Caenorhabditis Elegans Germline Research Articles

Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation

Here we describe an invitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both invitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation invitro and invivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.

Regulation of wago‐1: An Examination of the Regulatory Architecture that Governs Argonaute Protein Expression in Caenorhabditis elegans

Across eukaryotes, thousands of small noncoding RNA molecules, in complex with Argonaute family proteins, silence or license gene expression through RNA interference (RNAi) and related processes. Since its discovery in 1998, RNAi has emerged as an invaluable tool for functional genomics, a critical player in regulation of endogenous gene expression, and a promising route to new therapeutics. Despite the important role RNAi plays in the regulation of gene expression, little is known about how the Argonaute effector proteins involved are themselves regulated. As a foray into understanding the regulation of the Argonaute protein network, we have chosen to examine the developing germ line of the nematode Caenorhabditis elegans, where at least five Argonautes are expressed in overlapping patterns. Presumably because the genome is being prepared for passage to the next generation and is relatively inaccessible to transcription factors, gene expression in the germ lineages of many animals is thought to rely heavily on posttranscriptional regulation. In C. elegans, a systematic study of 30 genes whose expression is known to be regulated in the germ line revealed that regulated gene expression in developing oocytes relied almost exclusively on sequences in the 3′ untranslated region (UTR), while sperm‐specific expression required promoter sequences but was largely independent of 3′ UTRs [2]. We therefore hypothesize that proper patterns of Argonaute expression in the germ line will depend both on promoter sequences and the 3′UTR. Using molecular cloning methods combined with CRISPR/Cas9‐mediated genome engineering based, we are using a GFP reporter transgene strategy to test this hypothesis. Thus far, I have generated a series of four reporter constructs that will allow me to individually assess contributions of the promoter and 3′UTR to regulation of the Argonaute protein WAGO‐1, which is expressed both in developing sperm and developing oocytes. I have designed and constructed CRISPR sgRNA sequences to guide integration of these transgenes into a defined locus on Chromosome II known to support germline expression, and am currently performing microinjection to establish transgenic worm strains. This research will provide the first insight into the mechanisms that regulate Argonaute protein expression during germ cell development, and will also advance methods for generating CRISPR‐integrated reporter transgenes in C. elegans.

The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

A Transport Model for Estimating the Time Course of ERK Activation in the C. elegans Germline

The Caenorhabditis elegans germline is a well-studied model system for investigating the control of cell fate by signaling pathways. Cell signals at the distal tip of the germline promote cell proliferation; just before the loop, signals couple cell maturation to organism-level nutrient status; at the proximal end of the germline, signals coordinate oocyte maturation and fertilization in the presence of sperm. The latter two events require dual phosphorylation and activation of ERK, the effector molecule of the Ras/MAPK cascade. In C. elegans, ERK is known as MPK-1. At this point, none of today’s methods for real-time monitoring of dually phosphorylated MPK-1 are working in the germline. Consequently, quantitative understanding of the MPK-1-dependent processes during germline development is limited. Here, we make a step toward advancing this understanding using a model-based framework that reconstructs the time course of MPK-1 activation from a snapshot of a fixed germline. Our approach builds on a number of recent studies for estimating temporal dynamics from fixed organisms, but takes advantage of the anatomy of the germline to simplify the analysis. Our model predicts that the MPK-1 signal turns on ∼30 h into germ cell progression and peaks ∼7 h later.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems.

Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch.

Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for facultative stem cell quiescence. We find that mitotically dividing germ cells--including germline stem cells--become quiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a block to M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, we demonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance: The signaling pathway required for stem cell maintenance under fed conditions--GLP-1/Notch signaling--becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence can itself maintain stem cells, independent of the signaling pathway otherwise essential for such maintenance.

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling.

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase's role in germline development in higher eukaryotes.

Transdifferentiation mediated tumor suppression by the endoplasmic reticulum stress sensor IRE-1 in C. elegans.

Deciphering effective ways to suppress tumor progression and to overcome acquired apoptosis resistance of tumor cells are major challenges in the tumor therapy field. We propose a new concept by which tumor progression can be suppressed by manipulating tumor cell identity. In this study, we examined the effect of ER stress on apoptosis resistant tumorous cells in a Caenorhabditis elegans germline tumor model. We discovered that ER stress suppressed the progression of the lethal germline tumor by activating the ER stress sensor IRE-1. This suppression was associated with the induction of germ cell transdifferentiation into ectopic somatic cells. Strikingly, transdifferentiation of the tumorous germ cells restored their ability to execute apoptosis and enabled their subsequent removal from the gonad. Our results indicate that tumor cell transdifferentiation has the potential to combat cancer and overcome the escape of tumor cells from the cell death machinery.

Control of Caenorhabditis elegans germ-line stem-cell cycling speed meets requirements of design to minimize mutation accumulation.

BackgroundStem cells are thought to play a critical role in minimizing the accumulation of mutations, but it is not clear which strategies they follow to fulfill that performance objective. Slow cycling of stem cells provides a simple strategy that can minimize cell pedigree depth and thereby minimize the accumulation of replication-dependent mutations. Although the power of this strategy was recognized early on, a quantitative assessment of whether and how it is employed by biological systems is missing.ResultsHere we address this problem using a simple self-renewing organ – the C. elegans gonad – whose overall organization is shared with many self-renewing organs. Computational simulations of mutation accumulation characterize a tradeoff between fast development and low mutation accumulation, and show that slow-cycling stem cells allow for an advantageous compromise to be reached. This compromise is such that worm germ-line stem cells should cycle more slowly than their differentiating counterparts, but only by a modest amount. Experimental measurements of cell cycle lengths derived using a new, quantitative technique are consistent with these predictions.ConclusionsOur findings shed light both on design principles that underlie the role of stem cells in delaying aging and on evolutionary forces that shape stem-cell gene regulatory networks.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0148-y) contains supplementary material, which is available to authorized users.

Emergent Stem Cell Homeostasis in the C. elegans Germline Is Revealed by Hybrid Modeling

The establishment of homeostasis among cell growth, differentiation, and apoptosis is of key importance for organogenesis. Stem cells respond to temporally and spatially regulated signals by switching from mitotic proliferation to asymmetric cell division and differentiation. Executable computer models of signaling pathways can accurately reproduce a wide range of biological phenomena by reducing detailed chemical kinetics to a discrete, finite form. Moreover, coordinated cell movements and physical cell-cell interactions are required for the formation of three-dimensional structures that are the building blocks of organs. To capture all these aspects, we have developed a hybrid executable/physical model describing stem cell proliferation, differentiation, and homeostasis in the Caenorhabditis elegans germline. Using this hybrid model, we are able to track cell lineages and dynamic cell movements during germ cell differentiation. We further show how apoptosis regulates germ cell homeostasis in the gonad, and propose a role for intercellular pressure in developmental control. Finally, we use the model to demonstrate how an executable model can be developed from the hybrid system, identifying a mechanism that ensures invariance in fate patterns in the presence of instability.

Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3

The Caenorhabditis elegans germ-line development defective (GLD)-2-GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2-GLD-3 acts by extending the short poly(A) tail of germ-line-specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3' end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3' end features of dormant cytoplasmic mRNAs.

New Insights into the Post-Translational Regulation of DNA Damage Response and Double-Strand Break Repair in Caenorhabditis elegans.

Although a growing number of studies have reported the importance of SUMOylation in genome maintenance and DNA double-strand break repair (DSBR), relevant target proteins and how this modification regulates their functions are yet to be clarified. Here, we analyzed SUMOylation of ZTF-8, the homolog of mammalian RHINO, to test the functional significance of this protein modification in the DSBR and DNA damage response (DDR) pathways in the Caenorhabditis elegans germline. We found that ZTF-8 is a direct target for SUMOylation in vivo and that its modification is required for DNA damage checkpoint induced apoptosis and DSBR. Non-SUMOylatable mutants of ZTF-8 mimic the phenotypes observed in ztf-8 null mutants, including reduced fertility, impaired DNA damage repair, and defective DNA damage checkpoint activation. However, while mutants for components acting in the SUMOylation pathway fail to properly localize ZTF-8, its localization is not altered in the ZTF-8 non-SUMOylatable mutants. Taken together, these data show that direct SUMOylation of ZTF-8 is required for its function in DSBR as well as DDR but not its localization. ZTF-8's human ortholog is enriched in the germline, but its meiotic role as well as its post-translational modification has never been explored. Therefore, our discovery may assist in understanding the regulatory mechanism of this protein in DSBR and DDR in the germline.

Syncytium biogenesis: It's all about maintaining good connections.

At the end of mitosis, cells typically complete their division with cytokinesis. In certain tissues however, incomplete cytokinesis can give rise to cells that remain connected by intercellular bridges, thus forming a syncytium. Examples include the germline of many species, from fruitfly to humans, yet the mechanisms regulating syncytial formation and maintenance is unclear, and the biological relevance of syncytial organization remains largely speculative. To better understand these processes, we recently used the germline of Caenorhabditis elegans as a model for syncytium development. Analysis of the germline syncytial architecture throughout development revealed that it arises progressively during larval growth and that it relies on the activity of 2 actomyosin scaffold proteins of the Anillin family. Our work also showed that the gonad can sustain elastic deformation when under mechanical stress and that this property may be conferred by the malleability of syncytial openings. We suggest that elasticity and resistance to mechanical stress constitutes a general property of syncytial tissues.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems.

A signaling-induced switch in dicer localization and function.

In this issue of Developmental Cell, Drake and colleagues (2014) report that Ras signaling results in Dicer phosphorylation, which induces its nuclear localization and modulates its function. This regulatory strategy, conserved in mammals, allows dynamic control of microRNA function required for Caenorhabditis elegans germline development and oogenesis.

The SET-2/SET1 Histone H3K4 Methyltransferase Maintains Pluripotency in the Caenorhabditis elegans Germline

Histone H3 Lys 4 methylation (H3K4me) is deposited by the conserved SET1/MLL methyltransferases acting in multiprotein complexes, including Ash2 and Wdr5. Although individual subunits contribute to complex activity, how they influence gene expression in specific tissues remains largely unknown. InCaenorhabditis elegans, SET-2/SET1, WDR-5.1, and ASH-2 are differentially required for germline H3K4 methylation. Using expression profiling on germlines from animals lacking set-2, ash-2, or wdr-5.1, we show that these subunits play unique as well as redundant functions in order to promote expression of germline genes and repress somatic genes. Furthermore, we show that in set-2- and wdr-5.1-deficient germlines, somatic gene misexpression is associated with conversion of germ cellsinto somatic cells and that nuclear RNAi acts in parallel with SET-2 and WDR-5.1 to maintain germline identity. These findings uncover a unique role forSET-2 and WDR-5.1 in preserving germline pluripotency and underline the complexity of the cellular network regulating this process.

C. elegans Anillin proteins regulate intercellular bridge stability and germline syncytial organization.

Cytokinesis generally produces two separate daughter cells, but in some tissues daughter nuclei remain connected to a shared cytoplasm, or syncytium, through incomplete cytokinesis. How syncytia form remains poorly understood. We studied syncytial formation in the Caenorhabditis elegans germline, in which germ cells connect to a shared cytoplasm core (the rachis) via intercellular bridges. We found that syncytial architecture initiates early in larval development, and germ cells become progressively interconnected until adulthood. The short Anillin family scaffold protein ANI-2 is enriched at intercellular bridges from the onset of germ cell specification, and ANI-2 loss resulted in destabilization of intercellular bridges and germ cell multinucleation defects. These defects were partially rescued by depleting the canonical Anillin ANI-1 or blocking cytoplasmic streaming. ANI-2 is also required for elastic deformation of the gonad during ovulation. We propose that ANI-2 promotes germ cell syncytial organization and allows for compensation of the mechanical stress associated with oogenesis by conferring stability and elasticity to germ cell intercellular bridges.

A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate

Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.

Protein kinase CK2 both promotes robust proliferation and inhibits the proliferative fate in the C. elegans germ line

Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. In this study, we describe a large-scale RNAi screen that identified kin-10, which encodes the β subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. We found that kin-10’s regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. We propose that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.

A role for WDR5 in TRA-1/Gli mediated transcriptional control of the sperm/oocyte switch in C. elegans

The hermaphrodite germline of Caenorhabditis elegans initially engages in spermatogenesis and then switches to oogenesis during late stages of larval development. TRA-1, a member of the Ci/Gli family of transcriptional repressors, plays an essential role in this switch by repressing genes that promote spermatogenesis. WDR5 proteins are conserved components of histone methyltransferase complexes normally associated with gene activation. However, two C. elegans WDR5 homologs, wdr-5.1 and wdr-5.2 are redundantly required for normal TRA-1 dependent repression, and this function is independent of their roles in histone methylation. Animals lacking wdr-5.1/wdr-5.2 function fail to switch to oogenesis at 25°C, resulting in a masculinization of germline (Mog) phenotype. The Mog phenotype is caused by ectopic expression of fog-3, a direct target of TRA-1 repression. WDR-5.1 associates with the fog-3 promoter and is required for TRA-1 to bind to fog-3 promoter. Other direct targets of TRA-1 are similarly derepressed in the double mutant. These results show that WDR5 plays a novel and important role in stabilizing transcriptional repression during C. elegans sex determination, and provide evidence that this important protein may operate independently of its established role in histone methyltransferase complexes.