No description available.

No description available.

Two fundamental problems of developmental biology are the understanding of cell fate specification, and the integration of broader environmental contexts into developmental programs. While cell fate specification is largely achieved by differential gene expression programs, environmental integration relies on cellular receptors. A predominant mechanism to mediate both processes utilizes nuclear hormone receptors (NHRs). However, it remains unclear how diverse the NHR’s modes of action are in regulating gene expression. This thesis utilizes the development of the C. elegans germ line as a model system to study a novel link that integrates cell fate specification and the nutritional environment. In C. elegans, germ cell fate specification is chiefly controlled by posttranscriptional mechanisms. Furthermore, overall germline development is influenced by the animal’s nutritional status. However, it remains unknown whether germline posttranscriptional control mechanisms and germ cell fate decisions are linked to nutrition, and if so, how this link may operate in molecular terms. This thesis reports the characterization of the nuclear hormone receptor nhr-114 and its crucial functions for germline development and fertility. Depending on the tissue of expression, nhr-114 regulates overall germline organization, germ cell proliferation and oogenesis. Importantly, all aspects of nhr-114 function are linked to diet. Feeding nhr-114 mutants with a specific E. coli strain, or a tryptophan-supplemented diet significantly reduces germline development defects and sterility. Based on mutant analysis, nhr-114 was found to have overlapping functions with gld-4 cytoplasmic poly(A) polymerase (cytoPAP). This thesis provides evidence that nhr-114 may function in germ cells in a posttranscriptional manner linked to gld-4 cytoPAP. Further evidence shows that NHR-114 interacts with GLD-4 cytoPAP. Together these findings suggest that NHR-114 may control gene expression by transcriptional and posttranscriptional mechanisms in a tissue-specific manner. This thesis proposes that NHR-114 ensures the input of tryptophan to allow germline development; and that this function integrates nutritional information into the germline gene expression programs according to the environment of the worm. Therefore, NHR-114 potentially provides a direct molecular link to how a developmental program is coordinated with the nutritional status of an animal.

Germ cells are a pool of cells that serve as a link between generations. These cells are separated from the somatic cells by specialized type of cytoplasm, called the germ plasm. Germ plasm contains, membraneless, electron dense subcellular structures, termed germplasm granules that contain numerous components of mRNA metabolism pathway. One of the most prominent protein families, commonly found in germplasm granules are DEAD-box helicases. While this protein family is currently heavily investigated, surprisingly little is known about their functions in germ plasm granules and the mechanisms of their association with the granules. This work identified novel biological and molecular roles of C. elegans’ LAF-1 in both somatic and germ cells. It reveals strong dependency of animal’s somatic, embryonic and post-embryonic development on LAF-1 activity, resulting in high penetrance developmental arrest phenotype. Moreover, this work documents requirement of LAF-1 for the fertility of the animal. Analysis of germ cells in the absence of LAF-1 activity reveals multilayered defects occurring at all stages of germ cell development and maturation. LAF-1 appears to be involved in the maintenance of proliferating potential of the germline stem cell pool and loss of LAF-1 significantly expands the region occupied by mitotic cells. Furthermore, loss of LAF-1 significantly affects expression of GLD-1, REC-8 and H3-S10P, implying that mitosis-to-meiosis boundary cannot be established correctly in the absence of LAF-1. This work solidifies previous conclusions that LAF-1 is a component of P granules, both in the adult germ cells and embryonic germ cell precursors and reveals that LAF-1 is required for correct assembly and dynamic behavior of P granules. Intrinsically disordered regions present in LAF-1 are indispensable for LAF-1’s association with P granules and its role in recruiting granule components. Lastly, LAF-1 associates with RNPs containing cytoplasmic polyA polymerases, indicating that LAF-1 might be involved in translational regulation. Altogether, the collected data describes biological functions of LAF-1 and elucidates the molecular mechanisms underlying these functions.

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.

Bei Säugerkeimzellen, Zygoten und Embryos in frühen Stadien kommt der epigenetischen Neuprogammierung eine außergewöhnlich wichtige Rolle in der Regulation der Genomfunktionen in entscheidenden Entwicklungsstadien zu. Die epigenetische Neuprogrammierung in Keimzellen löscht zuerst die Imprinting-Markierungen und Epi-Mutationen und stellt dann geschlechtsspezifische Markierungen (genomische Prägung) wieder her. Die vorliegende Arbeit bezieht sich auf das Löschen epigenetischer Modifikationen in primordialen Mauskeimzellen (primordial germ cells (PGCs)) zwischen dem 10.5 bis 13.5 Tag nach der Befruchtung. Entgegen früheren Annahmen zeigen unsere Ergebnisse, daß primordiale Mauskeimzellen (PGCs) beim Eintritt in die embryonalen Keimdrüsen noch immer DNS Methylierungsmarker besitzen, die ähnlich dem Marker in somatischen Zellen sind. Kurz nach dem Eintritt in die Keimdrüsen werden die DNS Methylierungsmarker, die in Verbindung mit geprägten und nicht geprägten Genen stehen, gelöscht. Für die Mehrzahl der Gene beginnt die Löschung der Marker in männlichen und weiblichen Embryos gleichzeitig und ist innerhalb eines Entwicklungstages abgeschlossen. Diese Kinetik deutet auf einen aktiven Demethylierungsprozess hin, initiiert durch ein somatisches Signal, ausgehend von der embryonalen Keimdrüse. Der Zeitpunkt der Neuprogrammierung in den primordialen Keimzellen ist entscheidend, da er sicherstellt, daß Keimzellen beiden Geschlechts einen epigenetisch äquivalenten Status erhalten, bevor sie geschlechtsspezifisch ausdifferenzieren und anschließend neu elterlich geprägt werden. Vollständiges Verständnis des Prozesses der Neuprogrammierung der Keimzellen ist nicht nur im Hinblick auf genomisches Imprinting wichtig, sondern auch für die Erforschung von Mechanismen für die Wiederherstellung von omnipotenten Zellen bei Klonierung und Stammzellenerhaltung. / Epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. Germ line epigenetic reprogramming assures erasure of all the imprinting marks and epi-mutations and establishment of new sex-specific gametic imprints. The presented work focuses on the erasure of epigenetic modifications that occur in mouse primordial germ cells (PGCs) between day 10.5 to 13.5 post coitum (dpc). Contrary to previous assumptions, our results show that as they enter the genital ridge the PGCs still possess DNA methylation marks comparable to those found in somatic cells. Shortly after the entry of PGCs into the gonadal anlagen the DNA methylation marks associated with imprinted and non-imprinted genes are erased. For most genes the erasure commences simultaneously in PGCs of both male and female embryos and is completed within only one day of development. The kinetics of this process indicates that is an active demethylation process initiated by a somatic signal emanating from the stroma of the genital ridge. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which, new parental imprints are established subsequently. Complete understanding of the germline reprogramming processes is important not only in the light of genomic imprinting but also for resolving other mechanisms connected with restoring cellular totipotency, such as cloning and stem cell derivation.

No description available.

Two fundamental problems of developmental biology are the understanding of cell fate specification, and the integration of broader environmental contexts into developmental programs. While cell fate specification is largely achieved by differential gene expression programs, environmental integration relies on cellular receptors. A predominant mechanism to mediate both processes utilizes nuclear hormone receptors (NHRs). However, it remains unclear how diverse the NHR’s modes of action are in regulating gene expression. This thesis utilizes the development of the C. elegans germ line as a model system to study a novel link that integrates cell fate specification and the nutritional environment. In C. elegans, germ cell fate specification is chiefly controlled by posttranscriptional mechanisms. Furthermore, overall germline development is influenced by the animal’s nutritional status. However, it remains unknown whether germline posttranscriptional control mechanisms and germ cell fate decisions are linked to nutrition, and if so, how this link may operate in molecular terms. This thesis reports the characterization of the nuclear hormone receptor nhr-114 and its crucial functions for germline development and fertility. Depending on the tissue of expression, nhr-114 regulates overall germline organization, germ cell proliferation and oogenesis. Importantly, all aspects of nhr-114 function are linked to diet. Feeding nhr-114 mutants with a specific E. coli strain, or a tryptophan-supplemented diet significantly reduces germline development defects and sterility. Based on mutant analysis, nhr-114 was found to have overlapping functions with gld-4 cytoplasmic poly(A) polymerase (cytoPAP). This thesis provides evidence that nhr-114 may function in germ cells in a posttranscriptional manner linked to gld-4 cytoPAP. Further evidence shows that NHR-114 interacts with GLD-4 cytoPAP. Together these findings suggest that NHR-114 may control gene expression by transcriptional and posttranscriptional mechanisms in a tissue-specific manner. This thesis proposes that NHR-114 ensures the input of tryptophan to allow germline development; and that this function integrates nutritional information into the germline gene expression programs according to the environment of the worm. Therefore, NHR-114 potentially provides a direct molecular link to how a developmental program is coordinated with the nutritional status of an animal.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.