Banse, Stephen A. et al. (2011) International Worm Meeting "rde-4 is epistatic to rde-4."

Hunter, Craig P., Banse, Stephen A.

[International Worm Meeting, 2011]

In C. elegans the introduction of dsRNA (double-strand RNA) down regulates genes in a process termed RNA interference (RNAi). RNAi acts via small interfering RNAs (siRNAs), which are generated by RDE-4/DCR-1 cleavage of long dsRNA. Interestingly, RDE-4 and a few other genes required for RNAi (e.g. rde-1) are also necessary for the inconvenient silencing of multi-copy transgenic arrays. In contrast, enhanced RNAi (eri) mutants show enhanced transgene silencing. These observations are consistent with a simple model whereby transgene derived dsRNA is processed into siRNAs by RDE-4/DCR-1 leading to RNAi-dependent silencing of the transgenic arrays. Phenotypically, the ERI proteins act directly or indirectly to inhibit transgene silencing. The ERI proteins physically interact and are required for the production or stability of small RNAs that appear to silence endogenously expressed genes. It has been proposed that this eri-dependent small RNA silencing pathway competes with the RNAi pathway for limiting silencing resources. Our analysis shows that the eri-induced enhancement of transgene silencing requires the nuclear argonaute NRDE-3, indicating that not only is transgene silencing a nuclear RNAi process, but that NRDE-3 may be a limiting silencing resource. Interestingly, in addition to RNAi, RDE-4 is necessary for the generation of NRDE-3 associated endogenous siRNAs. This leads to the prediction that rde-4 mutants should relieve competition for NRDE-3, and therefore be "Eri", if RDE-4 weren't also required for RNAi. Consistent with this seemingly contradictory prediction, our genetic analysis provides supports for RNAi promoting and RNAi inhibiting roles for RDE-4 in somatic transgene silencing. This conflict is resolved by spatial segregation of these functions, with RDE-4 repression of the NRDE-3-dependent somatic transgene silencing activity being limited to the germline of the previous generation in contrast to the known somatic role of RDE-4 in promoting transgene silencing. Our work shows that transgene expression in the soma is synergistically repressed by the canonical RDE-1/RDE-4 RNAi pathway acting in the soma and by a second, a NRDE-3 dependent pathway most active in the parental germline. This germline process is inhibited by ERI-1 and RDE-4 and functions to generate a silencing signal which is inherited by the soma. Although loss of germline RDE-4 produces this silencing signal, it is typically masked by a requirement for somatic RDE-4 for efficient transgene silencing.

Banse, Stephen A. et al. (2013) International Worm Meeting "Microfluidic devices for electrical measures of pharyngeal health."

Weeks, Janis C., Robinson, Kristin J., Willis, John. H., Phillips, Patrick C., Lockery, Shawn R., Banse, Stephen A.

[International Worm Meeting, 2013]

The conventional method of health span screening in C. elegans currently faces three critical barriers: the absence of rigorously standardized culture conditions, the difficulty of performing longitudinal studies on individuals, and the challenge of high-throughput quantification of feeding behavior, one of the most reliable measures of health and a predictor of longevity in the worm. Whereas new microfluidic technologies are being developed to provide controlled growth chambers that minimize the first two challenges, the technology to automate measurements of feeding behavior is lagging behind. In C. elegans aging research, pump rate (0-5 Hz) is currently recorded and analyzed manually by direct observation of slow-motion videos of single worms while feeding on agar plates. It currently takes 5 hours to record and analyze 10 minutes of video, a 30:1 ratio that has become a bottleneck in C. elegans aging research, particularly in health-span screens requiring large data sets. We have devised an alternative approach using a recently developed microfluidic device for recording the electrical activity of the pharynx - an electropharyngeogram (EPG). These recordings can be made on eight or more worms at once, and analyzed computationally to extract pump frequencies, as well as higher-order pump features not visible in videos. Consistent with traditional measures, EPGs reveal generalized decay in pharyngeal pumping with age, with effects revealing themselves as early as 5 days. Additionally, we observe that interventions that slow aging have clear EPG phenotypes. We believe that compared to traditional video analysis, this approach will be more amenable to automation, less subjective, and provide a more information-rich readout of pharyngeal health. Additionally, this technology presents the possibility of combining EPG with other microfluidic devices to create arrays of sealed, individually addressable culture chambers that permit longitudinal assays of feeding behavior and other established measures of C. elegans health in tens, and ultimately hundreds, of worms at a time.

Banse, Stephen et al. (2019) International Worm Meeting "The role of FMRFamide-like signaling in regulating lifespan in C. elegans."

Banse, Stephen, Coleman-Hulbert, Anna, Phillips, Patrick, Sedore, Christine, Johnson, Erik, Robinson, Kristin

[International Worm Meeting, 2019]

Neuronal control of behavior is potentiated through many different classes of signaling molecules. In vitro characterization of one such class, FMRFamide-like peptides (flp), has identified numerous peptides that can regulate the rate of pumping of the C. elegans feeding organ, the pharynx. Because slowed pumping can induce dietary restriction, a widely conserved intervention that extends lifespan, we performed a survey of the available flp-gene mutants to identify regulators of lifespan. Of the 22 mutants examined, three strains were long lived, defined by lifespans greater than 120% of N2. Four strains were short lived, defined by lifespans shorter than 90% of N2. Of the seven genes with biochemical data showing efficacious alteration of pump frequency at ?100nM, six exhibited altered lifespans. The one exception, flp-8, appears to have a developmentally restricted expression pattern and may not be expressed in adulthood. This analysis suggests that modulation of neuronal flp signaling is a candidate for pharmacological interventions seeking to extend lifespan.

Banse, Stephen et al. (2021) International Worm Meeting "All trans retinoic acid extends C. elegans lifespan in an aak-2 and hsf-1 dependent manner by modulating metabolism."

Johnson, Erik, Jones, E Grace, Willis, John, Phillips, Patrick, Coleman-Hulbert, Anna, Banse, Stephen, Segerdell, Erik, Sedore, Christine

[International Worm Meeting, 2021]

Aging is a pan-metazoan phenotype that significantly impacts human health and society. Across metazoans, the rate of aging is influenced by a zero-sum allocation of resources between maintenance and growth. We therefore sought to manipulate signaling components from conserved resource allocation systems to identify potential anti-aging interventions. Here we present the exploration of the endogenous vitamin A derivative all trans Retinoic Acid (atRA), and its role promoting gluconeogenesis, as a modulator of aging in Caenorhabditis elegans. We found that supplementation with atRA extends lifespan in a concentration dependent manner. As predicted based on the conserved pathway, atRA acts independently of daf-16, but it does require the conserved AMP sensor aak-2, consistent with a change in metabolism and energy storage. The observed shift in metabolism comes at a reproductive cost, consistent with a redirection of energy resources to somatic maintenance. This approach shows that the manipulation of a conserved metabolic regulatory circuit by co-opting endogenous signaling molecules can extend lifespan.

Chamberlain SH et al. (1994) Worm Breeder's Gazette "A Molecular Approach to Identify skn-1-Responsive Genes in the Early C. elegans Embryo"

Chamberlain SH, Bowerman BA

[Worm Breeder's Gazette, 1994]

A molecular approach to identify skn-1-responsive genes in the early C. elegans embryo. Stephen H. Chamberlain and Bruce Bowerman, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

Blue, Ben et al. (2015) International Worm Meeting "What are they Choosing? Linking Food Preference to Life-History Outcomes."

Banse, Stephen, Phillips, Patrick, Blue, Ben

[International Worm Meeting, 2015]

C. elegans is a bacteriovore capable of consuming a variety of bacterial species. When presented a choice between bacterial foods C. elegans exhibits dietary preferences that generally correlate with the ability to support fast developmental rates. These preferred foods have therefore been described as "good quality" foods. However, it is not clear how well these foods mimic the worms' natural diet, nor whether these models are relevant to human diets for health research. Common lab practices skew experimental conditions even further to extremes by exposing worms to a vast excess of food which can have unaddressed consequences on their metabolism. Our goal is to use different bacterial strains of variable quality to link C. elegans food preference with observed physiological outcomes. This dietary profile allows a better understanding on the internal calculus of resource acquisition behaviors and dietary choice as well as how resources become internally allocated. In order to create this profile we use an array of novel and traditional physiological assays that allow for fine resolution of life-history traits such as longevity, health, and reproduction.Through the integration of the Lifespan machine system developed by Stroustrup et al. and microfluidic technology we investigate the internal calculus that mediates the preferences shown by C. elegans. With Lifespan machines we automate the measurement of longevity from thousands of worms grown on foods of differing quality. By also integrating the Lifespan system with a microfluidic device that allows worms to experience and choose between different bacterial strains in a controlled environment, we have achieved a high-throughput method of food preference determination that eliminates the majority of extraneous signals. In addition, we use a series of physiological and behavioral recordings to look at how internal resources are allocated when individuals are provided different foods as adults. Physiological health is determined from the maximal pump rate of the pharynx as recorded by a microfluidic chip that measures the electrical activity given off by pharyngeal muscles. The nutritional effects on reproduction are determined via rates of egg-laying. In summation, we present the physiological profile of C. elegans grown on different bacterial diets as well as the technology used to collect such data. .

Archer, Heather et al. (2019) International Worm Meeting "High throughput assessment of natural variation in the response to adult starvation in C. elegans using microfluidics."

Blue, Ben, Banse, Stephen, Archer, Heather, Phillips, Patrick

[International Worm Meeting, 2019]

Caenorhabditis elegans typically feeds on rotting fruit and plant material in a fluctuating natural habitat, a boom-and-bust lifestyle. Moreover, stage specific developmental responses to low food concentration suggest that starvation-like conditions are a regular occurrence. In order to assess variation in the C. elegans starvation response under precisely controlled conditions and simultaneously phenotype a large number of individuals with high precision, we have developed a microfluidic device that, when combined with image scanning technology, allows for high-throughput assessment at a temporal resolution not previously feasible and applied this to a large mapping panel of fully sequenced intercross lines. Under these conditions worms exhibit a markedly reduced adult lifespan with strain-dependent variation in starvation response, ranging from <72 hours to ~120 hours. We performed genome-wide mapping and epistatic analysis of the responses of 7,855 individuals spread across 72 mapping lines, identifying a number of different chromosomal regions responsible for this variation. Genetic differences for a subset of lines were confirmed using a multi-generational introgression analysis using backcrossing with selection. Overall, there is a clear genetic basis for natural variation in the response to food availability within this species.

Robinson, Kristin et al. (2019) International Worm Meeting "The egg-counter: A microfluidic platform for analysis of egg-laying behavior."

Phillips, Patrick, Banse, Stephen, Jarrett, Cody, Blue, Benjamin, Robinson, Kristin

[International Worm Meeting, 2019]

For animals that do not provide parental care, when and where eggs are laid in the environment can have profound effects on the reproductive success of an individual. Because of its importance, it is not surprising that egg-laying is highly regulated in response to environmental cues. This regulation, coupled with the well described neural circuitry involved in the act of egg-laying, make it a great system for studying how animals interpret their environment and make behavioral choices. To facilitate this line of research we have developed a microfluidic "egg-counter" that allows 32 nematodes to reside in individual growth chambers while their egg-laying behavior is recorded at a sub-minute temporal resolution. The platform utilizes a perfusion-based feeding system that allows experimental control of the chemical environment as well as a built-in temperature control unit that allows the temperature to be patterned with 0.1 deg C accuracy without the use of incubators or temperature control rooms. Preliminary analysis of egg-laying behavior from wildtype animals in our microfluidic environment grossly fits that of the three-state model used to describe egg-laying on agar plates, with the log-tail distribution of egg-laying intervals exhibiting a bi-phasic distribution consistent with two interval types, inter and intra-cluster intervals. We will present a genetic characterization of the role of insulin signaling in regulating egg-laying behavior.

Coleman-Hulbert, Anna L et al. MicroPubl Biol

Sedore, Christine A, Phillips, Patrick C, Coleman-Hulbert, Anna L, Driscoll, Monica, Banse, Stephan A, Lithgow, Gordon J, Guo, Max, Johnson, Erik

[MicroPubl Biol, 2019]

The authors, Coleman-Hulbert, AL; Johnson, E; Sedore, CA; Banse, SA; Guo, M2; Driscoll, M3; Lithgow, GJ; and Phillips, PC, submit the following correction. The first reference in the methods paragraph should be (Lucanic et al., 2017; Plummer et al., 2017) and should not be letteredaccordingly. We assayed lifespan in response to imatinib mesylate exposure in three Caenorhabditis species in triplicate using our previously published workflow (Lucanic et al. 2017a; b). should be corrected to: We assayed lifespan in response to imatinib mesylate exposure in three Caenorhabditis species in triplicate using our previously published workflow (Lucanic et al., 2017; Plummer et al., 2017).

Timmermeyer, Nadine et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "THE LOW-COST WORM SORTER: MICROFLUIDICS AS A TOOL FOR EXPERIMENTAL EVOLUTION"

Lockery, Shawn R., Banse, Stephen A., North, Joseph H., Timmermeyer, Nadine, Sottile, Matthew, Phillips, Patrick C.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Experimental evolution is a powerful tools used in evolution research. Nematodes are especially suited for experimental evolution, with a demonstrated track record of being able to monitor and manipulate large populations. Still, there are a few constraints and obstacles if worms cannot simply be pipetted from one generation to the next, for example if the sex ratio needs to be manipulated or selection depends on a certain phenotype. The limitations of hand transfer lead to small population sizes, small numbers of replicates and/or fewer treatments, resulting in strong drift effects and low statistical power. One inexpensive solution to those problems is the use of microfluidic devices, in this case worm sorters, either controlled by an observer or a computer. Fabricated out of a silicon polymer, these devices are air permeable, hydrophobic and non-toxic and can be adjusted to the particular experiment. We present examples from our own work on sexual selection in Caenorhabditis nematodes for the application of three different sorter types that we optimized for their specific purpose as well as other applications of microfluidics in nematode work.