Suchold, Dana et al. (2015) International Worm Meeting "Genome engineering based protein tagging."

Sarov, Mihail, Ernst, Susanne, Suchold, Dana, Loester, Elisabeth

[International Worm Meeting, 2015]

Protein tagging is a generic and efficient way to explore the localization and physical interactions of any protein of interest in vivo. To facilitate the broad application of this approach we previously generated a genome wide resource of tagged fosmid transgenes (Sarov et al 2012), which covers about 70% of the genome. The advent of the of CRISPRs mediated genome engineering techniques has made it possible to tag the rest of the genome, which was previously not feasible due to the lack of suitable fosmid clones. The direct targeting of the endogenous copy has obvious advantages to the transgenic approach and the remarkable efficiency of homologous recombination in worms indicates that this approach should be scalable, but many questions still remain to be addressed. We are currently comparing the methods described in the literature and are working towards an efficient high-throughput pipeline, based on the experience from similar efforts in mice and other system where homologous recombination was routine even before the discovery of Cas9. We are specifically focusing on:1. Optimal Cas9/sgRNA delivery method (DNA vs. RNA vs. protein)2. The type of engineered allele (N vs. C terminal tagging, simple tagging vs. multipurpose alleles)3. Selected vs. non-selected insertions4. Ways to multiplex the engineering, isolation and validation of targeted strains.We propose the creation of an open genome engineering registry to aid a distributed, community-wide effort towards the ultimate tagging of the entire proteome.

Jane Shingles et al. (2006) European Worm Meeting "Progress of the Localization of Expression Mapping Project"

Marc Vidal, John Reece-Hoyes, Ian Hope, Jane Shingles, Denis Dupuy

[European Worm Meeting, 2006]

Jane Shingles1, John Reece-Hoyes1, Denis Dupuy2, Marc Vidal2 and Ian Hope1. The Promoterome library containing 6500 Caenorhabditis elegans promoter fragments has previously been generated and used to construct promoter ::GFP fusions to allow determination of gene expression patterns in C. elegans. Optimization of the C. elegans micro-particle bombardment transformation technique, to give a medium through-put system, allowed the generation of expression patterns for over 300 C. elegans promoter::GFP fusions. Promoter fusion constructs of transcription factors genes and genes with homology to human genes with no assigned function were selected for the initial analysis and the results are shown. Initial analysis of the results highlights several significant differences between the two sets of genes. Promoters from C. elegans homologues of human genes with no known function were far more likely to yield either no GFP expression (35% vs. 6%) or ubiquitous GFP expression (23% vs. 8%) under the standard laboratory conditions utilized. In contrast, promoters from C. elegans transcription factor genes were far more likely to drive neuronal expression (62% vs.16%).. Full results are presented on the Hope Laboratory Expression Pattern database URL:http://bgypc059.leeds.ac.uk /~web

Harterink, Martin et al. (2013) International Worm Meeting "Neuronal microtubule organization in C. elegans."

Hoogenraad, Casper, de Haan, Bart, Harterink, Martin, Van den Heuvel, Sander

[International Worm Meeting, 2013]

In polarized cells, such as neurons, the microtubule cytoskeleton offers tracks to transport various cargos along axonal and dendritic projections. Recent data indicate that differences in microtubule organization within axons and dendrites enable molecular motors to sort cargo to specific directions and establish and maintain neuronal polarity. However how these differences are established is hardly understood. C. elegans has relatively simple neurons which offer a great starting point to understand the basics of this microtubule organization. Using GFP tagged end-binding proteins to visualize microtubule growth we have compared several classes of neurons with different characteristics (such as ciliated vs unciliated and branched vs unbrached). Here we will present a preliminary analyses of the neuronal microtubule organization of wildtype as well as in several mutants.

Lehtela A et al. (2000) West Coast Worm Meeting "Characterization of a C. elegans Defecation Mutant"

Wong G, Lehtela A

[West Coast Worm Meeting, 2000]

Defecation in C. elegans is a tightly regulated and complex behavior. The defecation behavior is generated from neuronal signals and is transmitted into movement of multiple body muscles in a highly coordinated fashion. Defecation occurs in 3 steps: contraction of posterior body muscles (pBoc); contraction of anterior body muscles (aBoc); expulsion of intestinal contents (exp). The defecation cycle occurs at regular 50-60 second intervals when wildtype Bristol N2 worms are feeding. A C. elegans defecation mutant was identified in a mutagenesis screen. Six-thousand genomes were mutagenized with 50 mM ethyl methanesulfonate for 4 h. F2's were selected for resistance to 0.7 mM aldicarb, an acetylcholine esterase inhibitor. A single mutant, which appeared to be constipated, was cloned and then outcrossed to N2. The defecation mutant, in comparison to N2 per 30 min, had fewer pBOC (15.3 2 vs. 32.8 1), fewer exp (6.5 1 vs. 31.8 0), and fewer successful complete defecation motor program events (32 9% vs. 97 2%), respectively. When pBOC was followed by an exp event, the latency appeared longer in the mutant (8-10 sec) compared to N2 (2-3 sec). On the few occasions when the complete defecation motor program was intact in mutants, the interval was longer (80-90 sec) than N2 (55-60 sec). Genetic analysis has tentatively identified the location of the mutation on chromosome III. These results, describing a derangement of the defecation motor program, combined with identification of the gene mutation responsible for this phenotype, should provide insights into the control of complex neuronal and neuromuscular controlled behaviors.

Mosley, Matthew et al. (2021) International Worm Meeting "The transcriptional signature of long vs. short life is distinct from that of chronological age"

Stroustrup, Nicholas, Martin, Olivier, Pincus, Zachary, Kornfeld, Kerry, Schedl, Tim, Mosley, Matthew, Kinser, Holly

[International Worm Meeting, 2021]

What determines an individual's lifespan? The intuitive answers of "genetics" or "environment" do not tell the full story-even isogenic populations of C. elegans reared in a controlled environment show the same degree of inter-individual variation in lifespan as that seen in outbred human populations. One hypothesis is that individuals are driven toward different fates by stochastic differences in the expression of key regulatory genes. We recently showed that the expression levels of 10 out of the 22 microRNA promoter::GFP constructs we examined were sufficient to predict an individual's future lifespan. At least two of these GFPs predict lifespan independent of the activity of daf-16, and at least three report on lifespan redundantly despite being expressed in distinct tissues. We wondered whether these results might be evidence of a global gene expression state that determines (or is determined by) an individual's remaining lifespan. To address this, we set out to understand the global transcriptional states underlying high vs. low expression of these lifespan-predictive GFPs. We performed RNA-seq on populations sorted by the GFP intensity of several of these predictive markers, as well as intestinal autofluorescence, a phenomenological biomarker of aging. We found that, regardless of biomarker, the differentially expressed genes between prospectively long- vs. short-lived populations were extremely similar. However, we also noted that these differences overlapped significantly with genes that change over time during chronological aging. We reasoned that this was due to the "apparent age" of the worms; that is, worms predicted to be long-lived had gene expression reflective of chronologically younger animals, while worms predicted to be short-lived showed premature genetic hallmarks of aging. We then asked whether this accelerated or retarded "apparent age" was the only difference between prospectively long- vs. short-lived populations. By first aligning each population to a high-resolution transcriptional time course, we account for and remove the effects of "apparent age," revealing a signature that specifically reflects long vs. short future lifespan. Surprisingly, we found that, for all the predictive markers we tested, the expression of germline-related genes is highly related to future lifespan, even after controlling for the "apparent age" of the transcriptome.

Newman AP et al. (1995) International C. elegans Meeting "EGL-29 FUNCTIONS UPSTREAM OF LIN-12 IN THE #185# CELL INDUCTION PATHWAY"

Newman AP, Sternberg PW

[International C. elegans Meeting, 1995]

During ventral uterine (VU) pattern formation, the anchor cell (AC) induces adjacent VU intermediate precursor cells to adopt the pi cell fate. This induction is mediated by lin-12, which two generations earlier mediates lateral inhibitory signaling between the AC and the grandmother of the pi cells during the AC vs. VU decision. We assay pi cell fate by cell lineage and lin-11-lacZ expression. egl-29 is required for induction of the pi cell fate, but not for other lin-12-mediated cell fate decisions. Genetic epistasis experiments indicate that egl-29 functions upstream of lin-12 in the pi cell induction pathway. We have preliminary evidence that egl-29 is required for maintenance of lag-2 expression in the AC. lag-2, the apparent ligand in the AC vs. VU decision, may be re-utilized during pi cell fate induction. egl-29 may function in regulation of the ligand during the switch from lateral inhibitory signaling to induction. Toward the molecular cloning of egl-29, we have narrowed it down to the 100kb overlap between two rescuing YAC clones.

Hall DH et al. (1997) International C. elegans Meeting "SYNAPSES IN SUBLATERAL NERVE CORDS"

Hall DH, Duerr JS, Johnson CD, Rand JB

[International C. elegans Meeting, 1997]

Previous studies in Ascaris showed that sublateral nerve cords contain many synapses to bodywall muscle, esp. in the head, at least some of which are excitatory, as well as nerve-nerve synapses between sublateral neurons and passing motorneuron commissures (1,2,3). New immuno-fluorescence studies in C. elegans and Ascaris using several antibodies against synaptic proteins (CHA-1, SNT-1, UNC-17) suggest that the sublateral cords in C. elegans might be involved in similar contacts not described in "The Mind of the Worm" (4). Serial section EM of C. elegans shows periodic swellings of four classes of sublateral neurites. Most swellings form putative synapses: a large cluster of synaptic vesicles and one presynaptic density. These synapses usually lie near the border of two bodywall muscles in the same quadrant, and we presume that they are motor synapses. SIA contacts dorsal muscles; SIB contacts ventral muscles. SMB contacts dorsal muscles more often than ventral; SMD has the opposite preference. PLN and SDQL are involved in few synapses along the sublateral cords. If mechanisms for transmitter degradation are low in this vicinity, even synapses "pointing" towards hypodermis or other neurites may really target muscle. Sublateral nerves could globally modulate muscle activity or muscle tone, perhaps in reciprocal fashion via opposing classes of neurites (dorsal vs. ventral; e.g. SIA vs. SIB, and SMB vs. SMD). Synapses also occur in C. elegans where a circumferential motor commissure crosses a sublateral nerve. Some involve sublateral neurites synapsing onto commissures; others involve commissures synapsing onto sublateral neurites. 1. A.O.W.Stretton (1976) J. exp. Biol. 64:773-88. 2. C.D. Johnson & A.O.W. Stretton (1987) J Neurosci 7:223. 3. Manney, Donmoyer, Angstadt and Stretton, , pers. comm. 4. J.G. White et al. (1986) Phil. Trans. R.Soc.Lond. 314:1-340.

Ilbay, O. et al. (2017) International Worm Meeting "Integration of environmental stress signals into the heterochronic developmental timing pathway via DA-independent regulation of daf-12."

Ambros, V., Ilbay, O.

[International Worm Meeting, 2017]

C. elegans development is remarkably robust against environmental stresses. Two major environmental sources of stress, food (type and quantity), and the population density pheromones, alter the developmental trajectory by affecting the overall rate of larval development and/or cell fate choices at the end of the L2 stage (L3 fates vs quiescence). However, despite the striking changes in the developmental trajectory, the temporal patterning of developmental cell divisions and cell fates is robustly maintained by the developmental timing pathway. A key component of the developmental timing pathway that coordinates temporal cell fates with the developmental trajectory is the nuclear hormone receptor DAF-12. In response to an endocrine signal, dafachronic acid (DA), DAF-12 controls both the developmental trajectory (reproductive vs dauer-interrupted) and the expression level of certain let-7 family microRNAs (high vs low). Our recent data suggest that DAF-12 integrates environmental signals into the developmental timing pathway also in a DA and let-7 independent manner. First, the developmental timing defects of the daf-12(rh61) mutant, which has low let-7 levels due to its inability to bind the DA ligand, is suppressed by growth in the presence of dauer-promoting ascarosides, or by the presence of hypomorphic alleles of daf-7 or daf-2), demonstrating a DA-independent interaction between environmental signals and the developmental timing pathway. Second, this ascaroside-mediated suppression of heterochronic phenotypes does not affect the levels of let-7 family microRNAs and does not require any particular let-7 family microRNA, suggesting a let-7-independent change in the developmental timing control in response to ascarosides. Moreover, we observe that a half-dose of the lin-28 gene [lin-28(lf)/+] suppresses the developmental timing defects of the daf-12(rh61) mutant. This suppression requires lin-46, which was shown to perform developmental timing functions specific to the dauer-interrupted developmental trajectory. We propose that environmental stress signals are integrated into the developmental timing pathway via down-regulation of lin-28 in a DA and let-7 independent manner.

Aimee Jaramillo-Lambert et al. (2008) Development & Evolution Meeting "Analysis of sex-specific differences in C. elegans meiosis"

Aimee Jaramillo-Lambert, JoAnne Engebrecht

[Development & Evolution Meeting, 2008]

Meiosis is essential for all sexually reproducing organisms and consists of a single round of DNA replication followed by two rounds of nuclear division and specialized cell divisions that result in the production of two very distinct haploid gamete types, oocytes and sperm. In C. elegans, meiosis is restricted to occur in germ cells, which are set apart from somatic cells early in development. Germ cell nuclei are contained within a syncytial gonad, which allows for the introduction of small molecules by microinjection, and nuclei representing all stages of meiotic prophase are arranged in a temporal/spatial gradient within the gonad. We have developed an S phase labeling assay to investigate germ-line DNA replication, chromosome behavior, and meiotic prophase progression. By monitoring S phase labeled nuclei through the germ line we discovered that hermaphrodite meiotic prophase for oogenesis takes 54-60 hours whereas prophase for spermatogenesis in males is completed by 20-24 hours. To determine if the sex-specific difference in meiotic prophase timing is due to chromosome sex (XX vs. X0), somatic sex (female vs. male body) or germ-line sex (production of oocytes vs. sperm), we analyzed meiotic progression in a number of sex determination mutants. We found that meiotic prophase progression timing is dictated by the production of oocytes or sperm. Another sex-specific difference is the extent to which checkpoints monitor and safeguard the genome. The hermaphrodite germ line undergoes physiological apoptosis, but perturbation in chromosome synapsis or DNA damage causes a checkpoint activated increase in apoptosis. On the other hand, the male germ line has neither physiological nor checkpoint activated apoptosis. Analysis of the same sex determination mutants revealed that both physiological and checkpoint activated apoptosis are also dictated by germ-line sex. Surprisingly, we found that a single X chromosome does not activate the synapsis checkpoint in a female germ line competent for apoptosis. We are currently investigating the mechanisms underlying why a single unsynapsed chromosome does not activate the synapsis checkpoint.

Yihong Zhu et al. (2002) Mid-west Worm Meeting "Putting exc-5 into Those Blasted Worms"

Yihong Zhu, Margaret Snow, Matthew Buechner, Brendan Mattingly

[Mid-west Worm Meeting, 2002]

The EXC-5 guanine exchange factor regulates the amount of cytoskeleton devoted to the apical vs. basolateral surface in the excretory canal. In null mutants, the apical cytoskeleton is disrupted and the apical surface swells into large fluid-filled cysts. When exc-5 is overexpressed, however, the lumen retains its normal diameter, while canal extension fails. We are currently performing a noncomplementation screen to isolate other point mutations in the exc-5 gene. In addition, we would like to perform enhancer-suppressor screens to isolate other members in this signal transduction cascade. In order to perform this screen, we are trying to integrate a rescuing exc-5::gfp construct into the chromosome in a single copy.