The egg-counter: a novel microfluidic platform for characterization of Caenorhabditis elegans egg-laying.

Reproduction is a fundamental process that shapes the demography of every living organism yet is often difficult to assess with high precision in animals that produce large numbers of offspring. Here, we present a novel microfluidic research platform for studying Caenorhabditis elegans' egg-laying. The platform provides higher throughput than traditional solid-media behavioral assays while providing a very high degree of temporal resolution. Additionally, the environmental control enabled by microfluidic animal husbandry allows for experimental perturbations difficult to achieve with solid-media assays. We demonstrate the platform's utility by characterizing C. elegans egg-laying behavior at two commonly used temperatures, 15 and 20 °C. As expected, we observed a delayed onset of egg-laying at 15 °C degrees, consistent with published temperature effects on development rate. Additionally, as seen in solid media studies, egg laying output was higher under the canonical 20 °C conditions. While we validated the Egg-Counter with a study of temperature effects in wild-type animals, the platform is highly adaptable to any nematode egg-laying research where throughput or environmental control needs to be maximized without sacrificing temporal resolution.

The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background.

Aging is characterized by declining health that results in decreased cellular resilience and neuromuscular function. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of pharmacological anti-aging interventions. Here we assessed swimming performance as well as survival under thermal and oxidative stress across a nematode genetic diversity test panel to evaluate health effects for three compounds previously studied in the Caenorhabditis Intervention Testing Program and thought to promote longevity in different ways - NP1 (nitrophenyl piperazine-containing compound 1), propyl gallate, and resveratrol. Overall, we find the relationships among median lifespan, oxidative stress resistance, thermotolerance, and mobility vigor to be complex. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. In this study, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Notably, some compounds exert strong impact on some health measures without an equally strong impact on lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible anti-aging interventions, with data underscoring how personalized treatments might be required to optimize health benefits.

Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain-specific lifespan and health effects in Caenorhabditis nematodes.

The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17α-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies-for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.

The Egg-Counter: A novel microfluidic platform for characterization of Caenorhabditis elegans egg-laying.

Reproduction is a fundamental process that shapes the demography of every living organism yet is often difficult to assess with high precision in animals that produce large numbers of offspring. Here, we present a novel microfluidic research platform for studying Caenorhabditis elegans' egg-laying. The platform provides higher throughput than traditional solid-media assays while providing a very high degree of temporal resolution. Additionally, the environmental control enabled by microfluidic animal husbandry allows for experimental perturbations difficult to achieve with solid-media assays. We demonstrate the platform's utility by characterizing C. elegans egg-laying behavior at two commonly used temperatures, 15 and 20°C. As expected, we observed a delayed onset of egg-laying at 15°C degrees, consistent with published temperature effects on development rate. Additionally, as seen in solid media studies, egg laying output was higher under the canonical 20°C conditions. While we validated the Egg-Counter with a study of temperature effects in wild-type animals, the platform is highly adaptable to any nematode egg-laying research where throughput or environmental control needs to be maximized without sacrificing temporal resolution.

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS).

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

Transgenesis ­ the ability to insert foreign genetic material (known as transgenes) in to the genome of an organism ­ has revolutionized biological research. This approach has made it possible for scientists to study the role of specific genes and to produce animal models which mimic aspects of human diseases. For transgenes to be maintained and passed down to future generations, they must be introduced into germ cells which will go on to form the egg and sperm of the organism. However, despite advances in genetic engineering, this process (called 'specific transgenesis') is still laborious and time-consuming, and limits researchers to working with only a small number of known DNA sequences at a time. In contrast, 'exploratory transgenesis' ­ where dozens of transgenes from a library of DNA sequences are introduced simultaneously into multiple individuals ­ is more efficient and allows for more large-scale experiments. However, this approach can only be done with single-celled organisms like bacteria, and remains virtually impossible in laboratory animals like worms or mice. Stevenson et al. therefore set out to boost the efficiency of exploratory transgenesis in a commonly used laboratory animal, the roundworm Caenorhabditis elegans. To do this, they used the 'library' principle of exploratory transgenesis in order to develop a new resource called TARDIS (short for, Transgenic Arrays Resulting in Diversity of Integrated Sequences). First, Stevenson et al. genetically engineered worms to carry a 'landing site' for foreign DNA. Next, a library of transgenes and a mechanism which cuts pieces of DNA and pastes them into the landing site were introduced into the germ cells of these worms using traditional methods. The worms were then bred to generate a large population of offspring that had inherited this array of foreign DNA sequences. Finally, the 'cut and paste' mechanism was switched on and a random transgene was inserted into the landing site in the genome. This resulted in thousands of worms which each had a unique genetic modification that can be passed on to future generations. These results show for the first time that larger-scale transgenesis experiments are possible in multi-cellular animals. In the future, Stevenson et al. hope that TARDIS can be adapted to different organisms and allow researchers to carry out experiments that were not previously possible.

Genetic diversity estimates for the Caenorhabditis Intervention Testing Program screening panel.

The Caenorhabditis Intervention Testing Program (CITP) was founded on the principle that compounds with positive effects across a genetically diverse test-set should have an increased probability of engaging conserved biochemical pathways with mammalian translational potential. To fulfill its mandate, the CITP uses a genetic diversity panel of Caenorhabditis strains for assaying longevity effects of candidate compounds. The panel comprises 22 strains from three different species, collected globally, to achieve inter-population genetic diversity. The three represented species, C. elegans, C. briggsae, and C. tropicalis, are all sequential hermaphrodites, which simplifies experimental procedures while maximizing intra-population homogeneity. Here, we present estimates of the genetic diversity encapsulated by the constituent strains in the panel based on their most recently published and publicly available whole-genome sequences, as well as two newly generated genomic data sets. We observed average genome-wide nucleotide diversity (π) within the C. elegans (1.2e-3), C. briggsae (7.5e-3), and C. tropicalis strains (2.6e-3) greater than estimates for human populations, and comparable to that found in mouse populations. Our analysis supports the assumption that the CITP screening panel encompasses broad genetic diversity, suggesting that lifespan-extending chemicals with efficacy across the panel should be enriched for interventions that function on conserved processes that are shared across genetic backgrounds. While the diversity panel was established by the CITP for studying longevity interventions, the panel may prove useful for the broader research community when seeking broadly efficacious interventions for any phenotype with potential genetic background effects.

Metformin treatment of diverse Caenorhabditis species reveals the importance of genetic background in longevity and healthspan extension outcomes.

Metformin, the most commonly prescribed anti-diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti-aging drug-the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose-dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.

A simplified design for the C. elegans lifespan machine.

Caenorhabditis elegans (C. elegans) lifespan assays constitute a broadly used approach for investigating the fundamental biology of longevity. Traditional C. elegans lifespan assays require labor-intensive microscopic monitoring of individual animals to evaluate life/death over a period of weeks, making large-scale high throughput studies impractical. The lifespan machine developed by Stroustrup et al. (2013) adapted flatbed scanner technologies to contribute a major technical advance in the efficiency of C. elegans survival assays. Introducing a platform in which large portions of a lifespan assay are automated enabled longevity studies of a scope not possible with previous exclusively manual assays and facilitated novel discovery. Still, as initially described, constructing and operating scanner-based lifespan machines requires considerable effort and expertise. Here we report on design modifications that simplify construction, decrease cost, eliminate certain mechanical failures, and decrease assay workload requirements. The modifications we document should make the lifespan machine more accessible to interested laboratories.

Automated lifespan determination across Caenorhabditis strains and species reveals assay-specific effects of chemical interventions.

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach toward aging-intervention screening.

The Stress-Chip: A microfluidic platform for stress analysis in Caenorhabditis elegans.

An organism's ability to mount a physiological response to external stressors is fundamental to its interaction with the environment. Experimental exploration of these interactions benefits greatly from the ability to maintain tight control of the environment, even under conditions in which it would be normal for the subject to flee the stressor. Here we present a nematode research platform that pairs automated image acquisition and analysis with a custom microfluidic device. This platform enables tight environmental control in low-density, single-worm arenas, which preclude animal escape while still allowing a broad range of behavioral activities. The platform is easily scalable, with two 50 arena arrays per chip and an imaging capacity of 600 animals per scanning device. Validating the device using dietary, osmotic, and oxidative stress indicates that it should be of broad use as a research platform, including eventual adaptation for additional stressors, anthelmintic-drug screening, and toxicology studies.

The nuclear argonaute NRDE-3 contributes to transitive RNAi in Caenorhabditis elegans.

The Caenorhabditis elegans nuclear RNA interference defective (Nrde) mutants were identified by their inability to silence polycistronic transcripts in enhanced RNAi (Eri) mutant backgrounds. Here, we report additional nrde-3-dependent RNAi phenomena that extend the mechanisms, roles, and functions of nuclear RNAi. We show that nrde-3 mutants are broadly RNAi deficient and that overexpressing NRDE-3 enhances RNAi. Consistent with NRDE-3 being a dose-dependent limiting resource for effective RNAi, we find that NRDE-3 is required for eri-dependent enhanced RNAi phenotypes, although only for a subset of target genes. We then identify pgl-1 as an additional limiting RNAi resource important for eri-dependent silencing of a nonoverlapping subset of target genes, so that an nrde-3; pgl-1; eri-1 triple mutant fails to show enhanced RNAi for any tested gene. These results suggest that nrde-3 and pgl-1 define separate and independent limiting RNAi resource pathways. Limiting RNAi resources are proposed to primarily act via endogenous RNA silencing pathways. Consistent with this, we find that nrde-3 mutants misexpress genes regulated by endogenous siRNAs and incompletely silence repetitive transgene arrays. Finally, we find that nrde-3 contributes to transitive RNAi, whereby amplified silencing triggers act in trans to silence sequence-similar genes. Because nrde-dependent silencing is thought to act in cis to limit the production of primary transcripts, this result reveals an unexpected role for nuclear processes in RNAi silencing.

Vampiric isolation of extracellular fluid from Caenorhabditis elegans.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Reduced mismatch repair of heteroduplexes reveals "non"-interfering crossing over in wild-type Saccharomyces cerevisiae.

Using small palindromes to monitor meiotic double-strand-break-repair (DSBr) events, we demonstrate that two distinct classes of crossovers occur during meiosis in wild-type yeast. We found that crossovers accompanying 5:3 segregation of a palindrome show no conventional (i.e., positive) interference, while crossovers with 6:2 or normal 4:4 segregation for the same palindrome, in the same cross, do manifest interference. Our observations support the concept of a "non"-interference class and an interference class of meiotic double-strand-break-repair events, each with its own rules for mismatch repair of heteroduplexes. We further show that deletion of MSH4 reduces crossover tetrads with 6:2 or normal 4:4 segregation more than it does those with 5:3 segregation, consistent with Msh4p specifically promoting formation of crossovers in the interference class. Additionally, we present evidence that an ndj1 mutation causes a shift of noncrossovers to crossovers specifically within the "non"-interference class of DSBr events. We use these and other data in support of a model in which meiotic recombination occurs in two phases-one specializing in homolog pairing, the other in disjunction-and each producing both noncrossovers and crossovers.