Van Rompay, Liesbeth et al. (2013) International Worm Meeting "Molecular genetic deciphering of the reproductive pathway in Caenorhabditis elegans."

Van Rompay, Liesbeth, Schoofs, Liliane, Beets, Isabel, Frooninckx, Lotte, Janssen, Tom, Temmerman, Liesbet

[International Worm Meeting, 2013]

In vertebrates, reproduction is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis. A number of well studied G protein-coupled receptors (GPCRs) are involved in this process. Kisspeptin/GPR54 signaling is vital to puberal development and vertebrate reproduction by regulating the hypothalamic release of gonadotropin-releasing hormone, which stimulates the release of follicle stimulating hormone and luteinizing hormone from the anterior pituitary. These hormones in turn bind to their specific GPCRs in the gonads leading to the production of sex steroids, the stimulation of gametogenesis and gamete release. Although genome sequencing data show that in Ecdysozoa putative orthologs occur of the components of the vertebrate HPG axis, its counterpart in C. elegans has only been scarcely studied. To unravel the C. elegans reproductive signaling system in more detail, we search for putative upstream molecular players that influence the process of vitellogenesis by applying both a forward and reverse genetic approach. An EMS mutagenesis screen was conducted using a transgenic strain that is able to express the integrated GFP reporter under the control of the vit-2 promoter. A handful of promising mutants showing significantly altered expression of gfp were identified by means of a single-step SNP mapping strategy coupled with whole-genome sequencing. A list of possible variants, including a low density lipoprotein receptor-related protein, a glutamate receptor, and an unannotated hypothetical protein, was retrieved by use of the CloudMap pipeline to analyze the mutant genome sequences. To further unveil the involvement of neuropeptidergic signaling, we carried out an additional RNAi screen of candidate genes, including all predicted neuropeptide GPCR and neuropeptide precursor genes. Genes originating from both screens will be thoroughly characterized by use of a range of morphological and physiological assays.

Van Rompay, Liesbeth et al. (2015) International Worm Meeting "Studying genetic control of yolk protein synthesis in relation to overall reproduction in C. elegans."

Beets, Isabel, Van Rompay, Liesbeth, Caers, Jelle, Borghgraef, Charline, Schoofs, Liliane, Temmerman, Liesbet

[International Worm Meeting, 2015]

Vitellogenesis, the process by which maternal yolk is formed, is thought to be a critical step in reproduction of all egg-laying animals. In vertebrates, the well-characterized hormonal hypothalamus-pituitary-gonad regulatory system drives reproductive success, including yolk protein synthesis by the liver, in a time- and sex-dependent manner. Whether similar control mechanisms exist for invertebrate reproduction, is an open question in surprisingly unexplored scientific territory. It can be expected that genetic control of reproduction in invertebrates will to a certain extent be similar to the vertebrate system, conform genome sequencing data, whereas many invertebrate-, clade- or species-specific factors are thought to exist as well - e.g. depending on distinct reproductive cycles. Therefore, we reasoned intestinal yolk protein production to be a suitable downstream readout to unravel the nematode reproductive axis in more detail. Using a GFP-tagged yolk protein as a reporter, we performed a forward genetic screen in C. elegans, focusing on mutants overtly defective in yolk protein production. Via a combined mapping and whole-genome sequencing strategy we identified five mutant alleles, corresponding to three novel regulatory genes involved in C. elegans vitellogenesis. We further confirmed that indeed, mutations in genes encoding a vitellogenin regulating Caenorhabditis-specific, C. elegans homeobox and low-density lipoprotein receptor related protein disrupt yolk protein synthesis at the transcriptional and translational level. Our data on each of these molecular players support the view that parallel pathways, some of which contain evolutionarily unique regulators, exert control over vitellogenesis in C. elegans. As opposed to general assumptions, we show that a virtually absent yolk protein pool does not necessarily imply a severely affected reproductive potential. This raises questions on the relevance and quantitative needs of yolk for reproduction. Further research, e.g. concerning the consistency of the yolk-depleted eggs and other reproduction-related defects, will be important to further raise our understanding of invertebrate control of reproduction.

Wolf FW et al. (1998) West Coast Worm Meeting "Yes! you can suppress vab-8"

Wolf FW, Garriga G

[West Coast Worm Meeting, 1998]

vab-8 acts in posteriorly directed cell and axon growth cone migrations in a cell autonomous manner. vab-8 functions in posterior ALM cell migration but not in later anterior ALM axon migration, and continuous vab-8 expression in the ALM can reorient anterior ALM axon outgrowth posteriorly. This finding suggests that the direction of migration can be regulated at the level of vab-8 activity. vab-8 encodes two isoforms of a novel intracellular protein, and the opal null allele e1017 is predicted to remove both activities. Our analysis of vab-8 has led us to propose that there exists at least one mechanism for the global guidance of posterior migrations. To identify other genes that function in posterior guidance, we suppressed the highly penetrant CAN cell migration defect of vab-8(e1017) mutants. Extragenic suppressors could identify genes that act downstream of vab-8 as well as genes that act in parallel pathways. From screening over 20,000 genomes we isolated four partially penetrant van (variably abnormal normal) suppressors, each defining a distinct gene. van-1(gm128) is a semi-dominant suppressor of vab-8 cell and axon outgrowth defects that is allele non-specific but at least so far is gene specific. Alas, van-1 has no phenotype on its own. In dosage studies van-1(gm128) appears to alter gene function. We have mapped van-1 to LGI between stu-4 and unc-11 and transformation rescue experiments are underway.

van Schendel, R. et al. (2017) International Worm Meeting "Polymerase theta is a key driver of genome evolution, of CRISPR/Cas9-mediated mutagenesis and of mutagen-induced deletion allele formation."

Tijsterman, M., van Schendel, R.

[International Worm Meeting, 2017]

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. We have recently demonstrated for two of the most widely used mutagens in C. elegans, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs) 1, 2. This discovery allowed us to survey many thousands of available deletion alleles, which reveals a step-wise and versatile model for the in vivo mechanism of TMEJ, explaining the molecular nature of mutagen-induced deletion alleles 3. We also found that CRISPR/Cas9-induced genomic changes are exclusively generated through POLQ action, refuting a previously assumed requirement for NHEJ in their formation; Unlike somatic cells, which use NHEJ to repair DNA breaks, germ cells use an alternative route 4. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification. New insights into the mechanism of genome engineering, genome maintenance and evolution will be discussed. 1. Koole,W. et al. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 5, 3216 (2014). 2. Roerink,S.F., van Schendel,R., & Tijsterman,M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954-962 (2014). 3. van Schendel R., van Heteren J., Welten,R., & Tijsterman,M. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining. PLoS. Genet. 12, e1006368 (2016). 4. van Schendel,R., Roerink,S.F., Portegijs,V., van den Heuvel,S., & Tijsterman,M. Polymerase Theta is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat. Commun. 6, 7394 (2015).

van Luenen HGAM et al. (1995) International C. elegans Meeting "Mechanism of Tc3 transposition"

van Luenen HGAM, Plasterk RHA

[International C. elegans Meeting, 1995]

The strain Bristol N2 has no endogenous Tc3 transposition activity, but forced expression of Tc3 transposase results in somatic transposition of endogenous Tc3 elements as well as Tc3 elements located on a transgene (Van Luenen et al. 1993 EMBO J. 12:2513-2520). This provides us with an assay for the requirements of Tc3 transposition in vivo.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

Galvin BD et al. (2000) East Coast Worm Meeting "Two screens for new genes involved in programmed cell death"

Galvin BD, Bob Horvitz, Cameron S

[East Coast Worm Meeting, 2000]

Many genes involved in programmed cell death in C. elegans have been identified by screening for mutations that allow survival of cells that are normally destined to die. The opposite approach, screening for mutations that result in the deaths of cells, has been comparatively unexplored, in part because of a lack of efficient methods to identify such mutations. A lin-11::gfp reporter (see abstract by Cameron and Horvitz) expresses in Pn.aap cells. In wild-type animals, only the six Pn.aap cells of the P3-P8 lineages survive and express GFP, while in mutants defective for cell killing all 12 Pn.aap cells survive and express GFP. A strain carrying this reporter allows one to monitor the survival of cells using a dissecting microscope rather than Nomarski optics, greatly facilitating screening. In this way, I am performing a ced-4 suppression screen by looking for a reduction in the number GFP-positive cells and thus an increase in cell death in the ventral cords of partial loss-of-function ced-4 mutants. To identify substrates of the caspase CED-3, which is required for programmed cell death, I am using a modified two-hybrid screen. An active caspase is composed of two subunits proteolytically derived from an inactive procaspase. The active site of the caspase contains a cysteine, which when mutated prevents the cleavage activity of the enzyme. In this screen the two active subunits of the CED-3 caspase are expressed separately, with the active-site cysteine changed to a serine. This amino acid substitution should permit an interaction with substrates but prevent their cleavage, allowing for the identification of interacting proteins. Other groups have identified substrates of mammalian caspases using this approach (1, 2). Candidates from the screen will be analyzed by testing in vitro cleavage by CED-3 and assayed by RNAi to examine their roles in programmed cell death. 1. Van Criekinge, W., van Gurp, M., Decoster, E., Schotte, P., Van de Craen, M., Fiers, W., Vandenabeele, P., and Beyaert, R. Analytical Biochemistry. 263, 62-66 (1998). 2. Kamada, S, Kusano, H., Fujita, H., Ohtsu, M., Koya, R. C., Kuzumaki, N., and Tsujimoto, Y. Proc. Natl. Acad. Sci. USA. 95, 8532-8537 (1998).

Seidel, Hannah et al. (2011) International Worm Meeting "Questioning Adult Reproductive Diapause."

Kimble, Judith, Seidel, Hannah

[International Worm Meeting, 2011]

Upon starvation, some C. elegans L4 hermaphrodites do not 'bag' as adults but instead survive long-term, reportedly via a regulatory program termed adult reproductive diapause (ARD) (Angelo and Van Gilst, 2009). Key features of ARD include degeneration of most germ cells, embryonic arrest in utero (as evidenced by early-stage embryos visible 3-5 days after starvation), and the ability to regenerate a functional germline upon refeeding (Angelo and Van Gilst, 2009). We have confirmed most but not all of these observations. We removed food at ~2hr intervals from early- to late-stage L4 hermaphrodites, and over the next several days, we scored these animals for germline shrinkage, oocyte production, and embryo viability. After starvation of early L4s, germlines shrank and a fraction of animals survived long-term, as expected for ARD; also as expected, the depleted germlines regenerated after refeeding. However, two observations lead us to question the proposed embryonic arrest associated with ARD. First, in animals starved from early L4, oogenesis was severely delayed, with many animals producing their first oocyte only 3-5 days into adulthood. This delay in oogenesis suggests that the early-stage embryos visible 3-5 days after starvation in putative ARD animals may reflect recent fertilization events, rather than embryonic arrest. Second, the delayed oocytes made viable embryos in some cases but dead embryos in others. This increase in embryo lethality provides an alternate explanation for the ability of some animals to avoid the bagging fate and survive long-term. Furthermore, we found that germline shrinkage was not unique to animals in putative ARD: hermaphrodites starved from late L4 uniformly bagged as adults, yet prior to bagging, these animals nevertheless possessed shrunken germlines much like the germlines reported for ARD. From these observations, we propose that all L4 hermaphrodites respond to starvation equivalently: the oogenic germline degenerates and shrinks; the animal produces as many embryos as possible, given the nutrients available; and the viability of these embryos determines long-term survival. We propose that when starvation begins in late L4, embryos are typically viable, and the animal dies via bagging; when starvation begins earlier in L4, embryos are often inviable, and this inviability allows some animals to survive long-term. We are now performing additional experiments to test this possibility. [Angelo G, Van Gilst M (2009) Starvation protects germline stem cells and extends reproductive longevity in C. elegans. Science 326: 954-958.].

Gems DH et al. (1995) International C. elegans Meeting "MATING BUT NOT GAMETE PRODUCTION REDUCES LONGEVITY"

Riddle DL, Gems DH

[International C. elegans Meeting, 1995]

We have studied the effect of mating on the life span of males and hermaphrodites. Although male life span is unaffected by mating, hermaphrodite life span is reduced. These results are opposite to those reported by Van Voorhies (Nature 360:456-458, 1992), who observed a cost of reproduction to C. elegans males but not hermaphrodites. However, the strain used in that study appears to be a variant of the wild-type N2 strain with reduced longevity and reduced brood size. Using the wild-type CGC reference N2 and the conditions used by Van Voorhies, we observed that male life span was unaffected by sexual activity, whereas average hermaphrodite life span was reduced by 36% from 16.90.6 to 10.90.7 days. Hermaphrodite reproductive costs were also found to vary with the dosage of males. When the brood sizes of individual, briefly mated hermaphrodites were compared to their life spans, little correlation was seen between egg production and life span. A large decrease in hermaphrodite life span was also caused by mating with fer-6 males. (The fer-6 males copulate, but neither stimulate oocyte production nor transfer sperm.) We then tested whether internal hatching causes early hermaphrodite mortality. Mutant fog-2(q71) females produce no sperm, so mating with fer-6 males at 25C produces very few or no fertilized eggs. In two experiments using a male to hermaphrodite ratio of 2:1 a cost of mating was observed similar to that observed with N2 males, indicating that neither transfer of sperm nor internal hatching of larvae is the cause of reduced hermaphrodite life span. These results suggest that, as in fruit flies, male secretions which enhance male reproductive fitness may reduce life span in the opposite sex. However, they do not support the hypothesis that increased gamete production accelerates aging.

Rankin, Aysha C. et al. (2015) International Worm Meeting "Genes involved in embryonic motor neuron positioning along the AP axis."

Tanner, Matthew, Slatculescu, Cristina, Robinson, Claire, Rankin, Aysha C., Colavita, Antonio, Perkins, Theodore

[International Worm Meeting, 2015]

The six embryonically-derived DD motor neurons are born evenly spaced along the AP axis in newly hatched larvae and maintain this spacing into adulthood. We have found that components of a planar cell polarity (PCP)-like pathway that includes prkl-1/Prickle and vang-1/Van Gogh are important for properly positioning DD motor neurons in the ventral nerve cord. In prkl-1, and to a lesser extent, vang-1 mutants, DD neurons are shifted anteriorly and display distinctive cell spacing defects. These defects can be rescued by prkl-1 or vang-1 expression from a pan-neuronal promoter suggesting cell-autonomous roles. DA and DB neurons are also mispositioned in prkl-1 and vang-1 mutants. Neuronal positioning defects are present in newly hatched larvae suggesting an embryonic origin for the defect. DD1,3,5 and DD2,4,5, derived from left and right sides of the embryo, undergo cell intercalation during the comma stage to form a single line of DD neurons at the midline. prkl-1 and vang-1 mutants display DD intercalation defects that are suggestive of a delay in cell intercalation or improper cell-cell neighbor interactions during intercalation. In order to identify new genes involved in cell intercalation and the proper positioning of motor neurons along the AP axis as well as new insight into PCP-like signaling we are in the process of performing a forward genetic screen for DD neuron spacing defects. PCP signaling is known to play an important role in cell intercalation during neural tube and organ morphogenesis in vertebrates. Our findings show for the first time that a PCP-like pathway involving the core components Prickle and Van Gogh is involved in some aspects of cell intercalation during C. elegans development.