Casar, Jason et al. (2021) International Worm Meeting "Force sensitive upconverting nanoparticles as a direct, noninvasive assay for force generation by muscles"

Casar, Jason, Siefe, Chris, Dionne, Jennifer A., Lay, Alice, McLellan, Claire, Goodman, Miriam B.

[International Worm Meeting, 2021]

The digestive system relies heavily on muscles to breakdown, transport and expel material in order to provide the body the requisite nutrients to live. Additionally, activity of these muscles are key biomarkers of health status. Understanding the fundamental function of the digestive system and its dysfunction in aging and disease is hindered by a lack of suitable in vivo force sensors. Quantifying the forces generated in the alimentary lumen in vivo is currently not feasible through established mechanosensing platforms such as atomic force microscopy, traction force microscopy and Forster Resonant Energy Transfer (FRET)-based molecular strain sensors. To overcome that limitation, we are developing a microscale, biocompatible mechanosensing platform based on upconverting nanoparticles (UCNPs) that can rapidly and noninvasively readout micronewton scale forces within the intestinal and pharyngeal lumen of C. elegans. We synthesize lanthanide-doped ceramic nanoparticles (SrLuF:Yb,Er) embedded in polystyrene microspheres for efficient delivery. When excited in the near-infrared, these mechanosensing UCNPs have an emission spectrum that depends on pressure [PMID: 29927609; PMID: 31592655] and applied force. The ratiometric emission (red/green) increases by 25% with applied forces of 2 micronewtons as measured using dual confocal microscopy and atomic force microscopy. These particles are bright enough to image even the most rapid luminal pressure cycle in the worm (pharyngeal pumping, 5 Hz). We demonstrate the efficacy of using UCNPs to map luminal forces associated with feeding and digestion in wild-type adults. These measurements provide a time-resolved and direct indication of coordinated muscle function. We apply mechanosensing UCNPs concurrently with electrophysiological measurement of pharyngeal activity (ScreenChip), enabling assessment of the relationship between electrical activity and muscle function. We describe how these tools may be further developed into a high throughput platform for muscle function during aging and in C. elegans disease models, as well as how our UCNPs can be adapted to perform analogous measurements in other animals.

David Weinkove et al. (2002) European Worm Meeting "The roles of developmental arrest, phosphoinositides, Type II PIP kinase and DAF-16 in the larval response to oxidative stress."

David R Jones, David Weinkove, Nullin Divecha, Jonathan R Halstead, Ronald H A Plasterk

[European Worm Meeting, 2002]

Since the appearance of terrestrial oxygen, it has been essential to deal with oxidative stress. For a nematode, the important issue is to lay eggs without germline damage. Rather than study the response in adults, for which it is probably too late to do anything useful, we have investigated how L1 larvae respond (1) developmentally, (2) genetically and (3) biochemically to a burst of hydrogen peroxide.

A Madi et al. (2002) European Worm Meeting "Proteome analysis reveals distinct molecular differences in developmental stages of wild type Caenorhabditis elegans"

S Mikkat, A Madi, B Ringel, M Ulbrich, M O Glocker, H-J Thiesen

[European Worm Meeting, 2002]

Caenorhabditis elegans is a well-known model organism of complex molecular processes such as programmed cell death of mammalian cells because of evolutionary conserved pathways. Our interests is to investigate normal development in wild type worms by proteome analyses to characterize stage-specific proteins and lay the ground for further comparisons and mutation studies. Up to now only proteome maps of populations with mixed stages have been constructed [1-3].

Koelle MR et al. (1995) International C. elegans Meeting "egl-10 CONTROLS THE FREQUENCIES OF SEVERAL BEHAVIORS AND MAY REGULATE G PROTEIN SIGNAL TRANSDUCTION."

Koelle MR, Horvitz HR

[International C. elegans Meeting, 1995]

C. elegans lays eggs more frequently in the presence of a bacterial lawn than in its absence. To learn how the activities of the egg-laying neurons and muscles are regulated by environmental cues, we are studying two classes of mutants in which this regulation is disrupted: 1) mutants that fail to lay eggs despite having an apparently morphologically normal egg-laying system; 2) mutants that lay eggs more frequently than does the wild type. egl-10 loss-of-function mutants fail to lay eggs and also have reduced rates of locomotion and foraging. By varying the dosage of the egl-10 gene we found that the rates of these behaviors are proportional to the number of functional copies of egl-10. For example, animals carrying extra copies of the gene lay eggs more frequently and move faster than wild- type animals. We cloned egl-10 and determined its expression pattern using antibodies and GFP reporters. EGL-10 is present in the processes of many neurons, including the HSN neurons that control egg laying. The protein is also present in the vulval muscles, which control egg laying, and in body wall muscles, which control locomotion. Within muscle cells, EGL-10 protein is localized to what appears to be the sarcoplasmic reticulum. EGL-10 shows a distant sequence similarity to the yeast Sst2 protein. Sst2 is induced by and negatively regulates the G protein mediated signal transduction pathway that responds to mating pheromone. Genetic evidence suggests Sst2 may directly interact with the yeast G alpha subunit. By analogy, we postulate that EGL-10 may regulate a C. elegans signal transduction pathway that controls the frequencies of egg laying and other behaviors by modulating neural and muscle activity. Using degenerate PCR, we amplified nine egl-10 homologs from rat brain cDNA. EGL-10 thus appears to be a member of a large family of proteins that may regulate many G protein mediated signal transduction pathways.

Sawin ER et al. (1991) International C. elegans Meeting "THE MODULATION OF EGG-LAYING RATES BY ENVIRONMENTAL STIMULI."

Horvitz HR, Sawin ER

[International C. elegans Meeting, 1991]

C. elegans modulates its rate of egg laying in response to a variety of environmental conditions. We have developed an assay that measures the effect of food (ie., bacteria) on egg laying rates. Young adult hermaphrodites are starved for one hour and then placed individually on agarose assay pads in humidified chambers for an additional hour. If a lawn of E. coli is present on the surface of the assay pad the worrns lay several times as many eggs as they do the absence of bacteria. In the absence of bacteria the average number of eggs layed per hour ranges from less than one to as many as four, while in the presence of bacteria wildtype hermaphrodites on average lay from eight to twelve eggs per hour. This modulatory response to the presence of bacteria appears to depend on chemosensory cues rather than on the nutritional state of the animal. First, the modulation can be observed within several hours and does not require prolonged starvation. In addition, the response occurs even when the worms are separated from the bacteria by an agarose barrier, implying that diffusible molecules are involved. Finally, mutants with chemosensory defects show an abnormal modulation of egg-laying rates in the presence of bacteria. For instance, several mutants with abnormal sensory cilia, including che-2(elO33J, cke-12(el812J and che-lO(el809J, lay eggs at approximately the same low rate whether or not bacteria are present. This response to bacteria is dependent on the HSNs, the motor neurons that innervate the egg-laying musculature. egl-l(n986) animals, which lack HSNs, are able to lay a small number of eggs (3-4 eggs per hour), but the rate of egg laying by this mutant is not influenced by the presence of bacteria. This observation suggests that chemosensory inputs modulate the activity of the egg-laying muscles via a neural pathway that involves the HSN. In addition to bacteria, other environmental stimuli also modulate the egg-laying behavior of C. elegans. For instance, high osmolarity depresses the rate of egg laying in the presence of bacteria, while the volatile attractant isoamyl alcohol stimulates egg laying in the absence of bacteria. To understand how sensory information is integrated and processed to regulate behavior, we would like to deflne the neural circuitry that controls the modulation of egg laying. Currently, we are using a laser microbeam to ablate chemosensory neurons and assaying the egg-laying behavior of the operated animals.

C M Loer et al. (2005) International Worm Meeting "Egg laying behavior in C. elegans social vs. solitary strains"

C M Loer, M Maxwell, M Ailion, K Kent

[International Worm Meeting, 2005]

Some wild isolates of C. elegans feed together ("social" strains) whereas "solitary" strains - including the standard wildtype N2 - do not1,2. A single amino acid difference in the NPR-1 neuropeptide receptor mediates this profound behavioral difference; reduction or loss-of-function results in social behavior 2. Social strains in the lab not only clump together, but also "border," spending most of their time in the thicker edge of a bacterial lawn. Solitary strains engage in much less bordering. Bordering is at least partly a consequence of social strains' preference for the lower oxygen concentration of the lawn edge3. We are currently investigating whether social strains display other behaviors in association with clumping and bordering. In particular, we predicted that social strains might display specific egg laying behaviors that would be advantageous in association with social behaviors. The clumping behavior of social worms results in high local density of conspecifics, which likely results in intense competition for food between individual worms. We investigated whether social adults might compensate by laying eggs away from the border of the lawn where the worms aggregate. We allowed individual young adult worms to lay eggs for 4 hours, and then examined the spatial distribution of eggs. Under 21% O2, both wild isolate social strains (RC301, CB4856) and npr-1 mutants with an N2 background (DA650, IM222) lay >90% of eggs at the border whereas N2 lays 48% of eggs at the border. Therefore, social strains do not disperse eggs, but lay them at the border, where they spend most of their time. Low O2 (7%) reduces the percentage of border eggs in social strains to that of N2 (53%). We have also confirmed that wild social strains lay eggs at a earlier stage of development, and retain fewer eggs in the uterus, whereas npr-1 mutants with an N2 genetic background do not4. For example, 92% of eggs laid by the Hawaiian social strain CB4856 are at early gastrulation or younger versus 8% for N2. As a young adult, CB4856 averages about 5 eggs in the uterus whereas N2 averages about 18 eggs. The mutant npr-1(ad609) is like N2, laying only 4% early eggs and carrying on average 13 eggs in the uterus. Preliminary genetics suggests that the early egg laying trait is semi-dominant and located on LG X. How the suite of behavioral traits associated with social behavior affect individual reproductive success and population dynamics warrants further investigation. Refs: 1Hodgkin & Doniach (1997) Genetics 146:149. 2de Bono & Bargmann (1998) Cell 94:679. 3Gray et al. (2004) Nature 430:317. 4Ailion & Thomas (2001) WBG 17(1):39.

Truong T et al. (1997) International C. elegans Meeting "Comparative Neuroanatomical and Behavioral Analysis of Egg-laying"

Truong T, Schafer WR

[International C. elegans Meeting, 1997]

Genetic, anatomical, and pharmacological studies suggest a simple neural circuit that controls egg-laying. Moreover, our recent quantitative behavioral analysis has provided insights into the temporal pattern of egg-laying. These observations have gradually defined the roles of specific neurons involved in regulating the egg-laying behavior of C. elegans. Given this wealth of information and the conserved structure of nematodes, we have begun a comparative neuroanatomical and behavioral analysis of egg-laying behavior between C. elegans and nematodes in the order Rhabditia. These studies should provide insight into the molecular and cell mechanism for behavioral diversity. Initially, we surveyed the behavior of a number of species from the family Rhabditidae (includes C. elegans) for differences in egg-laying behavior. These include two species each from the genera Caenorhabditis, Oscheius, and Rhabditella and one each of the genera Teratorhabditis and Rhabditis. They exhibit varied patterns of egg retention. Caenorhabditis remanei, Oscheius guntheri, Oscheius sp., Rhabditella octopleura, and Teratorhabditis palmarum all lay early eggs. C. elegans and Rhabditella axei retain eggs longer, and they lay them in clusters. Finally, Rhabditella blumi retain embryos until very late in development. These observations suggest that egg-laying behavior varies among related species. We think these differences have a neural basis. We plan to investigate the cellular differences between species with different behavior by comparative neuroanatomical studies.

Bany IA et al. (2000) East Coast Worm Meeting "A Genetic Screen for Components of the Goalpha Signaling Pathway in C. elegans"

Bany IA, Koelle MR

[East Coast Worm Meeting, 2000]

Neurotransmitters can mediate neural communication by activating heterotrimeric G proteins that signal through poorly understood pathways. GOA-1, a G protein expressed in the C. elegans nervous system, is 80% identical to human Goa, the major G protein of the brain. Neural signaling defects in goa-1 mutants result in hyperactivity of several behaviors, including feeding, locomotion, and egg laying. To identify components of the GOA-1 signaling pathway, we screened 39,000 mutagenized haploid genomes for mutations that confer a phenotype similar to that of goa-1 loss-of-function mutants. Like goa-1, these hyperactive egg-laying mutants lay their eggs soon after fertilization, presumably due to the hyperstimulation of the egg-laying machinery. This results in worms that accumulate very few eggs within their uterus and eggs that are laid at an early stage of development. In the past, it has been difficult to identify mutants with this phenotype (thin and empty of eggs) because they resemble both young adults and the partially sterile animals that appear at a high frequency following mutagenesis. To overcome this difficulty we developed an efficient selection for the early-stage eggs laid by hyperactive egg-laying mutants that is followed by a visual rescreening for animals resembling goa-1 mutants. This procedure allows for the identification of mutants with neurotransmitter signaling defects due to the altered function of proteins both upstream and downstream of GOA-1. This screen has isolated mutants with the desired hyperactive egg-laying phenotype. We are currently characterizing 17 isolates that lay greater than 70% of their eggs at the 8-cell stage or earlier. The strength of this phenotype distinguishes these mutants from previously described "Egl-C" mutants which, in a limited survey, typically lay a much lower percentage of early-stage eggs. Only loss-of-function mutations in goa-1 and eat-16, components of G-protein signaling pathways that control egg-laying behavior, have comparably strong phenotypes. Interestingly, all of the mutations analyzed from this screen show some degree of semi-dominance, as do goa-1 (n363) and eat-16(ad702). We plan to clone and characterize the genes identified in this screen to understand GOA-1 signaling at a molecular level.

Ringstad, Niels et al. (2009) International Worm Meeting "Inward Rectifier Potassium Channels Inhibit C. elegans Egg Laying and Locomotion."

Horvitz, Bob, Ringstad, Niels

[International Worm Meeting, 2009]

Inward-rectifier K+ (IRK) channels function to regulate membrane excitability in neurons and neuroendocrine cells. Three C. elegans genes, irk-1, irk-2 and irk-3, are predicted to encode potassium channel subunits similar to mammalian Kir1-7, which form tetrameric inward-rectifier potassium channels that are activated by diverse intracellular signals, including G proteins, ATP and phosphoinositides. To study the function of C. elegans inward rectifier K+ channels, we isolated deletion alleles of irk-1, irk-2 and irk-3. irk-1 mutants move faster than the wild type and lay early-stage eggs. irk-1 deletion also suppresses the egg-laying defect caused by a gain-of-function mutation in the EGL-6 G protein-coupled receptor for FMRFamides that activate inhibitory G protein signaling in the HSN egg-laying motor neurons. We did not observe gross behavioral defects of irk-2 and irk-3 mutants. An irk-1::gfp reporter transgene was expressed in a small number of neurons, including the HSNs. Expression of ChannelRhodopsin using the irk-1 promoter caused animals to lay eggs and accelerate in response to a blue-light stimulus, indicating that irk-1 functions to inhibit neurons that promote egg laying and locomotion. Xenopus laevis oocytes expressing irk-1 showed inward-rectifying currents in high-potassium saline. We are currently testing whether signaling pathways known to modulate IRK channels in other organisms function to modulate IRK-1 in vitro and in vivo.

Brewer, Jacob et al. (2015) International Worm Meeting "The HSN command neuron co-releases serotonin and NLP-3 neuropeptides to coordinate egg-laying activity."

Brewer, Jacob, Koelle, Michael R.

[International Worm Meeting, 2015]

It remains unclear exactly how serotonin regulates neural circuit activity in the brain or how changes in serotonin signaling might cause mental disorders. The C. elegans egg-laying system is well-suited for studying the basics of how serotonin functions in a circuit. The serotonergic HSN motor neurons initiate ~2.5 minute egg-laying active phases that occur every ~20 minutes. The evidence for this is that rhythmic HSN Ca2+ activity measured using a GCaMP reporter always accompanies egg-laying active phases, and optogenetic activation of HSN neurons is sufficient to stimulate activity of the egg-laying circuit.We knocked out serotonin biosynthesis and showed that optogenetically stimulating the HSNs still caused animals to lay eggs. We hypothesized that the HSNs release a signal in addition to serotonin to stimulate egg laying. We found that neuropeptides encoded by the nlp-3 gene, which is known to be expressed in the HSNs, are such a signal. Knocking out either nlp-3 or serotonin biosynthesis alone caused mild egg-laying defects, but knocking out both caused a strong egg-laying defect that phenocopies that of animals lacking HSNs. Interestingly, co-release of serotonin and a neuropeptide also occurs in the mammalian brain, although the function of such co-release is unknown.Serotonin stimulates egg-laying through the G-protein coupled receptors (GPCRs) SER-1 and SER-7 that are expressed on the egg-laying muscles.1,2,4 Consistent with this previous work, we found that double-mutant worms lacking NLP-3 and either SER-1 or SER-7 show a strong synthetic egg-laying defect. NLP-3 neuropeptides were previously reported to act in an aversive response circuit through the GPCR NPR-17.3 Using a fosmid-based GFP reporter, we observed additional previously unreported expression of NPR-17 in cells of the egg-laying circuit.We found that animals lacking HSNs, serotonin, or NLP-3 neuropeptides, which all lay eggs less frequently than wild type, surprisingly all show increased activity of the egg-laying circuit as measured using GCaMP recordings in freely-behaving animals. We therefore hypothesize that co-release of serotonin and NLP-3 from the HSNs is not essential for activity of the circuit, but is required to coordinate muscle activity, allowing the animal to productively lay eggs.1Carnell et al. (2005) J. Neurosci. 25:10671-81; 2Hapiak et al. (2009) Genetics 181:153-63; 3Harris et al. (2010) J. Neurosci. 30:7889-99; 4Hobson et al. (2006) Genetics 172:159-69.