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[
International C. elegans Meeting,
1999]
C27H5.1 is a weak homologue of UNC-119, a gene involved in axon guidance and outgrowth. We first became interested in C27H5.1 because of its similarity to UNC-119. We have since learned that there is a mammalian homologue of C27H5.1; rod phosphodiesterase delta subunit PDE6D. These two proteins are very highly conserved exhibiting 70% identity and 85% similarity. UNC-119 also has a close mammalian homologue; retinal protein HRG4/RRG4. Both C27H5.1 and UNC-119 are pan-neuronal in C. elegans while HRG4 and PDE6D are expressed in the mammalian retina. The conservation of these genes has led us to speculate that they may constitute a new protein family. While UNC-119 has been fairly well characterized, very little is known about C27H5.1. Based upon its conservation with UNC-119 and PDE6D, we suspect that C27H5.1 is playing an important role, perhaps in nervous system development. However, C27H5.1 may play a more general role. Unlike UNC-119, C27H5.1 expression is not limited to the nervous system. There is also expression in pharyngeal and vulval muscle. To determine this role, we have performed RNA interference against C27H5.1. Unfortunately, no phenotype was observed. Subsequently we performed RNAi against GFP under the control of the C27H5.1 promoter. There was no reduction in fluorescence suggesting that the expression of C27H5.1 is not affected by RNAi. We are currently screening for a deletion in C27H5.1. Although we have not yet been successful in determining the C27H5.1 mutant phenotype, studies with its mammalian homologue, PDE6D, suggest a possible role for this protein. PDE6D has been shown to bind to and solubilize rod phosphodiesterase (PDE). Since PDE6D expression is not limited to the retina, PDE d may regulate the membrane binding of a variety of proteins. Interestingly, C27H5.1 also binds human rod phosphodiesterase, suggesting that there is at least some functional conservation between PDE d and C27H5.1.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
We fabricated and tested a high-throughput microfluidic platform to study nerve regeneration in C. elegans. The device consists of an array of small chambers in a parallel fluidic circuit allowing for simultaneous trapping of dozens of C. elegans worms in individual visualization chambers for in-vivo imaging and laser ablation of fluorescently labeled axons. With proper liquid nutrients, the animals can easily survive in the microfluidic chambers for three days or more for monitoring nerve regeneration. This device could serve as the optical and fluidic interface for automated genome-wide nerve regeneration studies using femtosecond laser nano-axotomy and fluorescence microscopy. Using our device and conventional methods, we investigated the regenerative capacity of the oxygen sensory neuron, PQR. This neuron is located in the left lumbar ganglion on the posterior-lateral side of the worm's body, and has only two processes emerging from the cell body – a dendrite extending posterior toward the tip of the tail and an axon extending anterior joining the ventral nerve cord. We looked at regeneration rates in animals in which either only one or both neurites were severed. We observed that the dendrite process regenerated with a higher frequency when the axon was simultaneously severed. This result suggests that the molecular machinery responsible for regeneration is more efficiently recruited in a given process when there is additional damage to other parts of the neuron.
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[
International Worm Meeting,
2015]
Time-lapse microscopy has proven indispensible for studying dynamic processes in living organisms and has uncovered significant individual-to-individual variability at the cellular level. However, time-lapse microscopy is currently rarely used to study C. elegans post-embryonic development. The major obstacle is the need to restrict movement of animals for imaging, as drug-induced or mechanical immobilization precludes the animal's feeding and typically leads to growth arrest of larvae within hours. Here, we circumvent this problem by constraining the movement of animals in ~200x200x10mum chambers made of polyacrylamide hydrogel, with each chamber containing bacteria as food source to sustain development. We found that in such chambers C. elegans animals develop normally, as measured by body length extension, timing of the molting cycle and egg laying. Using arrays of microchambers, combined with fast image acquisition, we could perform fluorescence microscopy of developmental dynamics at the single-cell level and with ~10 minute time resolution for the full duration of post-embryonic development, in up to 50 animals simultaneously. We demonstrated the power of our setup in capturing a number of key developmental processes in C. elegans that span a significant fraction of the ~48 hours of post-embryonic development and hence have so far been inaccessible for time-lapse microscopy. In particular, we (i) studied the temporal regulation of seam cell divisions by imaging all cell divisions of the seam cell lineages, (ii) measured the trajectory of the distal tip cells during their entire ~30 hour migration and (iii) quantified the oscillatory gene expression dynamics of molting genes during all four larval stages. We observed significant variability, both between individuals and on the single cell level, in all these processes. Our setup should make it possible to use time-lapse microscopy as a routine tool to study C. elegans post-embryonic development.
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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.
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[
European Worm Meeting,
2006]
Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.
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[
International Worm Meeting,
2015]
*Equal ContributionsThe use of C. elegans in neuroscience has flourished due to advances in microfluidics. However, traditional microfluidic assays require time-consuming design and fabrication and have limited flexibility during operation. We present a platform for dynamically building microfluidic assays by in situ photopatterning of a bio-compatible hydrogel on NGM plates. This method adds flexibility to the workflow, enabling the researcher to incorporate new features in the assay based on observations as the experiment proceeds. To validate the technique, we first study whether in situ photofabrication of micropillars around swimming C. elegans would influence the worms' velocity and found that going from the open frame, to the micropillar array, to the rippled microchannel, the worm's maximum speed increases 270%. Our method eliminates the need to load worms into a device one by one and sort them into individual chambers, as the chamber can be fabricated in situ around each worm. Next, we fabricate a free-floating lever around a pin anchored to the NGM plate. Worms confined within a frame surrounding this mechanism actively interact with the lever. A variety of mechanisms can be fabricated within the culture environment (one-way gates, floating microparticles, movable isolation chambers, gears) as means to investigate more complicated behaviors. In addition, the researcher can generate freeform input using a tablet, resulting in real-time modification of the assay. Using custom-built LabVIEW, the time between when the pen touches the pad and final PEG-DA photopolymerization is less than 2sec. Last, we demonstrate how worms can be tracked in custom-made mazes to determine their ability to locate food. In the absence of food, the worms choose the left or right ends with equal probability, whereas, when food is placed in one end of the maze, worms choose the leg containing the food almost twice as often. Our photopatterning technique enables rapid and flexible experimentation via micro-scale confinement of model organisms, and in the future could incorporate image analysis and machine learning techniques to acquire large datasets and accelerate breakthroughs in understanding the behavior of model organisms such as C. elegans.
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Schneider, Martin, Krzyzanowski, Michelle, Bethke, Mary, Nagpal, Jatin, Gottschalk, Alexander, Woldemariam, Sarah, Ferkey, Denise, L'Etoile, Noelle
[
International Worm Meeting,
2015]
cGMP is a ubiquitous second messenger implicated in many important biological processes. In neurons, cGMP dynamics can regulate the function of ion channels and kinases, resulting in physiological changes. In the context of learning and memory, these changes result in short-term and long-term behavioral changes based on the organism's experience. We attempt to understand the molecular basis for long-term plasticity by studying the behavioral responses of the nematode C. elegans. Along with our collaborator Michelle Krzyzanowski from the Denise Ferkey lab in SUNY Buffalo, we are interested in how food might modulate behaviors. One food-modulated behavior is repulsion from quinine. This repulsion is mediated by the ASH neuron and it is down regulated by food withdrawal and the cGMP-dependent protein kinase EGL-4. This poses a conundrum since no guanylyl cyclases, which produce cGMP, are expressed in ASH. Genetic evidence suggests that guanylyl cyclases in other neurons are required for the food-modulated repulsion from quinine in ASH and that gap junctions are required for the transmission of cGMP from these neurons to ASH. In order to understand how cGMP dynamics in these neurons are modulated, we need a tool to visualize cGMP. To this end, we are using a cGMP sensor that will allow us to image cGMP dynamics in ASH and other neurons in the living behaving animal in the presence and absence of food.
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[
International Worm Meeting,
2021]
Despite sleep being essential and conserved, an understanding of its mechanisms are lacking. We recently described a role for NLP-14/orcokinin neuropeptides during the regulation of sleep, most notably during stress-induced sleep (SIS). Orcokinins are found in ecdysozoan animals and have been shown to regulate circadian rhythms and molting in insects. Despite their conservation, their downstream mechanisms are not well understood. Taking advantage of the observation that over-expression of
nlp-14 induces a strong sleep phenotype, we conducted a forward genetic screen for suppression of the sleep phenotype. Our experimental approach uses 3D-printed chambers which allow for the sorting of animals using gravity. Mutagenized animals are induced to fall asleep by
nlp-14 over-expression. Those who fell asleep were trapped in the lower chamber, while the rare suppressors swam against gravity to the upper collection platform. We have isolated multiple mutants, which we call orcokinin suppressors (orcs), and have sequenced their genomes. Currently, we are identifying the causative alleles. Two of these unmapped mutants orcs(327) and orcs(330) display drastically reduced levels of SIS. These new mutants may represent novel sleep genes essential for SIS regulation.
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[
C.elegans Neuronal Development Meeting,
2008]
We present on-chip technologies for high-throughput screening of C. elegans, and the use of these technologies for study of neural regeneration in C. elegans. We demonstrate high speed microfluidic sorters, which can rapidly isolate and immobilize awake C. elegans in a well-defined geometry for screening phenotypic features at sub-cellular resolutions non-invasively . We show use of these technologies for three-dimensional two-photon imaging and femtosecond laser nano-surgery on awake C. elegans for high-throughput neural degeneration and regeneration studies. We show integrated chips containing individually addressable screening-chambers for incubation and exposure of individual worms to RNAi/compounds, and high-resolution time-lapse imaging of many immobilized animals on a single chip without anesthesia. We report devices for delivery of compound libraries in standard multi-well plates to microfluidic devices, and also for rapid dispensing of screened animals into multi-well plates. When used in various combinations, these devices facilitate a variety of high-throughput assays including mutagenesis, RNAi and drug screens at sub-cellular resolution on awake animals, as well as large-scale on-chip in vivo neural degeneration and regeneration studies using femtosecond laser microsurgery. We will report the recent studies and advances we made using these technologies. www.rle.mit.edu/Yanik.
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[
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.