Goetting, Desiree et al. (2017) International Worm Meeting "Food deprivation suppresses sleep via AMP kinase and circumvents the lethal effect of sleep loss in C. elegans."

Soto, Rony, Van Buskirk, Cheryl, Goetting, Desiree

[International Worm Meeting, 2017]

A major challenge in understanding the function and evolution of sleep lies in identifying the mechanisms that offset the vulnerabilities that come with it, such as the inability to forage or escape. The nematode C. elegans has recently been identified as a useful model for the dissection of sleep function and evolution, as animals experience a primitive sleep-like state that is triggered by conditions that cause cellular damage. In response to noxious environmental conditions such as extreme heat, animals enter a state of behavioral quiescence characterized by a reduction in sensory responsiveness and a cessation of feeding and locomotion. This recovery sleep, or RS, appears to be beneficial under certain conditions, as sleepless mutant animals are impaired for survival following noxious heat exposure. We wished to investigate how the decision to enter into RS may be influenced by additional environmental inputs that could potentially alter the physiological benefit to be gained from sleep. Here we show that food deprivation suppresses RS, and that this effect is exacerbated as population density increases. In addition to suppressing sleep drive, food deprivation protects against the lethality associated with sleep loss, suggesting that food-deprived animals have a reduced need for sleep. We show that suppression of sleep drive during periods of food deprivation requires AMP kinase. Additionally, we show that competence to engage in RS is dependent on the neuroendocrine signal DAF-7/TGF- beta , activating a previously identified neural circuit that shifts several aspects of development and metabolism from conservation to utilization of energetic resources. These data suggest that recovery sleep in C. elegans is an energetically costly activity that can be suppressed when environmental conditions are unfavorable and animals are required to compete for resources.

Shen X et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Biomechanical analysis of C. elegans motility"

Arratia P E, Krajacic P, Lamitina T, Purohit P, Shen X

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

Previous studies of C. elegans motility have focused mainly on simple quantitative assays, such as the ''thrashing'' assay. More recently, quantitative approaches have been used to study nematode kinematics. Such quantitative approaches could provide a new level of phenotypic resolution to the study of C. elegans motility. We recently developed a simple mathematical model to describe the C. elegans swim gait. This model provides estimates for the biomechanical properties of motility including force, power, tissue viscosity, and Young''s modulus. Here, we used high-speed video as well as image analysis and particle tracking methods to experimentally measure the forces imparted on the surrounding fluid by a swimming C. elegans. Analysis of the local forces (propulsive and lateral) over the wave period is computed by performing a simple force balance and by coupling the fluid velocity field with the nematode body postures. The propulsive force shows a sharp peak as the lateral force approaches zero. The magnitude of the peak propulsive force (i.e. thrust) and the average mechanical power of a swimming nematode in M9 solution are approximately 3.0 nN and 2.0 pW. By comparison, values of force and power estimated using theoretical models are approximately 3.5 nN and 5.0 pW. These data provide experimental validation for our model of motility and demonstrate that biomechanical phenotyping can provide a sensitive and quantitative phenotypic metric for the analysis of C. elegans motility.

David HE et al. (1997) International C. elegans Meeting "APPLICATIONS OF LACZ/GFP STRAINS OF TRANSGENIC STRESS-INDUCIBLE CAENORHABDITIS ELEGANS."

de Pomerai DI, Dennis JL, Jones D, Candido EPM, Mutwakil MHAZ, David HE, Power RS, Nowell MA

[International C. elegans Meeting, 1997]

Transgenic stress-inducible strains of Caenorhabditis elegans have been evaluated in biomonitoring applications. Two transgenic lacZ-reporter strains have been utilised:- strain CB4027 (heterologous Drosophila hsp70 promoter) and strain PC72 (homologous hsp 16 promoter). The stress response in exposed worms is detectable by in situ staining for ss-galactosidase, and can be quantified using enzymatic assays. Stress responses to heavy metals have been demonstrated in laboratory tests and in water samples taken from a metal-polluted river system in south-west England. Surfactants amplify the effects of most metal ions tested. This biomonitoring test is being adapted for use in soils with the PC72 strain. In LUFA 2.2 soil, the stress response to Cd2+ and Hg2+ is proportional to their concentrations in centrifugally extracted soil water, which are much less than those originally added because of metal sorption to soil constituents. Immunological stress responses are also being evaluated in strain PC72 as a model for the effects of immune responses on parasitic nematodes. Antiserum raised against CTAB-stripped surface antigens gave specific surface labelling of worms. Although this antiserum evoked a significant stress response in PC72 worms, so too did naive rabbit serum. Specific immuno-fluorescence tests using naive rabbit serum and FITC-conjugated anti-C3 antibody suggest that the surface of C.elegans is itself capable of fixing complement, which may mediate a stress response. Future work will involve the development of novel hsp/GFP fusion strains to enable continuous dynamic monitoring of transgene product levels. This will necessitate the use of GFP variants in order to reduce the interference from gut autofluorescence.

Delia Garigan et al. (2001) International C. elegans Meeting "Characterization of a Short-Lived C. elegans Mutant"

Delia Garigan, Cynthia Kenyon

[International C. elegans Meeting, 2001]

Our lab screened 20,000 haploid genomes for long- and short-lived mutants and found at least ten short-lived mutants. We would like to know whether these animals are aging more rapidly than normal, or whether they are simply sick and unhealthy due to their mutations. Preliminary characterization of short-lived mutants reveals one candidate that appears healthy in early life but then dies early. We are using high-power microscopy and DNA microarray analysis to determine if this mutant has an accelerated progression of age-related tissue degeneration and biomarkers.

Xu M et al. (2010) Biology of the C. elegans Male, Madison, WI "Digital Alignment of Electron Microscopic Images for Nervous System Reconstruction"

Xu M, Hood G, Bloniarz A, Emmons S, Wetzel A

[Biology of the C. elegans Male, Madison, WI, 2010]

It is important to have electron microscopic images aligned before nervous system reconstruction. Alignment corrects the translation, rotation, magnification and distortion that occur during E.M. acquisition. A well-aligned E.M. dataset facilitates reconstruction using our program ELEGANCE, making it easier to identify neuron profiles and trace their progress through the EM stack. Moreover, alignment smooths the neuron process through sections to get better and more accurate maps. Alignment between high power series and low power series makes it possible to create a global coordinate system for neuron reconstruction across the whole worm. Alignment software created at the Pittsburgh Supercomputing Center is being used in our male nervous system reconstruction project. This software, which utilizes parallel computing algorithms, corrects misalignments including image warping. This program is installed on the Albert Einstein College of Medicine's Rock version computer cluster server with 65 nodes and 520 processors. We are currently using it to montage and align our new EM dataset of the male head, which when completed will consist of tens of thousands electron micrographs necessary to reconstruct the male anterior nervous system across 5000 serial sections. The software can also be applied to our already completed reconstruction of the male posterior nervous system to improve the accuracy of neuron maps.

Breimann, Laura et al. (2021) International Worm Meeting "Precise quantification of mRNAs across all C. elegans embryonic stages through a microscopy and machine learning-based approach"

Breimann, Laura, Bahry, Ella, Preibisch, Stephan, Harrington, Kyle I S, Epstein, Leo, Lionnet, Timothee, Ercan, Sevinc, Kolyvanov, Klim

[International Worm Meeting, 2021]

Precise quantification of mRNA transcripts in space and time throughout embryogenesis is essential for understanding gene regulation, a process critical for embryogenesis in all animals, including C. elegans. We developed an imaging approach using 3D widefield microscopy-based single-molecule RNA fluorescence in situ hybridization (smFISH) to quantify mRNA transcripts. To count individual single-molecule mRNA spots, we developed RS-FISH, a fast 3D spot detection method that we implemented in Fiji that combines radial symmetry and RANSAC outlier removal. To assign each fixed, imaged C. elegans embryo to its developmental stage, we used advanced machine learning-based image classification that relies on the concept of auto-encoders. Currently, we are applying our methods to understand the role of condensins in chromosome compaction and transcription regulation. In C. elegans, an X-specific condensin binds to and represses X chromosomes in XX hermaphrodites by 2-fold for dosage compensation. In our study, we want to understand condensin DC's effect on transcript numbers and dynamics in single embryos across development. We obtained thousands of smFISH images for a set of condensin DC-regulated and control genes and extracted mature and nascent RNA counts in 3D, which we use to determine transcription burst characteristics throughout embryonic development. The distribution of total transcripts in wild-type and condensin DC-depleted embryos shows that single genes on the X chromosome are downregulated ~2-fold. Our machine learning approach to separate embryo images by development stage allowed us to observe the timing of condensin DC-mediated transcription repression, which occurs from the 100-cell stage on. RS-FISH is freely available as a Fiji plugin, and details for installation can be found at https://github.com/PreibischLab/RadialSymmetryLocalization and described at https://doi.org/10.1101/2021.03.09.434205

Wada K et al. (2000) Japanese Worm Meeting "SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development."

Hisamoto N, Wada K, Hagiwara M, Matsumoto K, Kuroyanagi H, Kimura T

[Japanese Worm Meeting, 2000]

SR protein kinases (SRPKs) and their substrates, the serine/arginine-rich pre-mRNA splicing factors are key components of splicing machinery, and well conserved across phyla. These factors in metazoa have been well characterized through biochemical experiments, however, their physiological functions in multicellular organisms are still unclear. Here, we cloned a C. elegans SR protein kinase homologue, SPK-1, and one of its substrate, CeSF2. SPK-1 binds directly to and phosphorylates RS domain of CeSF2 in vitro. In situ hybridization analysis of adult hermaphrodite showed that both spk-1 and CeSF2 are predominantly expressed in gonads. Double-stranded RNA interference (RNAi) revealed that SPK-1 and CeSF2 play essential roles in the embryonic stage and that SPK-1 is also required for germline development of both hermaphrodites and males in C. elegans. We will also show that RNAi by soaking L1 larvae is also feasible for studying function of genes required for germline development.

Santos S I C O et al. (2008) European Worm Meeting "Ultra-fast optics: a novel tool to study C. elegans"

Mathew M, Santos S I C O

[European Worm Meeting, 2008]

In the last decade nonlinear techniques using ultra-fast optics has emerged. as a novel tool for biological investigation. We present the use of these. techniques in studying C. elegans viz. Two Photon Microscopy (TPEFM),. Harmonic Generation Microscopy (SHGM & THGM) and Ultra-fast laser Induced. Nanosurgery.. Nonlinear imaging techniques are superior to commonly used techniques with. respect to penetration depth, photobleaching and signal to noise ratio,. owing to the use of very high intensity low average power ultra-short near. infra red (NIR) pulses.. In the last few years these high intensity NIR pulses have also been used. to precisely target, cut and manipulate sub-cellular structures with sub-. micrometer resolution. This offers the possibility of in vivo surgery. without collateral damage.

Yukinobu Arata et al. (2007) International Worm Meeting "Quantitative measurement of the timing of cell division in C. elegans embryo."

Hitoshi Sawa, Yukinobu ARATA

[International Worm Meeting, 2007]

During animal development, cell cycle progression seems to be regulated tightly. In this study, we quantitatively measured cell volume and cell cycle duration. We found that cell cycle duration was in inverse proportion to the 0.26 (~1/4) th power of cell volume in the AB and MS lineages and the 0.34 (~1/3) th power of cell volume in the C lineage, but was independent of cell volume in the E and D lineages. These findings indicate that cells divide in size-dependent and -independent manners during the development, and suggest that cells possess unidentified mechanisms to regulate cell cycle progression, depending on cell volume. In addition, we also observed a relationship between cell division timing and cell position in the embryo, and showed that most cell divisions occur semi-synchronously throughout the embryo; anteriorly-positioned cells tended to divide slightly earlier than those in the posterior. This phenomenon seems to correspond to the mitotic wave that has been reported in Drosophila</I>, Xenopus</I> and zebrafish development. Is cell division timing affected by cell division in adjacent cells? We changed the relative positions of cells in embryos cultured in vitro</I>. Although most cells divide in the same order as in the embryo in egg shell, the division order of E cell daughters was earlier. This result suggests that cell division timing is affected by the embryonic microenvironment. Given these findings, we propose that cell cycle progression is regulated by size- and position-dependent mechanisms in the C. elegans</I> embryo.

Itskovits, Eyal et al. (2015) International Worm Meeting "A new multi-worm tracker reveals unprecedented insights on worm chemotaxis."

Itskovits, Eyal, Zaslaver, Alon

[International Worm Meeting, 2015]

We have developed a novel Multi Worm Tracker (MWT) that is capable of tracking over a hundred worms simultaneously. The tracker employs a machine learning classification approach to identify the behaving worms. The system produces a long informative track for each individual worm, and generally maintains tracking despite frequent animal collision events. This MWT provides unprecedented statistical power revealing subtle, yet significant, behavioral features. Here we present results that challenge the prevalent "biased random walk" strategy of worms' chemotaxis. Moreover, we readily obtain data with satisfactory statistical significance following a single chemotaxis assay. Our system includes a suite of solutions for acquisition, tracking, and statistical analyses via a friendly user interface that is easy to operate with minimal programming skills.