Regulation of totipotency in the "Caenorhabditis elegans" germ line

The fertilization of an oocyte with sperm leads to the formation of a zygote, which has the unique ability to differentiate into any cell type. This specific ability is defined as totipotency. Germ cells differentiate into highly specialized cells, oocytes and sperm, but germ cells also have an underlying totipotency, as totipotent cells can be derived from germ cells. However the mechanisms that allow germ cells to establish/maintain germline identity and to specialize, while maintaining an underlying totipotency, are not understood. In C.elegans, germ cells in the gld-1, and gld-1, mex-3 mutants fail to progress through meiosis and instead form a germline tumor. Recently Dr. Rafal Ciosk found that germ cells in the gld-1, and gld-1, mex-3 germline tumor lose their germline identity and instead acquired a somatic fate, a phenotype that is reminiscent to a special human germline tumor, called teratoma. This finding provided us with a genetic model system that allowed us to investigate the mechanisms that are required to maintain germline identity, and totipotency. To address these questions, we first needed to understand how teratoma formation occurs in C.elegans. What is the etiology of the cells undergoing teratoma formation? To address this question we used a compound mutant background in which the major mitotic and meiotic pathways were deleted and the gonad was lacking a distal to proximal orientation. As cells within this gonad showed a synchronized development we could follow the different cell cycle stages preceding teratoma formation. After an initial phase of proliferation germ cells enter meiosis, however fail to progress through meiosis, re-enter proliferation and undergo germ line to soma transition. This knowledge allowed us to reveal the cells that lead to teratoma formation in the simplest genetic background, the gld-1 mutant. This analysis showed us that the germline tumor in the gld-1 mutant is formed by two major populations of cells, a central and proximal tumor. As already the loss of GLD-1 alone leads to teratoma formation we sought to identify GLD-1 targets. In this analysis we could define core cell cycle factors as new GLD-1 targets, namely cyclin E and Cyclin Bs. Genetic experiments showed that ectopic expression of Cyclin E together with CDK-2 promotes the re-entry into mitosis and tumor initiation in the central region of the gld-1 gonad. This re-entry into mitosis leads to loss of germ line identity and unexpectedly to a change in the transcriptional program of the cells, preceding expression of markers of terminally differentiated cells. Furthermore we found that ectopic expression of a known GLD-1 target, GLP-1, promotes proximal tumor formation and suppresses germ line to soma transition in these cells. Taken together this study revealed that different cell populations lead to the formation of the heterogeneous germline tumor in the gld-1, or gld-1, mex-3 mutant, and identified its major regulators. Further this study provides a first mechanism promoting germline to soma transition. We propose that the loss of GLD-1 leads to ectopic expression of its targets, such as Cyclin E and the somatic determinant PAL-1/Caudal. Ectopic expression of Cyclin E promotes re-entry into mitosis and a change in the transcriptional profile of the cell, which creates an environment that allows a somatic determinant to promote germ line to soma transition. The importance of this finding is that it is not only the loss of translational control that leads to teratoma formation, but also a change in the transcriptional competence of the cells, and it emphasizes the importance of cell cycle control during meiosis as a fundamental mechanism to maintain germline identity

Pathway to Totipotency: Lessons from Germ Cells

Oocytes and sperm are some of the most differentiated cells in our bodies, yet they generate all cell types after fertilization. Accumulating evidence suggests that this extraordinary potential is conferred to germ cells from the time of their formation during embryogenesis. In this Review, we describe common themes emerging from the study of germ cells in vertebrates and invertebrates. Transcriptional repression, chromatin remodeling, and an emphasis on posttranscriptional gene regulation preserve the totipotent genome of germ cells through generations

RNA Recognition by the Caenorhabditis elegans Embryonic Determinants MEX-5 and MEX-3: A Dissertation

Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development. MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly. This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development

Conversion of germ cells to somatic cell types in C. elegans

The potential of a cell to produce all types of differentiated cells in an organism is termed totipotency. Totipotency is an essential property of germ cells, which constitute the germline and pass on the parental genetic material to the progeny. The potential of germ cells to give rise to a whole organism has been the subject of intense research for decades and remains important in order to better understand the molecular mechanisms underlying totipotency. A better understanding of the principles of totipotency in germ cells could also help to generate this potential in somatic cell lineages. Strategies such as transcription factor-mediated reprogramming of differentiated cells to stem cell-like states could benefit from this knowledge. Ensuring pluripotency or even totipotency of reprogrammed stem cells are critical improvements for future regenerative medicine applications. The C. elegans germline provides a unique possibility to study molecular mechanisms that maintain totipotency and the germ cell fate with its unique property of giving rise to meiotic cells Studies that focused on these aspects led to the identification of prominent chromatin-repressing factors such as the C. elegans members of the Polycomb Repressive Complex 2 (PRC2). In this review, we summarize different factors that were recently identified, which use molecular mechanisms such as control of protein translation or chromatin repression to ensure maintenance of totipotency and the germline fate. Additionally, we focus on recently identified factors involved in preventing transcription-factor-mediated conversion of germ cells to somatic lineages. These so-called reprogramming barriers have been shown in some instances to be conserved with regard to their function as a cell fate safeguarding factor in mammals. Overall, continued studies assessing the different aspects of molecular pathways involved in maintaining the germ cell fate in C. elegans may provide more insight into cell fate safeguarding mechanisms also in other species

Induced neurons from germ cells in Caenorhabditis elegans

Cell fate conversion by the forced overexpression of transcription factors (TFs) is a process known as reprogramming. It leads to de-differentiation or trans-differentiation of mature cells, which could then be used for regenerative medicine applications to replenish patients suffering from, e.g., neurodegenerative diseases, with healthy neurons. However, TF-induced reprogramming is often restricted due to cell fate safeguarding mechanisms, which require a better understanding to increase reprogramming efficiency and achieve higher fidelity. The germline of the nematode Caenorhabditis elegans has been a powerful model to investigate the impediments of generating neurons from germ cells by reprogramming. A number of conserved factors have been identified that act as a barrier for TF-induced direct reprogramming of germ cells to neurons. In this review, we will first summarize our current knowledge regarding cell fate safeguarding mechanisms in the germline. Then, we will focus on the molecular mechanisms underlying neuronal induction from germ cells upon TF-mediated reprogramming. We will shortly discuss the specific characteristics that might make germ cells especially fit to change cellular fate and become neurons. For future perspectives, we will look at the potential of C. elegans research in advancing our knowledge of the mechanisms that regulate cellular identity, and what implications this has for therapeutic approaches such as regenerative medicine

Gene Regulation: A Tale of Germline mRNA Tails

SummaryGene regulation often plays by different rules in the germline compared to the soma. In Caenorhabditis elegans, the spatial and temporal expression of germline genes is controlled post-transcriptionally via the 3′ UTR rather than transcriptionally via the promoter

Conserved Nucleosome Remodeling/Histone Deacetylase Complex and Germ/Soma Distinction in \u3cem\u3eC. elegans\u3c/em\u3e: A Dissertation

A rapid cascade of regulatory events defines the differentiated fates of embryonic cells, however, once established, these differentiated fates and the underlying transcriptional programs can be remarkably stable. Here, we describe two proteins, MEP-1, a novel protein, and LET-418/Mi-2, both of which are required for the maintenance of somatic differentiation in C. elegans. MEP-1 was identified as an interactor of PIE-1, a germ-specific protein required for germ cell specification, while LET-418 is a protein homologous to Mi-2, a core component of the nuc1eosome remodeling/histone deacetylase (NuRD) complex. In animals lacking MEP-1 and LET-418, germline-specific genes become derepressed in somatic cells, and Polycomb group (PcG) and SET domain-related proteins promote this ectopic expression. We demonstrate that PIE-1 forms a complex with MEP-1, LET-418, and HDA-1. Furthermore, we show that the overexpression of PIE-1 can mimic the mep-1/let-418 phenotype, and that PIE-1 can inhibit the Histone deacetylase activity of the HDA-1 complex in COS cells. Our findings support a model in which PIE-1 transiently inhibits MEP-1 and associated factors to maintain the pluripotency of germ cells, while at later times MEP-1 and LET-418 remodel chromatin to establish new stage- or cell-type-specific differentiation potential

The endogenous mex-3 3 UTR is required for germline repression and contributes to optimal fecundity in C. elegans

RNA regulation is essential to successful reproduction. Messenger RNAs delivered from parent to progeny govern early embryonic development. RNA-binding proteins (RBPs) are the key effectors of this process, regulating the translation and stability of parental transcripts to control cell fate specification events prior to zygotic gene activation. The KH-domain RBP MEX-3 is conserved from nematode to human. It was first discovered in Caenorhabditis elegans, where it is essential for anterior cell fate and embryo viability. Here, we show that loss of the endogenous mex-3 3 UTR disrupts its germline expression pattern. An allelic series of 3 UTR deletion variants identify repressing regions of the UTR and demonstrate that repression is not precisely coupled to reproductive success. We also show that several RBPs regulate mex-3 mRNA through its 3 UTR to define its unique germline spatiotemporal expression pattern. Additionally, we find that both poly(A) tail length control and the translation initiation factor IFE-3 contribute to its expression pattern. Together, our results establish the importance of the mex-3 3 UTR to reproductive health and its expression in the germline. Our results suggest that additional mechanisms control MEX-3 function when 3 UTR regulation is compromised

Characterizing a role for CoREST (SPR-1) in regulating the function of LSD1 (SPR-5)

Mutations in histone modifying enzymes have dramatic effects on transmitting epigenetic information between generations. In C. elegans, the H3K4me2 demethylase, spr-5, and the H3K9me2 methyltransferase, met-2, synergize at fertilization to reprogram the epigenetic landscape and prevent the inappropriate expression of germ specific genes in the early zygote. If either enzyme is lost, developmental defects occur; both spr-5 and met-2 mutants display a progressive sterility phenotype. This phenotype is even more exacerbated in spr-5; met-2 double mutants, which exhibit maternal effect sterility and a severe developmental delay after a single generation. In mammals, orthologs of SPR-5, LSD1, and SPR-1, CoREST, physically associate, and in the absence of CoREST, LSD1 demethylase activity is impaired. In C. elegans, SPR-5 and SPR-1 interact in vitro, and worms deficient in SPR-5 or SPR-1 rescue the egg-laying defect observed in se1-12 mutants. These findings suggest that a more complex co-regulatory mechanism may exist where SPR-1 interacts with and regulates SPR-5 function. We hypothesize that this interaction is required at fertilization to ensure proper epigenetic reprogramming and development of the zygote. To test this hypothesis, we first compared progressive sterility in spr-1 vs. spr-5 mutants. Unlike spr-5 mutants, spr-1 worms do not become progressively sterile over many generations nor do they display any obvious morphological defects. Taking advantage of the severe developmental delay and sterility phenotypes observed in spr-5; met-2 mutants, we assessed whether these same phenotypes were present in met-2; spr-1 mutants. Interestingly, similar to spr-5; met-2 progeny, met-2; spr-1 progeny display sterility and developmental delay in a single generation, albeit not as severe as spr-5; met-2 mutants. While ~ 20% of met-2; spr-1 progeny are sterile in a single generation, we observe an increase to ~ 60% by generation 8, and this increase in sterility is accompanied by a decline in the total number of progeny. By generation 10, we observe an increase in the number met-2; spr-1 adult germlines that phenocopy the disorganized and under developed germline morphology that we observe in spr-5; met-2 mutants. Taken together, our data suggest SPR-1 regulates SPR-5 function, but also leaves open the possibility SPR-5 may interact with other proteins/complexes to program the epigenetic landscape at fertilization. With this, spr-1 emerges as an epigenetic player with implications for regulating epigenetic reprogramming and development

Multiple RNA-binding proteins function combinatorially to control the soma-restricted expression pattern of the E3 ligase subunit ZIF-1

AbstractIn C. elegans embryos, transcriptional repression in germline blastomeres requires PIE-1 protein. Germline blastomere-specific localization of PIE-1 depends, in part, upon regulated degradation of PIE-1 in somatic cells. We and others have shown that the temporal and spatial regulation of PIE-1 degradation is controlled by translation of the substrate-binding subunit, ZIF-1, of an E3 ligase. We now show that ZIF-1 expression in embryos is regulated by five maternally-supplied RNA-binding proteins. POS-1, MEX-3, and SPN-4 function as repressors of ZIF-1 expression, whereas MEX-5 and MEX-6 antagonize this repression. All five proteins bind directly to the zif-1 3′ UTR in vitro. We show that, in vivo, POS-1 and MEX-5/6 have antagonistic roles in ZIF-1 expression. In vitro, they bind to a common region of the zif-1 3′ UTR, with MEX-5 binding impeding that by POS-1. The region of the zif-1 3′ UTR bound by MEX-5/6 also partially overlaps with that bound by MEX-3, consistent with their antagonistic functions on ZIF-1 expression in vivo. Whereas both MEX-3 and SPN-4 repress ZIF-1 expression, neither protein alone appears to be sufficient, suggesting that they function together in ZIF-1 repression. We propose that MEX-3 and SPN-4 repress ZIF-1 expression exclusively in 1- and 2-cell embryos, the only period during embryogenesis when these two proteins co-localize. As the embryo divides, ZIF-1 continues to be repressed in germline blastomeres by POS-1, a germline blastomere-specific protein. MEX-5/6 antagonize repression by POS-1 and MEX-3, enabling ZIF-1 expression in somatic blastomeres. We propose that ZIF-1 expression results from a net summation of complex positive and negative translational regulation by 3′ UTR-binding proteins, with expression in a specific blastomere dependent upon the precise combination of these proteins in that cell