89 Works

A new deletion allele of sma-4

Alexandra N. McKillop, Herong Shi & Jun Liu
sma-4 encodes the co-Smad of the BMP pathway in C. elegans, which is also known as the Sma/Mab pathway (Savage et al. 1996). Null mutations in core components of this pathway, including the BMP ligand DBL-1, the receptors SMA-6 and DAF-4, and the R-Smads SMA-2 and SMA-3, all result in a small body size phenotype without significantly compromising viability (Gumienny and Savage-Dunn 2013). However, we found that even after multiple rounds of out-crossing, two deletion...

Application of the red-shifted channelrhodopsin Chrimson for the Caenorhabditis elegans cGAL bipartite system

Mengyi Cao, Cynthia Chai, Jonathan Liu & Paul W. Sternberg
Channelrhodopsins are light-gated ion channels that serve as photoreceptors in photosynthetic microbes and have been applied as crucial optogenetic tools in genetic model organisms. When expressed in animals, they enable light-inducible control of ionic membrane permeability, which directly manipulates the activity of neurons expressing the protein. The application of channelrhodopsin-based optogenetics is particularly powerful when used in conjugation with the cGAL (GAL4-UAS) bipartite system (Wang, 2017). The mating of neuron-specific GAL4 driver lines to new...

Rapamycin-induced protein dimerization as a tool for C. elegans research

Sriyash Mangal, Jeffrey Zielich, Eric J. Lambie & Esther Zanin
Induced protein dimerization is an invaluable tool in cellular biology to study protein function. Induced dimerization has been widely used to modulate enzymatic activity as well as expression and localization of proteins (Voß et al., 2015; DeRose et al., 2013). A popular method employs the chemical dimerizer rapamycin to induce binding between the FRB domain of the mTOR kinase and the FKBP12 protein (FK506 binding protein 12 kDa) (Putyrski and Schultz, 2012). Rapamycin-induced protein dimerization...

Extending the CRISPR toolbox for C. elegans: Cpf1 as an alternative gene editing system for AT-rich sequences

Annabel Ebbing, Peng Shang, Niels Geijsen & Hendrik C. Korswagen
The CRISPR/Cas9 system has become a powerful tool for genome-editing in C. elegans. Sequence specificity of the system is determined by a guide RNA which targets the Cas9 endonuclease to the genomic region of interest. In addition, Cas9 needs to interact with a so-called protospacer-adjacent motif (PAM) next to the target sequence. In case of Cas9 from Streptococcus pyogenes (SpCas9), the most commonly used Cas9, this sequence is NGG. Because the C. elegans genome sequence...

Novel deletion alleles of a C. elegans gene Y73E7A.1, named as tm6429 and tm6475

Yuji Suehiro, Sawako Yoshina, Sayaka Hori & Shohei Mitani
We report tm6429 and tm6475 as novel deletion alleles of the gene Y73E7A.1 that is a homologue of mammalian Coiled-coil domain containing 124 (Ccdc124)1. The Ccdc124 is a conserved gene from invertebrates to human. In human cell lines, Ccdc124 is a component of the centrosome during interphase and at the G2/M transition. During cell division, Ccdc124 relocates to the midbody at telophase and acts as an essential molecular component in cytokinesis2. The alleles were isolated...

Nuclear localization of a C. elegans CCCH-type zinc finger protein encoded by T26A8.4

Takashi Koyama & Chisato Ushida
T26A8.4 encodes a CCCH-type zinc finger protein. The amino acid sequence of this protein is partially similar to that of S. cerevisiae Caf120p, which has no zinc finger domain. T26A8.4-encoded RNA was detected more abundantly in germ cells than in other tissues of L4-adult worms according to the NEXTDB data. When expressed specifically in the germline under control of the mex-5 promoter, the T26A8.4-encoded protein localized to germ cell nuclei in the adult hermaphrodite and...

Precision deletion of the entire coding sequence of the mod-5 locus causes increase in pharyngeal pumping frequency

Trisha Brock, Stelian Pop, Chandler Bradford, Jenn Lawson, Lauren Resch & Christopher Hopkins
​The C. elegans mod-5 gene encodes a serotonin transporter. The putative null allele contains a 1,688bp deletion leading to a premature stop codon in the resulting sequence (Ranganathan, 2001); however, unspliced the gene is 12,557bp in length from the start to the stop codon. We used CRISPR/Cas9 technology to create a 12,775bp deletion that eliminates all of the mod-5 coding sequence from the genome (Figure 1A). The mod-5(knu383) animals were measured for pharynx pumping frequency...

A quantitative trait locus for nictation behavior on chromosome V

Jun Kim, Daehan Lee & Junho Lee
Lee et al., 2017 showed that CB4856 has a lower nictation ratio (the number of nictating dauers over the number of moving dauers on a micro-dirt chip) than N2, and that a genetic locus (nict-1) on chromosome IV mediates this phenotype difference. Although this paper had no evidence of any other genetic loci for nictation behavior, we have studied other quantitative trait loci (QTL) using different linkage-based mapping strategies. Here, we report another genetic locus...

Trehalose Extends Healthspan in C. elegans

Kristen J Robinson & Kathryn McCormick
Adult worms were reared and tested in M9 medium plus 5 mM trehalose, which has previously been shown to extend lifespan and increase pump frequency late in life in C. elegans (Honda et al., 2010). Using an alternate electrophysiological readout of pumping, electrophayrngeograms (EPGs), we examined pump frequency at earlier timepoints than previously reported. Pumps were stimulated with 10mM 5HT in M9, recorded as EPGs in a microfluidic device, and analyzed using NemAnalysis software (NemaMetrix)....

A novel null allele of C. elegans gene ceh-14

Emily A. Bayer & Oliver Hobert
We have generated a novel null allele, ot900, of the C. elegans LIM homeodomain gene ceh-14. All existing deletion alleles of ceh-14 were generated using a Tc1 transposon insertion, one of which, ch3, is a putative null based on the resulting frame-shift and a reduction in detectable mRNA and protein levels (Cassata et al. 2000). However, in our hands we have noticed a propensity of the ch3 allele to revert to wild-type after several generations,...

Loss of eif-2alpha phosphorylation on S49 (mammalian S51) associated with the integrated stress response hastens development in C. elegans​

Jarod Rollins, Noah Lind & Aric N. Rogers
The gene eif-2alpha (Y37E3.10) in C. elegans encodes for the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). The eIF2 trimer is required for delivering charged tRNAMet to the ribosome in a reaction requiring the hydrolysis of GTP to GDP (Sokabe et al., 2012). The phosphorylation of eif2-alpha on its 49th serine residue (Nukazuka et al., 2008) prevents replenishing GDP with GTP and thus reduces translation initiation by preventing delivery of tRNAMet. The phosphorylation...

Immunofluorescence reveals neuron-specific promoter activity in non-neuronal cells

Sierra K. Lear, Alakananda Das & Miriam B. Goodman
Four transgenes designed to drive expression of Cre recombinase and a nuclear-localized blue fluorescent protein (::Cre::F2A::BFP::NLS::tbb-2 3'UTR) were expressed under the control of neuron-specific promoters. All transgenes were inserted into the ttTi5605 transposon insertion site in chromosome II of wild-type C. elegans animals (N2) using CRISPR/Cas9-mediated genome editing, equalizing any potential positional effects. We observed very faint BFP signal in live worms from the signal copy transgene insertions. Additionally, the signal was further masked by...

Novel deletion alleles of a C. elegans gene Y48E1C.1, named as tm5468, tm5625 and tm5626

S; Suehiro Hori
We report tm5468, tm5625 and tm5626 as novel deletion alleles of the gene Y48E1C.1 that is the only ortholog of human calmodulin-lysine N-methyltransferase (CAMKMT)1. CAMKMT encodes an evolutionarily conserved enzyme class I protein methyltransferase that acts in the formation of trimethyllysine in calmodulin for calcium-dependent signaling2. CAMKMT mutation is associated with Hypotonia-cystinuria syndrome in human2,3. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV4. In the screening, all the alleles...

daf-2 modulates regeneration of mechanosensory neurons I

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
Caenorhabditis elegans (C. elegans) possess the ability to spontaneously regenerate injured axons via a highly efficient mechanism known as axonal fusion (Ghosh-Roy, et al., 2010; Neumann et al., 2011; Neumann et al., 2015; Abay et al., 2017). Following laser axotomy, regrowth from the proximal axon segment (still attached to the cell body) reconnects and fuses with its separated distal segment (Fig. 1A). We recently demonstrated that the level of axonal fusion increases with age (Abay...

Expression pattern of endogenous PAR-4A & C after CRISPR/Cas9-mediated genome editing

Vincent Roy, Olivier Gagné, Karim Hamiche, Jean-Claude Labbé & Patrick Narbonne
PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al....

Delta-9 Fatty Acid Desaturase Mutants Display Increased Body Size

James F.; Savage-Dunn Clark
C. elegans contain three Δ9 fatty acid desaturases (FADs), fat-5, fat-6, and fat-7, responsible for the initial desaturation step in the synthesis of complex polyunsaturated fatty acids (PUFAs). Production of PUFAs is vital for survival, as simultaneous loss-of-function in all three genes results in synthetic lethality (Brock et al. 2006). Animals containing mutations in any one of the three Δ9 FADs, fat-5, fat-6, and fat-7, display an increase in body length. fat-5 mutants display a...

Endocannabinoid 2-arachidonoyl glycerol increases the transcription of daf-7 in ASI neurons

Celina Galles, Gastón M Prez & Diego De Mendoza
daf-7 expression in ASI neurons is increased when nematodes are grown in the presence of endocannabinoid 2-arachidonoyl glycerol (2-AG). Left upper panel: representative image of reporter strain FK181 ksIs2 [Pdaf-7::GFP+rol-6(su1006)] captured under GFP epifluorescence microscopy. Right upper panel: same individual showing bright field microscopy together with GFP signal. Gray/black triangles mark ASI neurons expressing daf-7. Scale bars correspond to 0.03 mm. Lower panel: point plot of the adjusted GFP intensity/ASI neuron values gathered from worms...

New allele of C. elegans gene spch-3 (T27A3.4), called xc2

Nicholas R. Munoz, Dana T. Byrd & Diana Chu
We created a new mutant allele, named xc2, of the gene spch-3 (T27A3.4). spch-3 encodes one of three C. elegans small, highly basic proteins that resemble invertebrate protamines and associate with sperm meiotic chromosomes (Chu et al., 2006). The alleles were isolated from gene mutations generated by Non-Homologous End Joining (NHEJ) mediated repair of Cas9-generated breaks (Dickinson et al., 2013; Ran et al., 2013). The alleles were discovered using PCR with the following primers 5’-...

Efficient FLP-mediated germ-line recombination in C. elegans

Javier Macías-León & Peter Askjaer
​Recombinases are very useful enzymes for spatiotemporal control of gene expression (Hubbard 2014). Through binding to specific DNA sequences, they can be used to excise or exchange DNA fragments. We have previously generated a series of Caenorhabditis elegans strains that stably express an efficient FLP recombinase in specific tissues and reported how they enable precise regulation of gene expression, cell ablation or conditional gene knockout (Muñoz-Jiménez et al 2017). However, these strains are restricted to...

Localization of tbg-1 mRNAs and GFP::TBG-1 protein in Early C. elegans Embryos

Lauren DeMeyer & Mi Hye Song
tbg-1 encodes gamma-tubulin, a ubiquitous and highly conserved component of centrosomes in eukaryotic cells (Strome et al, 2001). Using smFISH we determined the localization of tbg-1 transcripts (red). tbg-1 transcripts are detected within distinct foci throughout the cytoplasm during both the first (A) and second (B) mitosis. tbg-1 transcripts are not enriched at centrosomes or either blastomere. In contrast, GFP tagged TBG-1 proteins (green signal; arrowheads) localize at centrosomes, as previously shown (Strome et al,...

Reduced pharyngeal pumping rates observed in tph-1 mutants using microfluidic electropharyngeogram (EPG) recordings

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In Caenorhabditis elegans, serotonin (5-HT) activates and controls pharyngeal pumping in response to food (Horvitz et al., 1982; Sze et al., 2000; Song and Avery 2012). Tryptophan hydroxylase, the enzyme required for serotonin biosynthesis, is encoded by the gene tph-1. Worms with a tph-1 deletion mutation exhibit phenotypes associated with a lack of serotonin-signaling, including reduced pharyngeal pumping (Sze et al., 2000; Avery and Horvitz 1990; Song and Avery 2012). We used a microfluidic electropharyngeogram...

The protein kinase G orthologs, EGL-4 and PKG-2, mediate serotonin-induced paralysis of C. elegans

Andrew C. Olson & Michael R. Koelle
The C. elegans protein kinase G ortholog, EGL-4, has been demonstrated to be involved in C. elegans egg laying and the initiation of dwelling, a behavioral state characterized in part by low rates of locomotion (Trent et al. 1983; Hao et al. 2011). Serotonin regulates C. elegans egg laying through all five identified serotonin receptors, and serotonin promotes the dwelling state through the mod-1 serotonin receptor (Trent et al. 1983; Hapiak et al. 2009; Flavell...

Basal pharyngeal pumping elevated in C. elegans mod-5 mutants

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In C. elegans, the reuptake of serotonin (5-HT) is facilitated by mod-5, which encodes a 5-HT transporter that is orthologous to a human 5-HT transporter (SLC6A4). mod-5 has been shown to effect both feeding and locomotion in C. elegans (Ranganathan et al., 2001; Jafarau et al. 2011). We obtained and analyzed EPG data using a microfluidic device (NemaMetrix) for mod-5 null mutant strains, mod-5(n3314) (A, Exp 1 n=15; Exp 2 n=32) and mod-5(n822), (B, Exp...

wrt-2 expression oscillates during larval development

Ana G. Vera-Cruz, Siavash Amon, Sabih Rashid & Lesley T. MacNeil
wrt-2 encodes a hedgehog-like protein that is weakly required for molting (Kouns et al., 2011). As monitored using a transcriptional GFP reporter, wrt-2 is expressed in seam and hypodermal cells throughout development (Apock et al., 1999). However, wrt-2 transcript levels oscillate with a period consistent with the timing of the molting cycle (Hendriks et al., 2014). Because standard GFP reporters do not capture rapid changes in gene expression, we generated a wrt-2 transcriptional reporter driving...

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