Yun-Seong Jeong et al. (2001) International C. elegans Meeting "Enzymatic and in vivo functional characterization of a C.elegans RecQ DNA helcase"

Yun-Seong Jeong, Hyeon-Sook Koo, Myon-Hee Lee, Yool-Ie Kang

[International C. elegans Meeting, 2001]

The Caenorharbditis elegans E03A3.2 gene product is a member of the RecQ family DNA helicase, which includes Sgs1 in S. cerevisiae and RecQL, Bloom's Syndrome protein (BLM), Werner's Syndrome protein (WRN), Rothmund-Thomson Syndrome protein (RecQ4), and RecQ5 in human. Deficiency in either of Sgs1 and three human homologs (BLM, WRN, RecQ4) results in phenotypes suggesting their common roles in the maintenance of genomic stability. To examine enzymatic characteristics of the E03A3.2 protein which is most similar to human RecQ5 among 4 RecQ homologs in C. elegans , the recombinant protein was overexpressed in E. coli cells as a fusion protein with glutathione-S-transferase. The purified protein exhibited DNA-dependent ATPase and helicase activities. As a helicase, the RecQ5 protein preferentially displaced a primer with a 5'-overhang than a primer with a 3'-overhang, both of which had been annealed to M13 single-stranded DNA, indicating a 3' to 5' directionality in the helicase movement. To study the in vivo function, in situ mRNA hybridization and reporter gene expression, and double-stranded RNA interference were carried out. The mRNA was expressed in the gonad, oocytes, and early embryos and was localized in gut cells of the late embryos. This intestinal localization was also observed for a reporter protein (GFP) expression induced by the promoter from the late embryonic to adult stage. dsRNA i of the recQ5 gene expression resulted in a reduction of life-span by 2.3 days, suggesting that the RecQ5 protein may be involved in a cellular reaction affecting the aging of C. elegans .

Shah, Pavak et al. (2015) International Worm Meeting "Real-time tracking and optical manipulation: Single-cell perturbations in the embryo of Caenorhabditis elegans."

Bao, Zhirong, Shah, Pavak, Santella, Anthony

[International Worm Meeting, 2015]

While in toto imaging and image analysis methods have advanced the study of multicellular phenomena in development at single-cell resolutions, not much progress has been made in the design of tools to perturb embryos with comparable spatial and temporal resolution. Both classical techniques such as laser ablation and new technologies built around light-activated proteins offer significant promise in filling this need. Their use to-date, however, remains limited by a need for cell-specific promoters or completely manual operation. We have developed a platform for the real-time segmentation and tracking of cells in the C. elegans embryo to enable more reproducible perturbations at higher throughput and without a need for cell-specific markers. The platform consists of three components: 1) Automated cell detection and tracking. 2) An interface for curating detection and tracking results. 3) Laser control for carrying out a pre-defined perturbation protocol when a target cell identity is detected (ie. cell killing by ablation with a pulse laser).The performance of the cell detection pipeline exhibits little dependence on the number of cells present in the embryo; segmentation requires only 3 s per volume on a high-performance workstation. The time required to track detected cells at each time-point, however, is strongly dependent on the number of cells present; currently matching an average of 7.6 cells / s. For an imaging period of 75 s, the on-line segmentation pipeline executes faster than the imaging rate through the 500-cell stage and thus potentially up until twitching begins. More heavily parallelizable strategies for cell tracking are also being pursued to enable perturbations later and in larger model systems. This platform should prove valuable for performing single-cell ablations in the late embryo (ie. for the ablation of individual neurons following their terminal division) and could be easily adapted for other perturbations such as the induction of photoactivatable signaling proteins or even to use in other model systems.

Do Hyun Kim et al. (2006) East Asia C. elegans Meeting "The Study of Dolichol Phosphate Mannose Synthase I (DPM1) Homolog Y66H1A.2 in C.elegans"

Do Hyun Kim, Jin Won Cho

[East Asia C. elegans Meeting, 2006]

Dolichol phosphate mannose synthase is one of the multiple enzymes involved in N-glycan assembly in ER. It catalyses the synthesis of dolichylphosphatemannose(dol-P-Man) from GDP-mannose and dolichylphosphate. This enzyme is composed of a catalytic subunit DPM1 and regulatory subunits DPM2, DPM3. It was reported that partial deficiency in human DPM biosynthesis causes CDG(Congenital disorders of glycosylation) type Ie. We found that dpm1 homolog(y66h1a.2) in C.elegans was mainly expressed in hypodermis and intestine. Functional block of dpm1 homolog by RNA interference caused developmental delay, formation of huge embryos or unfertilized oocytes, abnormal cleavage of embryos, egg-laying defect, and enlargement of intestinal lumen. The intestinal lumen enlargement was defined to be associated to dysfuntion of digestion. we confirmed problems on ovary occurs when the funtion of DPM1 is inhibited ; by mating wild type male and hemaphrodite, they could not produce any progenies. These results indicates that C.elegans DPM1 have important roles involved development.

Lee, Hanee et al. (2011) International Worm Meeting "Dissection of the LATS kinase pathway in C. elegans."

Kang, Junsu, Lee, Junho, Lee, Hanee

[International Worm Meeting, 2011]

The Hippo(Hpo) pathway is a conserved signaling pathway regulating organ size homeostasis via controlling cell proliferation and apoptosis. We previously reported that wts-1, the C. elegans LATS homolog which is a core gene in the Hpo pathway, showed an early larval arrest phenotype due to the defective functions of the intestine(1). In this study, we tried to find the downstream genes of wts-1 by using EMS random mutagenesis. As a result, we obtained 18 suppressor lines that suppress the larval arrest phenotype of wts-1. We identified yap-1 and egl-44, the C.elegans homolog of YAP/Yki and TEAD respectively, as suppressors of wts-1 by SNP mapping. Mapping of the remaining mutations is in progress. Because it is known that LATS kinase acts through YAP/yki, a transcription co-activator, which interacts with the TEAD transcription factor, our results suggest that the core components of the Hpo pathway and their genetic interaction are conserved in C.elegans. Therefore, C. elegans is a good model organism to study the Hpo pathway. To delineate the Hpo pathway in C. elegans, we are looking for upstream and downstream genes using RNAi screening and microarray, respectively. Our work will contribute to further understanding of the Hpo pathway. (1)Kang J, et al. (2009), Lats kinase is involved in the intestinal apical membrane integrity in the nematode Caenorhabditis elegans. Development 136(16), 2705-15.

Matthew P Klassen et al. (2005) International Worm Meeting "Mechanisms Regulating Presynaptic Maturation in HSN"

Matthew P Klassen, Kang Shen

[International Worm Meeting, 2005]

Animals critically depend on synapses to compute appropriate behavioral responses to environmental stimuli. The assembly of these synapses requires the production of multiple synaptic proteins, including regulators of the synaptic vesicle cycle. However, it is not understood how the transcription/translation of these synaptic proteins are coordinated during synaptogenesis. We use the C. elegans hermaphrodite-specific neuron (HSN) as a genetic model system to study the regulatory mechanisms of presynaptic maturation. Preliminary results suggest that the expression of the rab-3 GTPase, a facilitator of synaptic transmission, and unc-57/endophilin, a protein involved in endocytosis are expressed late in the differentiation of HSN during synaptogenesis. Conversely, the SNARE-protein synaptobrevin (snb-1) and unc-18 appear to be upregulated following terminal mitosis. This differential regulation appears to be controlled intrinsically by the POU-domain transcription factor UNC-86. Unc-86(e1416) mutants exhibit precocious expression of rab-3 and unc-57. Furthermore, preliminary results suggest that regulated exocytosis is required for the expression of rab-3 in HSN although this maturation appears independent of the balance of the egg-laying apparatus (ie. Vulva, vulval muscles, VC4,5 neurons, and the gonad). In conclusion, we hope to provide insight into the relative contributions of intrinsic and extrinsic signals that regulate the maturation of the presynaptic terminal.

Plunkett JA et al. (1991) International C. elegans Meeting "IMMUNO-EM ANALYSIS OF THE DD AND VD MOTONEURONS."

Simmons RB, Walthall WW, Plunkett JA

[International C. elegans Meeting, 1991]

The embryonic DD motoneurons eIhibit different target specificities at different stages of development (White, et al., Nature, 1978). John White has determined that the period of respecification is closely associated with the molt from Ll to L2 and simultaneous with the completion of morphological differentiation of the postembryonic VD motoneurons. Morphologically the two classes of D motoneurons are very similar, eacb having a process in the dorsal nerve cord, a process in the ventral cord, a connecting commissure and a ventrally positioned cell body. However they exhibit different patterns of synapses. Currently only one gene has been identified that affects one class of D neuron and not the other. Interstingly that gene, unc-ff, alters the pattern of synapses of the VD motoneurons so that it becomes identical to the DD pattern. Are the VD motoneurons in unc-ff mutants following the same sequence as described for the DD motoneurons (ie transiently forming one pattern of synapses and then switching)? To answer this question we are developing EMimmuno methods using antisera to the D motoneuron neurotransmitter GABA. Although we have no answer at this time, the technique does appear to be working and is of enough general applicability to warrant discussion. Worms, encased in agar, are fixed in I % glutaraldehyde and 4% paraformaldehyde. They are then dehydrated in alcohol and embedded in LR Gold resin and cold polymerized (4 for 16 hours (o/n)) under UV light. Tissue was processed according to procedures detailed in Okamoto and Thomson (J. Neurosci.1985). An antirabbit secondary antiserum conjugated tol O angstrom colloidal 801d partides makes it possible to view the antigen antibody complex at the EM level.

Peter Nagele et al. (2002) Mid-west Worm Meeting "Behavioral Effects of laughing gas (nitrous oxide, N 2 O) in C. elegans"

Peter Nagele, Laura Metz, C Michael Crowder

[Mid-west Worm Meeting, 2002]

The molecular mechanism of action of the general anesthetic nitrous oxide (N2 O) is unknown. However, recent results indicate that the N-methyl-D-aspartate (NMDA) glutamate subtype receptor might be a potential target for nitrous oxide. In this study we first wanted to see if N2 O has behavioral effects in C. elegans and, if so, were the effects consistent with antagonism of NMDA receptors. Recently, a worm NMDA-receptor knock-out strain nmr-1(ak4) was isolated and found to confer a distinctive behavioral phenotype, namely a marked increase in the time spent moving forward and, of course, a decreased amount of reverse movement. To test the effects of nitrous oxide on locomotion, worms were picked to unseeded agar plates and put into gas-tight glass chambers containing either room air or a mix of 70% nitrous oxide/30% oxygen. After a 30-min equilibration, we scored the rates of forward and backward movement during a 7-minute period for each worm. Wild-type worms had an average duration of forward movement of 78.5+/-33.1 s (mean+/-SD) in air and 213.7+/-110.9 s in 70% nitrous oxide, p < 0.01. The average rate of reversal during the observation period was 3.2+/-0.5 per minute in air and 0.6+/-0.3 per minute in nitrous oxide. p < 0.01. We are currently testing nmr-1(ak4). Unlike for volatile anesthetics, nitrous oxide had no effect on the overall locomotion rate (ie, body bends/min). Our results show that nitrous oxide does produce a behavioral effect in C. elegans. Moreover, the effect is similar to that produced by nmr-1(ak4), consistent with an NMDA antagonism mechanism for nitrous oxide. Finally, the different behavioral effects of volatile anesthetics (drugs like ether and halothane) and nitrous oxide suggests that the mechanism of action of these two classes of general anesthetics differ.

Timpano, Autumn et al. (2009) International Worm Meeting "Identification of the circadian gene homologs sur-6/PP2A, gsp-1/PP1, gsp-2/PP1, kin-2/CKIIa and kin-10/CKIIb as developmental timing regulators of cell fate during C. elegans post-embryonic development."

Timpano, Autumn, Banerjee, Diya, Chen, Xin

[International Worm Meeting, 2009]

The development of multicellular animals is a complex process, which requires that molecular and cellular events occur in specific locations and in a specific sequence. Both spatial and temporal patterning of cell fate during animal development is genetically controlled. The most extensively investigated developmental timing pathway is the C. elegans heterochronic Pathway, which controls the timing of cell fate determination during post-embryonic, larval development. The heterochronic genes lin-42, kin-20 and tim-1 are the C. elegans homologs of the core circadian genes period, casein kinase Ie, and timeless, respectively. These three circadian genes are core components of the circadian clock, a molecular timing mechanism that regulates gene expression and behavior over the 24-hour day/night cycle. We have previously shown that the C. elegans circadian gene homologs regulate the timing of cell fate differentiation to occur during the larval to adult stage transition. These circadian gene homologs that have developmental timing function thus define a molecular and functional parallel between the circadian and developmental clocks, and suggest the hypothesis that the two types of timing pathways utilize a conserved mechanism of temporal regulation. To test this hypothesis, we have identified the C. elegans homologs of all known Drosophila and mammalian circadian timing genes and we are investigating the possible function of these homologs in control of developmental timing, using RNA interference (RNAi) to knock-down gene expression. We will present evidence that the C. elegans Casein Kinase II genes, kin-3 and kin-10, the Protein Phosphatase regulatory subunit gene, sur-6, and two Protein Phosphatase 1 genes, gsp-1 and gsp-2, interact genetically with the heterochronic pathway and thus may function as developmental timing genes.

Ardiel, Evan L. et al. (2013) International Worm Meeting "Response to repeated activation of ASH requires glutamate, dopamine, and neuropeptide signaling."

Giles, Andrew C., Rabinowitch, Ithai, Lindsay, Theodore, Lockery, Shawn, Ardiel, Evan L., Schafer, William, Rankin, Catharine H.

[International Worm Meeting, 2013]

The objective of this research was a comparative analysis of habituation in two neural circuits that synapse onto the same interneurons. Previous work in the lab has focused on habituation to a non-localized mechanical stimulus - a plate tap. Here we studied the response to repeated activation of the ASH sensory neurons, which detect a variety of aversive stimuli. A strain expressing ChR2 exclusively in ASH (Ezcurra et al., 2011) was used for consistent and discrete delivery of simulated aversive stimuli to worm populations being tracked by real-time computer vision software (Swierczek et al., 2011). In addition to increasing throughput, this optogenetic approach allowed us to prevent sensory adaptation and specifically activate ASH. Whole-plate blue light illumination elicited backward crawling (reversal) in the majority of animals. As with the tap-withdrawal response, the magnitude of ASH-mediated reversals decremented with repeated stimulation in a manner dependent on the stimulus intensity and frequency and recovered to baseline in minutes. Electrophysiological recordings of repeatedly activated photocurrents in ASH demonstrated that the decrement was not caused by ChR2 desensitization. Furthermore, a tap could dishabituate decremented responding. Cross-modal habituation was evident in only one-direction, ie the tap-withdrawal response was decremented by repeated activation of ASH, thereby localizing the site of plasticity to the shared circuitry. Although their magnitude decremented over the trial, ASH reversals persisted far longer than tap-induced reversals. This response maintenance was dependent on glutamate and dopamine signaling. Repeated ASH activation also led to increased speed of forward locomotion and a greater suppression of spontaneous reversals in the periods between stimuli, as compared to tap. This change was dependent on neuropeptide signaling. These distinct behavioural strategies are hypothesized to facilitate escape from a hazardous area.

Stefano Canevascini et al. (2001) International C. elegans Meeting "Cloning of negative regulators of the RAS/MAPK pathway in C. elegans."

Stefano Canevascini, ALEX HAJNAL, Erika Froehli, Gopal Battu, Amit Dutt, Thomas Berset

[International C. elegans Meeting, 2001]

The EGF/RTK/RAS/MAPK signal transduction pathway has been conserved during evolution. It controls cell differentiation in organisms as different as insects, worms and vertebrates. Its activity must be tightly controlled for the correct development of the organism and its incorrect activation can lead to tumour formation in vertebrates. In C. elegans, an EGF/RTK/RAS/MAPK signalling pathway is involved in vulval development. The anchor cell expresses the lin-3 ligand, a TGF-a homolog that activates the RTK/RAS/MAPK pathway in the P6.p cell, one of the six equivalent vulva precursor cells (VPC). In turn, the P6.p cell recruits the two neighbouring VPCs P5.p and P7.p through a lateral signal. The three cells then differentiate and form a functional vulva. Constitutive activation of the RTK/RAS/MAPK causes all six VPCs to be activated resulting in a multivulva (Muv) phenotype. Mutations that inactivate the RTK/RAS/MAPK pathway cause a vulvaless (Vul) phenotype because the VPCs remain uninduced and fuse with the hypodermis. We are cloning new factors involved in the negative regulation of the EGF/RTK/RAS/MAPK pathway in C. elegans. We carried out an EMS mutagen screen in a gap-1(0) background and selected for multivulva animals in the F2 generation. Gap-1 is a negative regulator of the RAS pathway that does not have a phenotype as single mutant. The new mutants look normal in a wild type background and show the multivulva phenotype only in a gap-1(0) background. For this reason we named them "multivulva enhancer of gap-1O(meg). We isolated six mutations that fall into four complementation groups, ie meg-1, meg-2, meg-3, meg-5 and meg-4 (see also presentation of Gopal Battu). We will report about the cloning of meg-3 that maps on chromosome II between the unc-4 and unc-53 markers.