Khivansara, Vishal et al. (2009) International Worm Meeting "The Cold Shock Domain Protein, CEY-3, regulates miR-1 target expression and AIN-1 localization to P bodies in the body muscle."

Kim, John, Moresco, Jim, Khivansara, Vishal, Wei, Dongping, Yates, John

[International Worm Meeting, 2009]

The microRNA-induced silencing complex (miRISC) guides microRNAs to the 3'' UTR of target mRNAs through partial base pairing and leads to translational inhibition and/or target degradation. However, the steps of target recognition and binding, and the role of miRISC and other RNA-associated factors remain active areas of investigation. We have identified a novel protein complex that binds microRNAs and their targets in the body muscle of C.elegans. This complex includes AIN-1, a conserved member of miRISC, as well as three highly conserved RNA binding proteins with Cold Shock Domains (CSD): CEY-2, CEY-3, and CEY-4. To elucidate the function of the CSD proteins, and in particular, CEY-3, in the regulation of microRNA targets, we have focused our studies on miR-1 rcontrol of its mRNA target, mef-2, in the body muscle (1). RNAi inactivation or deletion (tm2839) of cey-3 causes a significant increase in GFP::mef-2 3''UTR reporter expression, compared to vector control RNAi, and was similar to GFP reporter expression observed after RNAi-inactivation of ain-1, which is essential for the regulation of microRNA targets (2-3). Secondly, the P-body localization of AIN-1::GFP was completely abolished in the cey-3(tm2839) deletion mutant and by cey-3 RNAi. Endogenous levels of AIN-1 were also significantly reduced when cey-3 was inactivated, suggesting that cey-3 is required for AIN-1 expression and recruitment to P-bodies. All CSD proteins possess two conserved motifs (RNP1 and RNP2) within the cold-shock domain that are essential for binding RNA substrates. Substitutions of the following residues of CEY-3 to Ala: Tyr76 and Phe78 of RNP1 and Phe90 and His92 of RNP2 disrupt the recruitment of CEY-3::GFP to P bodies, especially during later stages of development, suggesting that RNA-substrate binding is a requirement for CEY-3 localization to P bodies. Current analysis is focused on determining whether miR-1 targets are substrates of CEY-3 and whether CEY-2,- 3 and -4 form distinct AIN-1 complexes in the body muscle. We expect that these studies will provide insights into the conserved mechanisms of how microRNA targets are recognized and designated for translational inhibition and/or degradation. 1.Simon, D.J. et al., Cell 133: 903-915 (2008). 2.Ding, L. et al., Mol Cell 19:437-447 (2005). 3.Zhang, L. et al., Mol Cell 28: 598-613 (2007).

Crittenden, Sarah L. et al. (2013) International Worm Meeting "Progress on developing the Q system to study GSCs and their control."

Crittenden, Sarah L., Kimble, Judith, Mohanty, Ipsita

[International Worm Meeting, 2013]

The Q system (Potter et al., 2010) permits inducible gene expression in C. elegans (Wei et al., 2012) and is similar in principal to the widely used GAL4-UAS system. The QF transcriptional activator directs transcription via an upstream activating sequence (QUAS). But in addition, another protein, QS, can inhibit QF activation and QS repression can be relieved by a small molecule, quinic acid or QA. QA is non-toxic and can be fed to worms (Wei et al., 2012). This inducible system has the potential to control target genes in both space and time. A chemically inducible method to control gene expression in specific cells at specific times would be tremendously useful for analyses of germline stem cells (GSCs) and their niche, the somatic distal tip cell (DTC). To this end, we are generating mosSCI insertion transgenes that rely on DTC-specific and GSC-specific regulatory sequences to drive QF expression. We are also generating a QUAS driven nuclear GFP (fused to H2B). Once we have the QF/QUAS pair working well for spatial regulation, we will add the QS/QA pair for temporal regulation. Preliminary experiments are promising and results will be shared at the meeting.

Driesschaert, Brecht et al. (2022) MicroPubl Biol

Driesschaert, Brecht, Temmerman, Liesbet

[MicroPubl Biol, 2022]

The Q system is a genetic tool developed to deliver spatiotemporal control over gene expression (Giles et al. 1991; Potter et al. 2010; Wei et al. 2012). Although it has already been adapted for use in C. elegans by Wei et al. in 2012, to date, the Q system has not been applied extensively in this nematode. In the relatively few available reports, it is mainly used to constitutively restrict gene expression in a spatial manner (e.g. Schild et al. 2014; Schild and Glauser 2015; Jee et al. 2016; Tolstenkov et al. 2018; Chiyoda et al. 2021), while but a handful of studies also explore the temporal aspect of the system (Matus et al. 2015; Yuan et al. 2016; Cottee et al. 2017; Hoang and Miller 2017). We aimed to apply this tool in the C. elegans nervous system to gain both spatial and temporal control over expression of a gene encoding a reporter protein that is targeted to the secretory pathway. Despite our efforts, we here report that in our hands, the Q system is not suitable for application in the neurons due to a lack of dynamic range.

Clovis Y et al. (2016) MicroPubl Biol "Mutations in KCNQ potassium channels cause pharyngeal pumping defects in C. elegans."

Roberts B, Webb A, Clovis Y, Turner C

[MicroPubl Biol, 2016]

Pumps were stimulated with 10mM 5HT in M9 recorded as electropharyngeograms (EPGs) for 2 minutes in a NemaMetrix ScreenChip, and analyzed using NemAnalysis software (NemaMetrix). The null mutant strains kqt-1(aw3) and kqt-3 (aw1) were kindly donated by Dr. Aguan Wei (Wei et al., 2002) A) Pump frequency in kqt-1(aw3) animals was significantly lower than in N2s, while kqt-3(aw1) worms showed an increase in pump frequency (*p<0.05; ***p<0.01; 1-tailed Mann-Whitney U-test; n = 21-23 worms in each strain). B) Microfluidic EPG recordings show that pumping pattern in kqt-1(aw3) mutants is arrhythmic, with frequent drops in frequency. C) Overlay of first 50 pumps of recordings show that pump duration is higher in kqt-1(aw3) and kqt-3(aw1) animals than in N2s. Pumps are showed aligned on E spikes, which occur when the pharynx is fully contracted. D) Duration histogram illustrating the probability of occurrence of inter-pump interval (E to E duration) for each mutant strain. Histograms were binned to 4 ms width and normalized to reach an area underneath the curve equal to 1 (duration 100% likelihood to occur). In kqt-1(aw3)) animals, the time between two pumps is significantly increased compared to N2s (p < 0.01).

Huang CG et al. (2006) Mol Biotechnol "Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening."

Lamitina T, Agre P, Strange K, Huang CG

[Mol Biotechnol, 2006]

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.

She, Xingyu et al. (2015) International Worm Meeting "Applying a Spatial-Temporal Controlled Gene Expression System to Aging Research."

She, Xingyu, Hansen, Malene

[International Worm Meeting, 2015]

C. elegans has been an effective model organism for identifying genes with important physiological functions, including in aging. However, to fully dissect gene function an effective spatial-temporal controlled gene expression system is needed. In C. elegans, several protocols have been put forward for this purpose, including driving temporal gene expression using heat shock inducible promoters (Bacaj et al. 2007), and achieving tissue-specific gene expression using site-specific recombination methods (Cre-loxP, FLP-FRT). However, none of these systems have been used for simultaneous temporal- and spatial control and it is uncertain how applicable they would be for aging studies (see Xu & Kim, 2011). While several chemicals (e.g., tetracycline, RU486) are utilized to induce gene expression in other systems, such protocols are not available in C. elegans. However, the Shen lab recently adapted the Q system to C. elegans, a system in which inducible gene expression is obtained by the chemical quinic acid (Wei et al. 2013). This binary system, originally discovered in the fungus Neurospora crassa, is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through the timing of quinic acid supplementation, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF (Wei et al. 2013). We are interested in applying the Q system for our research on genes with roles in aging. Towards this goal, we are focusing on three main aspects. First, we have tested the effects of quinic acid and the Q system on C. elegans lifespan and stress resistance and detected no apparent alterations on longevity or fitness, suggesting that the Q system is suitable for aging studies. Second, we are driving the elements of the Q system with tissue-specific promoters to test inducibility in major tissue types of adult animals. Preliminary results indicate that the Q system can be used to induce expression in selected tissues, including neurons and muscle. Third, we are creating a library of Gateway-compatible plasmids that contain the various elements of the Q system and tissue-specific promoters to expedite the generation of Q system transgenic strains for future gene-specific projects. We will present our current progress towards applying the Q system as a powerful tool for spatial- and temporal analysis of gene function in adult C. elegans. This work will also be discussed at the "Spatial and temporal analysis of gene function in C. elegans" workshop.

Thurman, Michellie et al. (2021) MicroPubl Biol

Sun, Haonan, Praitis, Vida, Kubica, Sam, Thurman, Michellie

[MicroPubl Biol, 2021]

Calcium signaling is known to play a critical role in cell migration (Ridley et al., 2003; Wei et al., 2012). In C. elegans, embryos with disruptions in the secretory pathway calcium ATPase gene pmr-1 show defects in cell migration that result in lethal phenotypes. Previous work has shown that pmr-1(lof) phenotypes can be suppressed by changes in the activity of the calcium channels IP3-receptor/ITR-1 and ryanodine receptor/UNC-68, indicating that cell migration defects are the results of changes in calcium homeostasis (Praitis et al., 2013). To identify additional genes important for cell migration during embryogenesis, we performed a genetic screen to isolate additional suppressors of the pmr-1(ru5) strain, reasoning that disruption of cell migration due to changes in pmr-1 activity could be suppressed by commensurate changes in other genes that regulate calcium levels or signaling. In this screen, we identified the kez8 allele. We confirmed that the kez8 allele suppresses the pmr-1(ru5) mutant phenotype by examining the percentage of viable progeny produced by pmr-1(ru5); kez8 strains when grown under restrictive conditions. The viability of the pmr-1(ru5); kez8 strain is 67%, significantly higher than the 3.9% viability observed in the pmr-1(ru5) control strain at 25C (Table 1; p&lt;0.01).

Zhang, Qing et al. (2011) International Worm Meeting "Dissecting the molecular pathway underlying IFT modeling."

Hu, Jinghua, zhang, yuxia, Wei, Qing, Zhang, Qing

[International Worm Meeting, 2011]

Cilia act as motile or sensory devices on the surfaces of most eukaryotic cells, and cilia dysfunction results in a variety of severe human pathologies, collectively termed ciliopathies. Phylogenetically conserved intraflagellar transport (IFT) machinery, which is composed of IFT-A and IFT-B subcomplexes, mediates the bidirectional movement of IFT cargos that are required for the biogenesis, maintenance, and signaling of cilia. However, an understanding of how IFT particles are assembled at the ciliary base and turned around at the ciliary tip remains elusive. Our previous data suggested that IFT-B component DYF-2 is the key factor involved in regulating IFT modeling at both the ciliary base and ciliary tip. From a mutagenesis screen for C. elegans mutants with defective IFT modeling at the ciliary tip, we identified jhu616, which encodes a mutant DYF-2 with a G361R alteration in the conserved WD40 domain. Further studies indicated that DYF-2 and BBS (Bardet-Biedl syndrome) proteins coordinate IFT assembly/reassembly at the ciliary base/tip (See Wei Q. et al's abstract from the Hu lab). To further dissecting the molecular pathway underlying IFT modeling, we performed a suppressor screen of the jhu616 allele. Dye-filling (Dyf) assay was used to assess cilia integrity. jhu616 animals are 100% Dyf. In ~300,000 haploids screened, we retrieved 7 independent strains that could rescue the Dyf phenotype of jhu616 allele. Sequencing results showed that no new mutations were introduced into dyf-2 locus, indicating that the suppressors contain mutations in other players involved in IFT modeling. We are mapping the suppressors and looking forward to fully dissecting the molecular pathway underlying IFT modeling and ciliogenesis.

She, X. et al. (2017) International Worm Meeting "Applying the Q system for spatiotemporal gene expression in adult C. elegans."

Hansen, M., She, X.

[International Worm Meeting, 2017]

While C. elegans has long been on the leading edge of aging research, a spatial-temporal controlled gene expression system is currently lacking to fully understand tissue- and adult-specific functions of genes modulating lifespan and healthspan in this organism. The Shen lab recently adapted the so-called Q-system for inducible gene expression in a subset of C. elegans neurons (Wei et al., Nature Methods, 2013). Originally discovered in the fungus Neurospora crassa, this binary system is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through quinic-acid supplementation to the media plates, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF. Here, we further developed the Q-system and tested it for its usability in C. elegans aging studies. First, we drove the elements of the Q-system with tissue-specific promoters expressing in several major tissue types to show the inducibility of the system in adult animals. Our results indicate that the Q-system can be used to induce expression in such tissues. Second, we tested the effects of quinic acid and Q-system components on C. elegans lifespan and stress resistance and detected no apparent effects on longevity or fitness, suggesting that the Q-system can be used for lifespan studies. We will present current progress towards applying the Q-system as a powerful system for spatial- and temporal analysis of gene function in adult C. elegans.

Jon Pierce-Shimomura et al. (2002) Japanese Worm Meeting "Behavioral responses to ethanol are mediated by a BK potassium channel in C. elegans"

Antonello Bonci, Steve McIntire, Hongkyun Kim, Jon Pierce-Shimomura, Tod R Thiele, Andrew Davies

[Japanese Worm Meeting, 2002]

We screened for mutant C. elegans that are resistant to intoxication by ethanol. We found that strong resistance is conferred by mutations in the gene slo-1 , which encodes a highly conserved ortholog of the human large conductance calcium-activated potassium (BK) channel 1-3 . Prevous work has shown effects of ethanol on vertebrate BK channels in vitro 4-9 . We hypothesized that intoxication in C. elegans may be primarily mediated by ethanol activating the SLO-1 channel; thus strong resistance to intoxication by ethanol results when the SLO-1 channel is absent. To test this idea, we first performed in vivo whole-cell voltage-clamp recordings of dopamine neurons to identify a SLO-1 current that was present in neurons in wild-type worms but absent in slo-1 mutants. Next, we found that ethanol selectively and reversibly increased the amplitude of the outward-rectifying current in neurons of wild-type animals at physiologically relevant concentrations (20 & 100 mM). Ethanol had no effect, however, on currents in neurons of slo-1 mutants. To determine whether ethanol can activate SLO-1 directly in the absence of cytosolic factors, we recorded the single-channel activity of SLO-1 in patches excised from dopamine neurons. Ethanol reversibly increased the probability of channel opening. Thus, we have demonstrated a direct relationship between the SLO-1 channel, a biochemical effect of ethanol and a genetic effect on behavior 1 Wei et al (1996) Neuropharm; 2 Wang et al (2001) Neuron; 3 Chu et al (1998) Mol Pharmacol; 4 Dopico et al (1998) J Pharmacol Exp Ther; 5 Dopico et al (1996) Mol Pharmacol; 6 Dopico et al (1999) J Physiol; 7 Gruss et al (2001) Eur J Neurosci; 8 Jakab et al (1997) J Membr Biol; 9 Walters et al (2000) Am J Physiol Cell Physiol