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Phd Thesis in the Laboratory of Dr Control of pluripotency during the oocyte-to-embryo transition in Caenorhabditis elegans Inauguraldissertation zur Erlangung der Würde einer Doktorin der Philosophie vorgelegt der Philosophisch-Naturwisschenschaftlichen Fakultät der Universität Basel von Cristina Tocchini aus Italien Basel, 2015 Original document stored on the publication server of the University of Basel edoc.unibas.ch This work is licensed under a Creative Commons Attribution 4.0 International License 1 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel auf Antrag von: Prof. Dr. Susan M. Gasser Dr. Rafal Ciosk Dr. Anne Ephrussi Basel, den 24 März 2015 Prof. Dr. Jörg Schibler (Dekan der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel) 2 Table of contents Summary…………………………………………………………………………………………………………………………. 5 Introduction……………………………………………………………………………………………………………………. 7 1. Pluripotency and stem cells……………………………………………………………………………………………… 8 1.1. Why studying stem cells……………………………………………………………………………………….. 8 1.2. Type of stem cells: an overview…………………………………………………………………………… 9 1.3. Pluripotency and germ cells………………………………………………………………………………… 10 1.3.1 Cytoplasmic factors controlling pluripotency in germ cells……………………………. 10 2. The oocyte-to-embryo transition ……………………………………………………………………………………… 12 2.1. Oocyte maturation……………………………………………………………………………………………. 13 2.2. Degradation of maternal factors………………………………………………………………... …......... 15 2.3. The embryonic genome activation………………………………………………………………………… 17 3. Caenorhabditis elegans as a model system to study germ cells……………………………………………. 22 3.1. Caenorhabditis elegans development……………………………………………………………………… 22 3.2. Development of the reproductive system………………………………………………………………. 23 3.2.1. During embryogenesis………………………………………………………………………….. 24 3.2.2. During the larval stages………………………………………………………………………… 24 3.2.3. During adulthood………………………………………………………………………………… 25 3.3. The embryonic genome activation as a marker for the onset of pluripotency…………………. 25 4. TRIM-NHL proteins and their roles in development and disease………………………………………….. 27 4.1. Domain structure of TRIM-NHL proteins………………………………………………………………… 27 4.1.1. The RING domain………………………………………………………………………………… 27 3 4.1.2. The two B-Boxes………………………………………………………………………………….. 28 4.1.3. The coiled-coil domain………………………………………………………………………….. 29 4.1.4. The immunoglobulin-NHL repeats domain………………………………………………… 29 4.2. TRIM-NHL proteins and their functions…………………………………………………………………… 29 4.2.1. Brat…………………………………………………………………………………………………… 30 4.2.2. Mei-P26…………………………………………………………………………………………….. 32 4.2.3. TRIM2 and TRIM3………………………………………………………………………………… 33 4.2.4. TRIM32……………………………………………………………………………………………… 35 4.2.5. LIN-41/TRIM71…………………………………………………………………………………….. 37 5. LIN-41/TRIM71 and the heterochronic pathway in Caenorhabditis elegans…………………………… 39 Results……………………………………………………………………………………………………………………………. 43 1. Manuscript: “The TRIM-NHL protein LIN-41 controls the onset of developmental plasticity in Caenorhabditis elegans” …………………………………………………………………………………………………… 44 2. Recent insights into LIN-41 function in the germline…………………………………………………………… 97 Discussion…………………………………………………………………………………………………………………….. 105 Appendix……………………………………………………………………………………………………………………….. 109 1. Features of early embryonic genes………………………………………………………………………………….. 110 2. Mutants with EGA-positive oocytes…………………………………………………………………………………. 117 3. GLD-1- and LIN-41-interacting factors play a role in controlling the EGA in the germline…………. 125 Materials and methods…………………………………………………………………………………………………. 131 References…………………………………………………………………………………………………………………….. 135 Acknowledgments…………………………………………………………………………………………………………. 155 Curriculum Vitae……………………………………………………………………………………………………………. 159 4 Summary 5 The germline of Caenorhabditis elegans has been used as a model system to study the control of pluripotency in germ cells: through the mean of a genetic screen, from which most of the results of this thesis derived, different factors involved in the control of pluripotency in growing oocytes have been found. It is interesting to note that all the factors identified in the screen are cytoplasmic RNA-binding proteins (RBPs) involved in different aspects of post-transcriptional gene regulation. Post-transcriptional gene regulation can therefore be considered the main mechanism through which a “quiescent” pluripotent state is maintained in oocytes until after fertilization. The oocyte-to-embryo transition has been shown to occur in the absence of polymerase II-dependent transcription, a fact which better clarifies the general importance of RBPs in this developmental context. This study has been the first one to provide the example of a factor, LIN-41, which regulates pluripotency specifically in developing oocytes. lin-41 mutant oocytes lose their germline identity, enter an embryonic and pluripotent state and terminally differentiate into somatic cells. Although LIN-41 was already known in C. elegans to be involved in a somatic developmental pathway, it appears to regulate different targets in the two developmental contexts. Not only lin-41 mutants, but also another class of mutants has been identified in the screen and started to be characterized. The oocytes of the mutants belonging to this class prematurely enter an embryonic state, but, despite that, they are not able to fully acquire pluripotent features and to differentiate into somatic cells. Overall this thesis has been able to shed some light on the factors and, at least in part, the post-transcriptional mechanisms controlling pluripotency during the oocyte-to-embryo transition. 6 Introduction 7 1. Pluripotency and stem cells 1.1. Why studying stem cells During eukaryotic sexual reproduction, the fusion of two highly specialized and haploid cells, the oocyte and the sperm (the gametes), gives rise to a single cell, the zygote. The zygote is a diploid cell which has the ability to give rise to any embryonic (and extra-embryonic for some species) cell of the three germ layers (endoderm, mesoderm and ectoderm), which will allow the proper development of the newly formed organism. To enable the maintenance and the propagation of the species, the great plasticity (named totipotency) possessed by the zygote has to be somehow retained in the cells that will originate the gametes, i.e., the germ cells. Understanding how a fully developed organism can arise from a single cell and how such a developmental information can be inherited and transmitted generation after generation constitute an intriguing challenge for biologists. The “potency” of a cell is defined based on its ability to differentiate into a certain range of differentiated cell types: as already mentioned, toti-potency, characteristic of the zygote, describes cells which are able to originate both embryonic and extra-embryonic cells; pluri-potency is defined as the ability of a cell to give rise to any differentiated embryonic cell, whereas multi- and uni-potency restrict their ability to a few (multi-) or one (uni-) cell types. In the last decades, scientists have developed more and more interest in the field of pluripotency, discovering that not only zygote and germ cells, but even in the adult organism stem cells can be found and they allow the physiological homeostasis of the organism as well as regenerative abilities (Pellettieri and Sànchez, 2007, for a review on the topic). In vitro, the use of cell culture allowed to indefinitely maintain cell pluripotency through the creation of cultured embryonic stem (ES) cells, which are able to self-renew without undergoing differentiation (Evans and Kaufman, 1981; Martin, 1981). The main questions scientists have been trying to unravel regard what factors and mechanisms underline this fundamental developmental process and how, in normal development, cell fate decision is made. Answering these basic biological questions wouldn’t be a simple satisfaction of understanding the “secret of life”, but it would be more practically useful for medical purposes: finding a clean and precise way to specifically and robustly drive a certain differentiation pathway, from undifferentiated stem cells, would provide an enormous contribution to regenerative medicine, allowing to bypass all the issues determined by heterologous transplantations (e.g., tissue rejection, immunosuppression etc.). 8 1.2. Types of stem cells: an overview The study of pluripotent stem cells started in the 1950s with teratocarcinomas, malignant germ cell tumors formed by embryonal carcinoma (EC) cells which can differentiate into somatic cells (Stevens and Little, 1954). Later on, in the 1970s, mouse EC cell lines could be propagated in vitro and were regarded as “in vitro caricatures of development” for their ability to mimic embryonic development in maintaining their undifferentiated state and being able to differentiate into any cell of the three embryonic germ layers, while restricting their developmental potential (Kahan and Ephrussi, 1970; Yu and Thompson, 2008). A step further was provided by the discovery that the transfer of early mouse embryo into extrauterine sites could cause teratocarcinoma formation, suggesting that these embryonic cells could retain pluripotent abilities for the first few cell cleavages (Solter et al., 1970; Stevens, 1970). From this discovery on, scientists have been able to derive in vitro cell lines from ES cells (Evans and Kaufman, 1981; Martin, 1981) which, differently from EC cells in being karyotypically normal, could give rise to a variety
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