropicalis tadpole's tail amputation in developmental stage 47 - 50. Transgenic XtiSCs culture expressing RFP was prepared to facilitate transplantation experiments. Transplantation experiments showed preferential migration of XtiSCs into the site of injury. XtiSCs transplantations in X. laevis tadpoles with downregulated NO synthases eNOS and nNOS revealed their migratory dependence on nitric oxide signalization. Imunocytochemical staining of XtiSCs in vitro showed positive iNOS, nNOS and Pax7 expression. Imunohistochemical staining of tadpole's tail vibratome...

Chez le xénope, le pronéphros constitue le rein fonctionnel du têtard. Le sang est filtré par le glomus et l'urine est modifiée lors de son transit dans le tubule. Au cours de ma thèse, nous avons analysé l'expression des quatre membres de la famille pou3f au cours du développement embryonnaire chez le xénope. Nous avons montré que leur expression neurale, otique ou encore épidermique est conservée entre le xénope et la souris, de même que l'expression rénale de pou3f3. Nous avons également mis en évidence une expression pronéphrique de pou3f4. Nous avons ensuite analysé le rôle de Pou3f4 au cours du développement du pronéphros. La perte de fonction de Pou3f4 entraine des défauts de différenciation terminale du tubule intermédiaire et distal. Sa surexpression aboutit au contraire à une augmentation de l'expression des marqueurs de différenciation slc12a1 et clcnkb1. Les morphants Pou3f4 présentent aussi des défauts de morphogenèse du tubule intermédiaire caractérisés par des circonvolutions réduites. Nos résultats montrent que Pou3f4 contrôle l'expression de l'ephrine efnA3 dans le tubule pronéphrique. La perte de fonction d'EfnA3 conduit à des défauts de morphogenèse du tubule, suggérant que Pou3f4, par la régulation de l'expression d'efnA3, est impliqué dans les mouvements cellulaires nécessaires à l'élongation antérieure du tubule. Enfin, nous avons observé que pou3f3 et pou3f4 ne se régulent pas l'un de l'autre. Nous avons en revanche mis en évidence une redondance fonctionnelle de ces gènes dans la morphogenèse et la différenciation du tubule puisque la perte de fonction combinée de Pou3f3 et Pou3f4 entraine un phénotype plus sévère que chaque simple perte de fonction. / In Xenopus, pronephros is the functional kidney at tadpole stage. The blood is filtrated by the glomus and the urine is modifed all along the tubule. Pou3f3 is required for intermediate and distal tubule formation in mouse. During my PhD, we have analyzed the expression of the four pou3f genes during Xenopus embryonic development. We found that neural, otic, or epidermic expression of the various pou3f genes is conserved between Xenopus and mouse. Pou3f3 expression in the pronephros is similar to that observed in mouse. We futher showed for the first time pou3f4 expression in the developping kidney. Then, we analyzed the role of Pou3f4 during pronephros development. Pou3f4 depletion inhibits the expression of terminal differentiation marker genes in the intermediate and distal tubule. Pou3f4 upregulates the expression of slc12a1 and clcnkb1 in the tubule. Moreoer, we found that Pou3f4 loss of function leads to intermediate tubule morphogenesis defects. While ephrin signaling pathway is largely described as playing crucial role in cellular movement, Pou3f4 controls efnA3 expression in the intermediate and distal tubule. EfnA3 depletion phenocopies Pou3f4 loss of function, suggesting that Pou3f4, by regulationg efnA3 expression, is implicated in cell movements necessary for anterior elongation of the tubule. Finally, we find that pou3f3 and pou3f4 do not regulate each other expression. However, they act redundantly in pronephros development since the combined Pou3f3 and Pou3f4 loss of function results in a more severe phenotype than each loss of function alone.

In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells

The study of Xenopus tropicalis testis-derived stem cells Nguyen Thi Minh Xuan Abstract The substances secreted by Sertoli cells (SCs) are crucial to determine male sex characteristics in embryos and regulate spermatogenesis in adulthood. The failure in SC maturation can cause sterility in men. Before puberty, SCs keep the ability to proliferate and have been considered as immature cells. They differ remarkably from mature cells in connection with their morphology and biochemical activity and thus they probably play a part in maintaining spermatogonia stem cells in an undifferentiated stage. The transient presence of cytokeratin in immature SCs has been reported in many species, but not in Xenopus yet. We investigated which molecules are expressing only in immature Sertoli cells of X. tropicalis testes. The regulation of cytokeratin and β-catenin was revealed by fluorescent immunostaining. Cytokeratin and membrane β-catenin co- expressed in X.tropicalis juvenile testes and in cultured SC progenitors, called XtiSCs, but they were absent in adulthood. There was no signal of cytokeratin in migrating SCs (pre-SCs) located outside the seminiferous tubules. The suppression of cytokeratin along with the breakdown of β-catenin-based cell contacts have been observed in XtiSCs after the treatment with a small...