Loose, Julia et al. (2019) International Worm Meeting "Impact of Meiotic-Gene Mutations on Lifespan and Health Span."

Ghazi, Arjumand, Yanowitz, Judith, Patil, Thayjas, Loose, Julia

[International Worm Meeting, 2019]

While the connection between increased maternal age and fertility loss is well established, it is unclear how the decline in germline integrity impacts aging. In addition, the mechanisms governing how germline fidelity impacts fertility loss and somatic aging have not been elucidated. These questions are challenging to address through human studies and vertebrate models. We are utilizing Caenorhabditis elegans to address this relationship. We screened 35 mutants impacting genes involved in different stages of meiosis, a germline-restricted phenomenon, for lifespan phenotypes. We found 24 mutants to be significantly short lived. To analyze the impact of meiotic fidelity on health span, the qualitative measure of rate of aging, we are assessing various physiological and behavioral parameters that decline with age, including neural deficits, in meiotic-gene mutants. We employed a widely-used chemotaxis assay to measure how short-term associative memory (STAM) changes with age and in the presence of various meiotic mutations. Compared to the wild type, all meiotic mutants showed a decreased ability to retain STAM from young adulthood (Day 1) to middle age (Day 5). Overall, our data indicates that mutations that govern meiotic fidelity in the germline impact health span, specifically, cognitive performance with age. Future studies will examine the specific mechanisms by which meiotic integrity influences somatic aging.

Loose, Julia et al. (2021) International Worm Meeting "Meiotic mutations impact lifespan and healthspan in C. elegans"

Patil, Thayjas, Loose, Julia, Yanowitz, Judith, Ghazi, Arjumand

[International Worm Meeting, 2021]

Meiotic chromosomal defects increase with age and are a major cause of miscarriages and age-related fertility loss in women. The link between maternal age and fertility has been well studied, however it is unknown if, and, how germline integrity impacts the rate of aging of the whole organism. We are utilizing the nematode model Caenorhabditis elegans to address the causative role of germline health on somatic aging. Specifically, we have examined genes that govern meiotic fidelity in the germline and addressed their role in somatic aging. C. elegans is uniquely suited to address this question as the somatic cells in the adult organism are post mitotic, allowing us to selectively disrupt germline integrity through meiotic gene mutations. We found that 14 of the 38 mutations we examined, in genes that govern different steps of meiosis, significantly reduced the lifespan of the animal. We also found that germline-specific RNAi knockdown of selected meiotic gene candidates, spo-11, dsb-2, and htp-3, also reduced lifespan. These genes have roles throughout meiosis including double strand break formation during the process of meiotic recombination (spo-11 and dsb-2) and the formation of the synaptonemal complex (htp-3). We also examined how meiotic gene disruption impacts the rate of aging by measuring healthpsan parameters. We found mutations in all of the selected candidate meiotic genes, exhibited deficits in at least one, and often more than one, healthspan feature, including loss of mobility, diminished pharyngeal muscle pumping, tissue integrity and neurological function. Overall, our data demonstrate that genes that govern meiotic fidelity in the C. elegans germline impact the physiological health of the somatic tissues and aging of the whole organism.

Amrit, Francis RG et al. (2021) International Worm Meeting "TCER-1-regulated alternative splicing promotes stress resilience"

Ghazi, Arjumand, Amrit, Francis RG, KUROYANAGI, Hidehito, Loose, Julia, Pfenning, Andreas

[International Worm Meeting, 2021]

Alternative RNA splicing augments proteomic biodiversity in eukaryotes, and has an emerging role in regulating longevity. This process has been shown to have various roles based on physiological demands including those in response to external stimuli. Here, we show that in the nematode Caenorhabiditis elegans, exposure to the Gram-negative pathogen, Pseudomonas aeruginosa, induces a shift in alternative splicing. In vivo analysis of transgenic reporters of splicing fidelity showed significant splicing changes in multiple tissues. Based on our recent discovery that TCER-1, worm homolog of the human transcription elongation and splicing factor, TCERG1, represses innate immunity in worms, we examined the role of TCER-1 in the modulation of splicing upon pathogen exposure. tcer-1 mutants exhibited aberrant splicing regulation in multiple tissues, and transcriptomic analysis identified 54 exon-skipping and 468 intron retention events upon tcer-1 inactivation. In vivo analyses of these events revealed a TCER-1 dependent splicing pattern that correlated with our stress resilience phenotypes. Further, CRISPR-mediated recreation of alternative splicing observed in tcer-1 mutants enhanced immunoresistance and motor function. Together our findings bring to the forefront the important role of alternative splicing in stress resilience and healthspan, and reveal TCER-1- to be a major regulator of this process.

Ben Lehner et al. (2006) European Worm Meeting "Systematic Mapping of Genetic Interactions in C. elegans Suggests a New Paradigm for Human Genetic Disease"

Catriona Crombie, Andrew G. Fraser, Ben Lehner, Angelo Fortunato, Julia Tischler

[European Worm Meeting, 2006]

Ben Lehner, Catriona Crombie, Julia Tischler, Angelo Fortunato and Andrew G. Fraser Most heritable traits, including disease susceptibility, are affected by the interactions between multiple genes. However, we still understand very little about how genes interact since only a minute fraction of possible genetic interactions have been explored experimentally. To begin to address this, we are using RNA interference to identify genetic interactions in C. elegans, focussing on genes in signalling pathways that are mutated in human diseases. We tested ~65,000 pairs of genes for possible interactions and identify ~350 genetic interactions. This is the first systematically constructed genetic interaction map for any animal. We successfully rediscover most components of previously known signalling pathways; furthermore, we verify 9 novel modulators of EGF signalling. Crucially, our dataset also provides the first insight into the global structure of animal genetic interaction maps. Most strikingly, we identify a class of highly connected ''hub'' genes: inactivation of these genes greatly enhances phenotypes resulting from mutations in many different pathways. These hub genes all encode chromatin regulators, and their activity as genetic hubs appears conserved across metazoans. We propose that these genes function as general buffers of genetic variation and that these hub genes will act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.

Donnelly J L et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Tyraminergic Inhibition of GABA Release Facilitates Turning Behavior in the C. elegans Escape Response"

Donnelly J L, Pirri J K, Alkema M J, Clark C M

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Gentle anterior touch induces an escape response in which the nematode reverses, suppresses head oscillations, and is often followed by a deep ventral bend (omega turn), and reinitiation of forward locomotion in the opposite direction. Tyramine plays a key role in the C. elegans escape response. We have shown that the tyramine-gated chloride channel, LGC-55, coordinates the suppression of head oscillations and the long reversal during the initial phase of the escape response. In a genetic screen for mutants that are resistant to the paralytic effects of exogenous tyramine, we isolated alleles of the Gαo signaling pathway, dgk-1 and goa-1. We therefore tested mutants for the three G-protein coupled tyramine receptors: SER-2, TYRA-2, and TYRA-3. Whereas lgc-55 mutants are resistant to the paralytic effects of exogenous tyramine on head movements, we found that ser-2 mutants are resistant to the paralytic effects on body movements. Genetic and pharmacological analysis indicates that SER-2 is coupled to the Gαo (GOA-1) pathway, which has been shown to inhibit neurotransmission. We found that SER-2 is expressed in the VD GABAergic motor neurons, indicating that SER-2 might inhibit GABA release onto the ventral body muscles. Omega turns are central to the escape response and may require the asymmetric contraction of ventral and dorsal muscle quadrants. Upon anterior touch ser-2 mutants initiate the escape response normally, but fail to fully contract the ventral side of their body, resulting in a loose omega turn. We further analyzed the role of GABAergic VD and DD motor neurons in locomotion and turning by laser ablation. Animals lacking VD motor neurons moved with a ventral bias and were able to fully contract the ventral musculature during an omega turn, while animals lacking DD motor neurons moved with a dorsal bias and could not relax the dorsal musculature, resulting in a loose omega turn. Our results indicate that the tyraminergic inhibition of GABA release on the ventral side allows the animal to engage in an efficient escape and steer away from the stimulus. This suggests that tyramine plays an essential role in the temporal control of the escape response through the synaptic activation of fast acting ionotropic receptor, LGC-55, and extra synaptic activation of slow acting metabotropic receptor, SER-2.

Carstensen, H. R. et al. (2017) International Worm Meeting "Characterization of a dauer constitutive mutant with dauer-specific molting defect in Pristionchus pacificus."

Villalon, R. M., Carstensen, H. R., Hong, R. L.

[International Worm Meeting, 2017]

In contrast to the unsheathed dauer larvae (DL) in Caenorhabditis species, the DL of parasitic nematodes often retain their old J2 cuticles, which may prevent desiccation outside of the host. Ensheathed DL are also found in Pristionchus pacificus, a necromenic nematode associated with beetles. To gain a better understanding of the mechanism coordinating dauer entry and the dauer-specific molt, as well as the evolutionary developmental changes between free-living and parasitic nematodes, we have characterized the dauer-constitutive mutant tu391. We found that tu391 is a temperature sensitive daf-c allele that exhibits a molting defect only in the J2-dauer larvae molt. The small fraction of tu391 mutants that are stuck within loose cuticles exhibit significantly slower pharyngeal pumping than J3s of either strain, suggesting that these immobilized animals are dauer larvae. Detailed examination of the molting-defective animals shows the old J2 cuticle is too loose to allow locomotion on OP50, in contrast to the J2 cuticle of active wild-type or daf-c DL that is attached tightly to the head and tail. Furthermore, amphid neurons in tu391 mutants are DiI-filling defective, suggesting that defects in amphid neuron development contribute to its daf-c phenotype. To determine if tu391 also affects the glial cells that support the ontology and function of the amphid neurons, we are now examining the expression of the amphid sheath marker, Ppa-daf-6p::rfp. A possible defect in the amphid organ may also affect sensing of host odors. The beetle sex pheromone (z)-7-tetradecen-2-one (ZTDO) paralyzes P. pacificus DL, an effect that is intensified in the ZTDO-hypersensitive mutant obi-1. To determine if ZTDO affects tu391 DL, we exposed DL to various concentrations of ZTDO mixed into nematode growth media. As expected, most wild-type DL exited the dauer stage while most obi-1 DL died with ZTDO exposure. At low ZTDO concentration, nearly all tu391 mutants remained active DL after two days, indicating that they are not as sensitive to ZTDO as obi-1 mutants and that DL per se are not more sensitive. Interestingly, DL of obi-1; tu391 double mutants are less sensitive to the lethal effects of ZTDO than obi-1 mutants, suggesting that tu391 may ameliorate the ZTDO-hypersensitivity of obi-1 mutants due to defects in the amphid sensilla. Because P. pacificus shares traits with both free-living nematodes and parasites, further characterization of its DL development may give us insight into how nematodes acquire pre-adaptations to parasitism.

Oren Schaedel et al. (2007) International Worm Meeting "Single worm imaging using a microfluidic platform."

Changhuei Yang, Paul Sternberg, Oren Schaedel, Xiquan Cui, Lap Man Lee, Cladiu Giurumescu

[International Worm Meeting, 2007]

Quantifying dynamic gene regulation can help understand the structure and the mechanism of action of gene regulatory networks. In population studies, individuals loose synchrony since they grow at different rates, resulting in a phase shift of data points. Capturing dynamic processes in single cell methods have proved efficient in bacterial cultures and in cell lines. Quantitative single animal methods are starting to emerge using confocal microscopy and time series imaging. Its transparency, made C. elegans a good model for dynamic quantitation of gene expression. Unlike bacterial and tissue cultured cells that stay immobilized, the natural motility of C. elegans raises a challenge in capturing single worm resolution images, without resolving to drug based methods. To overcome this challenge, we are developing a microfluidic device which acts as a micro-environment. Using PDMS as a construction substrate we are able to form flow and control layers, which trap liquids but are open to gas exchange. The confined environment maintains the worm directly under the microscope, yet lets it swim around freely. Upon imaging, the ceiling of the chamber is forced down by air pressure, immobilizing the worm, and releasing it when done. Sustainability and development of the worm is achieved by a steady state flow of Nematode growth buffer and E. coli while removing excretory products such as dauer pheromone. We will describe preliminary results in building this platform.

Bo Wang et al. (2006) European Worm Meeting "Multi-Parameter Axial Profiling of Transgenic C. elegans Expressing Fluorescent Proteins from Various Cell-Specific, Tissue Specific and Developmentally Regulated Promoters"

Bruce Holcombe, Julia Thompson, Yanping Zhang, Rock Pulak, Bo Wang, Michael Herman, Mariya Lomakina

[European Worm Meeting, 2006]

Bo Wang1, Julia Thompson1, Yanping Zhang2, Michael Herman2, Mariya Lomakina1, Bruce Holcombe1, Rock Pulak1 . The COPAS Biosort instrument automates the analysis, sorting, and dispensing of all stages of C. elegans, measuring the animals size and the intensity of expressed fluorescent markers. Once analyzed, animals can be selected according to user defined criteria, and then dispensed into multi-well plates for high throughput screening or collected in bulk for further analysis. With this technology, time required for large scale screening for certain changes in the optical properties of the animals, such as changes in the levels of expression of a fluorescent protein, can be dramatically reduced and human error minimized. Recent enhancements to an add-on module, called the Profiler II, have been tested for its ability to collect positional information of fluorescent expression. The instrument can simultaneously collect fluorescence information in three separate regions of the spectrum. Here we show that the instrument can analyze multi-colored transgenic animals and can be used to compare the amounts and relative positions of expression of two or three different colors of fluorescence. Furthermore, this technology can be used to screen for independent changes in the intensity or position of each reporter protein. We have tested various transgenic animals expressing green, yellow and/or red fluorescing proteins from a collection of promoters that include myo-2, str-1, egl-17, mab-5, and various others, separately and in certain combinations. We present some proof of principle examples of how these could be used in genetic screens.

Julia Tischler et al. (2006) European Worm Meeting "Analysing the Redundancy of Duplicated Genes and the Evolutionary Conservation of Genetic Interactions Using Combinatorial RNAi in C. elegans"

Ben Lehner, Julia Tischler, Andrew G Fraser

[European Worm Meeting, 2006]

Julia Tischler, Ben Lehner and Andrew G Fraser Systematic analyses of loss-of-function phenotypes have been carried out for almost all genes in S. cerevisiae, C. elegans, and D. melanogaster, and there are major efforts to make a comprehensive collection of mouse knockouts. While such studies greatly expand our knowledge of single gene function, they do not address redundancy in genetic networks, nor do they attempt to identify genetic interactions. Developing tools for the systematic mapping of genetic interactions is thus a key step for exploring the relationship between genotype and phenotype. We thus sought to establish protocols for targeting multiple genes simultaneously by RNA interference (RNAi) in C. elegans to provide a platform for the systematic identification of genetic interactions in this key animal model system.. We set up conditions for RNAi that allow us to target multiple genes in the same animal (combinatorial RNAi) in a high throughput setting and to detect the great majority of previously known synthetic genetic interactions. We then used this assay to test the redundant functions of genes that have been duplicated in the genome of C. elegans since divergence from either S. cerevisiae or D. melanogaster, and identified 16 pairs of duplicated genes that are at least partially functionally redundant. Intriguingly, 14 of these redundant gene pairs were duplicated before the split of C. elegans and C. briggsae 80-110 million years ago. Our data provide the first systematic investigation into the redundancy of duplicated genes in any organism and strongly support population genetics models, which suggest that redundancy can be maintained over substantial periods of evolutionary time.. Furthermore, we set out to test whether systematically compiled yeast genetic interaction data can predict genetic interactions in the worm. We will present these data.

Werner P et al. (1997) International C. elegans Meeting "HETEROTRIMERIC G PROTEINS OF C. ELEGANS: SEQUENCE ANALYSIS AND EXPRESSION STUDIES"

Plasterk RHA, Jansen G, Thijssen KL, Kato Y, Werner P, Hazendonk E

[International C. elegans Meeting, 1997]

Heterotrimeric G proteins regulate cellular effectors in reaction to extracellular signals. Each mammalian class of G-alpha subunits (Gs, Go, Gq, G12) is represented in C. elegans by one homologue. Four additional C. elegans G-alpha subunits (gpa-1 to gpa-3 and odr- 3) have been reported previously. The C. elegans genome sequencing project revealed 11 more G alpha candidate genes. Their predicted protein sequences share only 30 to 60 % of the amino acid residues with each other. These novel genes gpa-4 to gpa-11 and gpa-13 to 15 reside on chromosomes I, II, IV, V and X, individually or in loose clusters. We visualized their expression patterns in transgenic lines that carry translational fusions to green fluorescent protein. All 11 candidates show distinct although partially overlapping expression patterns. A common feature seems to be transcriptional activity in neurons, especially in amphid neurons. For all subunits, with the exception of gpa-7, a rather restricted expression pattern was observed. Other gpa-genes produced additional non-neuronal staining, e.g. in the vulva, spermatheca, gut, and the excretory cell. To further characterizate this gene family, we are currently screening for a complete set of gpa-deletion mutants and we are investigating the influence of overexpression and constitutively active variants on transgenic worms. The present number of 19 apparently active C. elegans G-alpha genes is high compared to other species and suggests a significant potential of differential signal processing in individual nematode cells.