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.

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.

Soto , Rony et al. (2017) International Worm Meeting "UNC-103/ERG potassium channel contributes to locomotor quiescence during sleep."

Soto , Rony, Van Buskirk, Cheryl, Sternberg, Paul W.

[International Worm Meeting, 2017]

In recent years the field of sleep research has been bolstered by studies in non-mammalian model organisms like C. elegans, promising to shed light on the long-standing mystery of the core function of sleep. C. elegans has been found to experience two distinct types of behavioral quiescence that each fulfill all of the behavioral criteria for sleep. The first to be discovered was developmentally-timed sleep (DTS), which occurs at the end of each of the four larval stages (Raizen et al., 2008). The other sleep state can be triggered at any time by exposure to damaging conditions such as noxious heat, tissue damage, and UV exposure, and is referred to as stress-induced sleep (SIS) or recovery sleep (RS) (Hill et al., 2014; Iannacone et al., 2016). We have shown that recovery sleep in C. elegans is dependent on EGF signaling, and that robust sleep can be induced at any time, in an EGFR-dependent manner, via forced expression of the EGF ligand LIN-3 (Van Buskirk and Sternberg, 2007). We have taken advantage of this 'forced-sleep' assay to uncover genes required for sleep regulation. We are particularly interested in determining which potassium channel genes, if any, contribute to C. elegans sleep, as K+ channel mutations produce short sleepers in Drosophila and zebrafish. An RNAi screen of K+ channel genes and their regulators identified UNC-103, an ERG-type potassium channel, as required for EGF-induced sleep. Here we show that UNC-103 is required for locomotor quiescence during recovery sleep (RS) and contributes to locomotor quiescence during lethargus (DTS) as well, indicating that these two types of sleep share common downstream effectors. We present evidence for developmental compensation by other K+ channels in the unc-103 null mutant, as short exposure unc-103 RNAi produces a greater sleep disruption than long exposure. We find that the application of ERG-blockers disrupts sleep in an UNC-103-dependent manner. Last, we present the results of our current site of action analyses aimed at determining in which neurons the widely expressed UNC-103 is required during sleep.

Makoto Kobayashi et al. (2001) International C. elegans Meeting "A SURVEY OF CHEMICALS AFFECTING HABITUATION"

Makoto Kobayashi, Hiroya Koshishiba, Ryuji Hosono, Seiichi Hashimoto

[International C. elegans Meeting, 2001]

C.elegans is a desirable model organism to inquire into the role of chemicals on the nervous system. Numerous chemicals influencing on C.elegans behavior have been studied. On the other hand response to mechanical touch or tap stimuli remains to be described. We are now developing a method to access the role by systematic survey of drugs affecting habituation and its retention. TV camera images of C.elegans behavior under a stereomicroscope are captured to a personal computer by using tapping pulses as tirgger. A future target is to develop the image processing of sequential images. We applied the apparatus to pharmacological screening affecting on tap response, especially habituation and recovery from the habituation by changing ISI, pulse or tap number. We have begun with the method to study actions of various drugs including baclofen, barbital, caffeine, chlorpromazine, chlorzoxazone, diazepam, haloperidol, imipramine, mephenesin, perphenazine, phenobarbital, theophylline and (RS)-CPP. Of these, we found that mephenesin causes rapid habituation but slow recovery from habituation. We are especially interested in the long term habituation. Therefore, in parallel with the work, we are now trying to extend time at the habituated state.

K Brouwer et al. (2004) European Worm Meeting "MUTATOR GENES THAT PREVENT MICROSATELLITE INSTABILITY IN C. ELEGANS"

J Pothof, R Plasterk, M Tijsterman, K Brouwer

[European Worm Meeting, 2004]

Proper functioning of DNA repair and signaling pathways that maintain the stability of the genome is essential in the prevention of cancer. To identify genes contributing to genomic stability, a frameshift reporter was developed in our lab, which was subsequently used in a genome-wide RNAi screen. This led to the identification of 61 genes that prevent an increase in genomic instability (1). Thirteen genes were shown to be specifically involved in maintaining the stability of microsatellites (short sequence repeats). Among these genes are the four genes known to be involved in the mismatch repair pathway and that are linked to the human cancer syndrome HNPCC (hereditary non-polyposis colon cancer). For three of the yet uncharacterized genes (Y76A2B.5, K09B11.2 and rev-1) we have isolated null alleles. We are currently working on the characterization of these genes by performing epistasis experiments with the mismatch repair pathway and analyzing the mutator phenotypes of the mutants in further detail. 1 Pothof J, van Haaften G, Thijssen K, Kamath RS, Fraser AG, Ahringer J, Plasterk RH, Tijsterman M., Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev. 2003 Feb 15;17(4):443-8.

T Kawano et al. (1999) International C. elegans Meeting "Analysis of SR protein genes in C. elegans"

H Sakamoto, T Kawano, M Fujita

[International C. elegans Meeting, 1999]

SR proteins contain one or two copies of RNA recognition motif (RRM) as RNA-binding domain and an arginine/serine-rich (RS) domain. Previous in vitro studies have revealed that mammalian SR proteins have essential roles in constitutive splicing of pre-mRNAs. In addition, some SR proteins have been shown to play an important role in splice site selection. To investigate the in vivo function of SR proteins, we first searched the C. elegans genome database and found five genes potentially encoding SR protein (ceSRp20, ceSC35, T28, W12.2 and W12.3). We next examined, using transgenic worms, the expression of these SR proteins fused with green fluorescent protein (GFP) under the control of their native promoters. All of these SR-GFP fusion proteins were localized in the nuclei of almost all somatic cells, consistent with that SR proteins are constitutive splicing factors as in mammalian cells. However, simultaneous suppression of two or more SR protein-coding genes by dsRNA-mediated interference resulted in drastic phenotypes, such as embryonic lethality, abnormal gonadal formation and decrease of fertility. These results raise the possibility that SR proteins also play important roles in the development and the germ cell differentiation in C. elegans . Disruption of genes encodong SR proteins and a search for target genes whose mRNA expression are affected in such mutants are currently in progress.

Ravichandiran Kumarasamy et al. (2002) European Worm Meeting "Dna polymorphism in Pristionchus pacificus"

Ralf J Sommer, Ravichandiran Kumarasamy

[European Worm Meeting, 2002]

To understand the evolution of developmental process at the microevolutionary level, it is important to study the biogeography and the evolutionary alterations of the developmental processes in detail. To acuminate this, we are working on the satellite model Pristionchus pacificus. The genus Pristionchus has a worldwide distribution and different strains of Pristionchus pacificus exist from Washington (PS 1843), Hawaii (JU 138), Ontario (AF 8130), Poland (RS 106) and the wild type strain from California (PS 312). Previous AFLP studies showed a high degree of polymorphisms between the strains from Washington, Hawaii and California. BAC end sequencing and SSCP analysis of BAC end fragments between the strains from California and Washington confirmed these original observations. To obtain a better picture of the distribution of polymorphisms between the strains of P. pacificus, we have chosen a random 5 Kb region on Chromosome III that contains no open reading frame. Our results showed a high degree of polymorphism (3.46%) in both Washington and Hawaii when compared to the laboratory strain California. There were ~ 0.7% transitions, ~ 0.5% transversions, ~ 0.3% insertions and ~ 0.1% deletions. Most of the polymorphisms observed were single base pair events except a few which were up to 5 bp . Further studies are underway to extend this study, comparing the ceh-20 and ceh-40 genes in these strains and to eventually include the different species' of Pristionchus namely P. maupasi and P. lheritieri.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Batchelder C et al. (1998) East Coast Worm Meeting "Transcriptional repression by C. elegans PIE-1"

Cassie C, Batchelder C, Choy B, Suh Y, Mello CC, Blackwell TK

[East Coast Worm Meeting, 1998]

In the developing C. elegans embryo, maternally-expressed PIE-1 protein is required for specification of germ cell precursors. In pie-1 (pharyngeal and intestinal excess) mutants, these cells instead form pharyngeal and endodermal cells by a pathway that is dependent on the skn-1 gene. Germ cell precursors normally appear to lack both embryonic mRNA transcription, and a particular phosphoepitope on the C-terminal repeat of RNA Polymerase II. PIE-1 is expressed specifically in these cells and is required for their transcriptional silencing, and thus for preventing them from being directed to differentate by SKN-1 or other transcription factors. The sequence of PIE-1 does not suggest an obvious model for how it might function. PIE-1 contains two predicted Cys(3)-His zinc finger motifs and an intervening RS-rich region. Other Cys(3)-His zinc finger proteins have been implicated in RNA processing, post-transcriptional gene regulation, and growth factor responses, but the roles of their zinc fingers remain undefined. An effect of PIE-1 on transcription could be mediated at any of a variety of levels, including signaling, an effect on expression or modification of a general transcription factor, or a direct repressive effect on the transcriptional machinery. We have used a mammalian cell transfection assay to investigate the last of these possibilities. Our experiments indicate that PIE-1 can repress transcription driven by various types of RNA Polymerase II promoters in mammalian cell transfection assays. By attaching portions of PIE-1 to the yeast GAL4 DNA binding domain, we have identified a region that can function as a powerful repressor domain when it is thus tethered to a promoter. Critical residues within this repressor domain are strikingly similar to the Pol II C-terminal repeat sequence, suggesting that PIE-1 might act on proteins that bind to this repeat structure. Experiments are in progress to test this model. Our findings demonstrate that PIE-1 contains sequences that can act directly at promoters to repress transcription. We hypothesize that PIE-1 acts on an evolutionarily conserved component of the transcriptional regulatory machinery, and brings to it residues that are capable of effecting repression.