Meyer, Michael et al. (2009) International Worm Meeting "NUD-1 Functions in C. elegans Sperm Development."

Large, Michael, Meyer, Michael, Gratz, Scott, Miskowski, Jennifer Anne

[International Worm Meeting, 2009]

The C. elegans NUD-1 protein is the structural and functional ortholog of the fungal NudC protein. NUD-1/NudC proteins are evolutionarily conserved and have been implicated in diverse processes such as asymmetrical cell division in yeast, nuclear migration in fungi, and neuronal migration in humans; however, their exact function is unknown. When NUD-1 levels are depleted in N2 hermaphrodites by RNAi, a variety of phenotypes are found, including sterility, which supports a role for NUD-1 in C. elegans gonad development. Analysis of NUD-1(RNAi) animals revealed abnormalities in both the somatic gonad and the germ line. To ascertain whether the observed germ line defects were the result of a direct role for NUD-1 in the germ line, rather then an indirect consequence of somatic gonad defects, NUD-1 RNAi was performed in rrf-1 mutants. Treated hermaphrodites were rendered sterile, although they sometimes made sperm and/or oocytes. rrf-1 males with depleted NUD-1 levels also made sperm. To test whether these sperm were viable, the RNAi-treated males were mated to Fog females, and a significant percentage of these matings yielded no progeny compared to control matings. The expression pattern of NUD-1 further supports its function in sperm development. Observation of transgenic worms expressing a full-length NUD-1::GFP construct revealed faint staining in sperm, in addition to other tissues. Furthermore, a number of immunofluorescence experiments have been performed using two different antibodies that were generated against a phosphorylated version of mammalian NUDC and cross-react specifically in C. elegans. Consistently, NUD-1 is localized in perinuclear puncta in primary spermatocytes with more diffuse staining in secondary spermatocytes and spermatids. NUD-1 colocalizes with fibrous-body membrane organelles (FBMOs), which are sperm-specific organelles. Like FBMOs, NUD-1 is asymmetrically segregated to spermatids and away from residual bodies and this localization pattern requires functional SPE-15/Myosin VI protein. To further explore the relationship between NUD-1 and FBMOs, the localization of FBMOs in NUD-1(RNAi) mutants is being explored.

Scheel JK et al. (1995) International C. elegans Meeting "SYNAPSE SPECIFICATION IN C. ELEGANS"

Hedgecock EM, Scheel JK

[International C. elegans Meeting, 1995]

We are interested in the molecular basis of synaptic specificity. Neurexins, presynaptic membrane proteins with large extracellular domains containing regions homologous to laminin G domains, are believed to be involved in cell-cell interactions at mammalian synapses. In mammals, there is a large variety of neurexin proteins created by a combination of alternative splicing and multiple genes.

Michael Hoffmann et al. (2006) European Worm Meeting "The Planar Cell Polarity Protein VANG-1 interferes with Intercalation Events during Epithelial Morphogenesis In C. elegans"

Michael Hoffmann, Christoph Segbert, Olaf Bossinger, Gisela Helbig

[European Worm Meeting, 2006]

Michael Hoffmann, Christoph Segbert, Gisela Helbig and Olaf Bossinger. C. elegans VANG-1 is the only homolog of the Drosophila planar cell polarity (PCP) protein Strabismus/Van Gogh. We originally identified VANG-1 as a putative binding partner of DLG-1 (Discs large), a MAGUK protein that acts as a molecular scaffold for the establishment of the apical junction in all epithelia of the C. elegans embryo. The phenotype observed in vang-1 mutant embryos show intercalation defects during epithelial morphogenesis of the intestine and the hypodermis. Similar phenotypes arise in lin-17 (frizzled) mutants or after RNAi against dsh-2 (dishevelled), indicating core components of the PCP pathway in other systems to be involved in cell intercalation in the C. elegans embryo. In addition, VANG-1 and DSH-2 do colocalize and the asymmetric distribution of VANG-1 in epithelia depends on DSH-2. Taken together these results suggests that planar cell polarity might exist in C. elegans.

Michael Shapira et al. (2005) International Worm Meeting "Large scale transcriptional and functional analyses reveal central roles for a GATA transcription factor in Caenorhabditis elegans innate immune responses toward Pseudomonas aeruginosa infection."

Michal Ronen, Min Jiang, Stuart Kim, Jiming Rong, Brigham Hamlin, Michael Shapira, Man-Wah Tan, Marianne Campbell

[International Worm Meeting, 2005]

The opportunistic human pathogen Pseudomonas aeruginosa infects the worm Caenorhabditis elegans, when ingested, and causes its death within 3 days. We performed genome-wide gene expression analyses at early time points during P. aeruginosa infection, followed by a large scale RNAi-based functional analysis, to identify genes that respond to and protect from this infection. Transcriptional responses were distinct from general stress responses as previously described (e.g. Huffman et al., 2004), although some overlap exists, suggesting that the bulk of this response is a bona fide immune response and not a secondary effect of cellular damage or impaired metabolism. Induced genes included known as well as yet unknown, or putative, members of all levels of the innate immune system. Among the latter, genes encoding proteins with the yet uncharacterized DUF141 domain were noticeable in their importance for protecting worms from infection. We further unraveled the participation of the Sma/TGFbeta pathway and the intestinal GATA transcription factor, elt-2, in the regulation of the immune response. RT-PCR and functional analyses in RNAi treated worms showed that elt-2 is crucial for gene induction during infection, slowing pathogen accumulation in the intestine and improving survival. These results provide direct evidence for elt-2 being a pivotal nuclear regulator of immune responses in C. elegans. Additional evidence suggests that elt-2 may function downstream to the Sma/TGFbeta pathway.

Zhao-Wen Wang et al. (2003) International Worm Meeting "Presynaptic Ryanodine Receptors Mediate Spontaneous Neurotransmitter Release at the C. elegans Neuromuscular Junction"

Lawrence Salkoff, Ding-Xia Shen, Zhao-Wen Wang, Michael L Nonet

[International Worm Meeting, 2003]

Spontaneous miniature postsynaptic potentials or currents (minis) are generally considered fusion events of individual synaptic vesicles. Consistent with this notion, minis at vertebrate neuromuscular junctions (NMJs) often appear unitary, and show a normal distribution in amplitude histograms. However, at many other synapses, such as synapses in the vertebrate central nervous system, amplitude histograms of minis are positively skewed due to the presence of numerous large-amplitude events. Little is known about the origin of large-amplitude spontaneous events. At the C. elegans NMJ, amplitudes of minis vary greatly. Although their average amplitude is about 25 pA, many are over 50 pA. We investigated the mechanism of large-amplitude minis at the C. elegans NMJ. Minis were recorded from body-wall muscle cells voltage-clamped at -60 mV. Large-amplitude minis persisted in the absence of external Ca2+, suggesting that they are not endogenous evoked responses. Experiments with nicotinic and GABAergic receptor blockers, and with mutants of GABAergic receptors (unc-49) suggest that large-amplitude minis are of both cholinergic and GABAergic origins. Large-amplitude minis were absent in unc-68(r1162), a null mutant of the ryanodine receptor (RyR). Inclusion of a RyR blocker in the recording pipette to selectively block postsynaptic RyRs had no effect on minis, whereas application of a membrane permeant RyR blocker to the NMJ preparation, which blocked both presynaptic and postsynaptic RyRs, produced effects similar to that of the unc-68 mutant, suggesting that large-amplitude minis are mediated by presynaptic RyRs. The frequency of minis was also severely reduced in the RyR mutant and in wild-type animals treated with a RyR blocker. The effect of RyRs appeared specific because large-amplitude minis remained in other mutants affecting intracellular Ca2+ mobilization (itr-1 and crt-1), or release of large dense core vesicles (unc-31). In the presence of extracellular Ca2+ (1-5 mM), mini frequency was high (~40 Hz). Elimination of external Ca2+ reduced mini frequency by ~80%. Preliminary results suggest that the effect of external Ca2+ on minis is mediated at least in part by presynaptic RyRs, perhaps through a process known as Ca2+-induced Ca2+ release.

Vecseri G et al. (1991) International C. elegans Meeting "AN UNUSUAL MUTANT STRAIN OF CAENORAHBDITIS BRIGGSAE (G16)."

Buza J, Fodor A, Vecseri G

[International C. elegans Meeting, 1991]

The founder animal of strain vg-182 was isolated as a Large Roller progeny of an EMS treated C. briggsae (G16) hermaphrodite. It has segregated only a few offsprings including Lons and animals of variable phenotypes. Lon hermaphrodites were singled and self-fertilized through subsequent generations and then crossed out several times. During this process 7 of 8 fertile lines segregated Lon males, Large Dpy hermaphrodites, Small Dumpies and Large Rollers. At the end, loci represented by one X-linked dominant lon, one autosomal recessive dpy and one independent, autosomal squat allele, respectively, could be identified and named temporarily as lon-2, dpy- 2 and sqt-l. lon-2(d) proved a dominant epistatic gene over dpy-2 and a semidominant epistatic one over sqt-l. (Worms of sqt-l; +/+; lon-2 genotype exhibited Large Roller phenotype while those of sqt-l/+; lon- 2/0 genotype were large, Non-Roller males.) The male--segregating (Him) phenotype could not be separated from lon-2(d). The lon-2 hermaphrodites kept in liquid nitrogene still keep their 'him' phenotype, those which were kept in laboratory conditions lost their male-segregating ability. The possibilities of getting a mutator strain of C. briggsae are discussed.

Alok, G. et al. (2019) International Worm Meeting "Developing a large-scale method for bulk segregant analysis of genetic variation underlying differential requirements for SKN-1 in endoderm specification."

Torres Cleuren, Y., Alok, G.

[International Worm Meeting, 2019]

The maternally supplied SKN-1 transcription factor initiates specification of the C. elegans endoderm. We have found that the requirement for SKN-1 in endoderm specification varies widely between genetically distinct isolates of the species; while some wild isolates show a nearly absolute requirement for this factor, the majority of embryos in others generate endoderm in its absence. GWAS and analysis of RILs allowed us to identify several regions of the genome responsible for this large variation in the requirement for SKN-1. To narrow in on the specific causal sequence differences responsible for this variation, we are developing a method for large-scale bulk segregant analysis by evaluating large numbers of embryos derived from many-generation bulk intercrosses. This approach relies on our ability to sort large numbers of individual embryos with a fluorescent-activated cell sorter that effectively separates embryos that undergo endoderm differentiation from those that do not. With this approach we can analyze thousands of recombinant embryos and hope to achieve fine-scale resolution mapping of causal loci responsible for evolutionary variation in the endoderm gene regulatory network.

Spooner, Patrick et al. (2011) International Worm Meeting "Immunoglobulin domain containing isoforms of UNC-89 (obscurin) are required for myofilament organization and calcium signaling in Caenorhabditis elegans."

Duran, M. Berenice, Spooner, Patrick, Benian, Guy, Bonner, Jennifer, Norman, Kenneth

[International Worm Meeting, 2011]

The force generating machinery in muscle is regulated by the influx of calcium ions into the muscle cytoplasm. Initially, depolarization of the sarcolemma leads to calcium entry via voltage gated calcium channels, which rapidly triggers the release of calcium from the sarcoplasmic reticulum via ryanodine receptors by calcium induced calcium release. Thus, calcium needs to be rapidly, accurately and reliably regulated and the calcium channels and contractile machinery must be maintained in a highly ordered arrangement for efficient and effective muscle contraction to occur. The mechanism underlying this highly organized configuration is not fully understood. Using a combination of genetic and cell biological techniques, we have found that the C. elegans ortholog of the giant muscle protein obscurin, UNC-89, is required for muscle cell organization. The unc-89 locus encodes at least 6 isoforms as large as 900 kDa, 4 large Immunoglobin (Ig) domain rich isoforms and two smaller tandem kinase containing isoforms (Small et al. 2004; Ferrara et al. 2005). From our analyses, we have found that large Ig domain rich isoforms of UNC-89 are critical for sarcomere and SR organization. Furthermore, we have found evidence that unc-89 mutants lacking the large Ig rich isoforms have defects in excitation-contraction coupling in the pharyngeal and body wall muscle, through the coordination of calcium influx. Thus, our data implicates the large Ig domain rich isoforms of UNC-89 in maintaining muscle cell architecture that is critical for efficient and effective muscle contraction.

Miwa Furuya et al. (2001) International C. elegans Meeting "Characterization of a deficiency mutant that produces abnormally large cell corpses in late embryogenesis."

Joel H Rothman, Asako Sugimoto, Miwa Furuya, Masayuki Yamamoto

[International C. elegans Meeting, 2001]

Programmed cell death (PCD/ apoptosis) is a common cell fate in most if not all multicellular animals. To understand its steps genetically, we screened for regions of the genome required zygotically for normal PCD using C. elegans chromosomal deficiency mutants, which collectively delete ~80% of the genome. By this screen, we identified 27 individual strains that abnormally increase or decreased the number of PCD, or tha exhibited abnormal corpse morphology. Among these, we characterized deficiency mutants tDf6 and nDf41 , which generated abnormally large corpses. We observed developing embryos of the tDf6 mutant by 4D time-lapse recording. We found that the number of PCD corpses was significantly decreased in this mutant and that most corpses appeared at the late stage of embryogenesis and were larger than those seen in the wild type. Because the ced-3 ; tDf6 and the egl-1 ; tDf6 double mutants showed neither large nor normal corpses, the large corpses appeared to be produced by the known PCD genetic pathway. By deficiency mapping and series of RNAi of the candidate genes in the region, we found that some cyk-4 ( RNAi ) embryos phenocopied tDf6 . The cyk-4 gene encodes a GTPase-activator protein (GAP) for Rho-like GTPase, and CYK-4 is thought to have a role to complete cytokinesis, as reported by Jantsch-Plunger V et al.. By temperature-shift experiments using the cyk-4 ( t1689ts ) mutant, we confirmed that the large corpses in tDf6 embryos were produced by the deprivation of zygotic CYK-4 in late embryogenesis. We also performed membrane and nuclei staining and found that terminally arrested tDf6 embryos had many large cells with 2-4 nuclei. Taken together, we conclude that the large corpses in tDf6 embryos are produced by deaths of multinucleate large cells. This phenotype of the tDf6 mutant was similar to that of another deficiency mutant nDf41 . By deficiency mapping and complementation tests, we found that zen-4 is responsible for the large corpses observed in nDf41 embryos. The zen-4 gene is reported to encode the motor protein kinesine and the mutant fails to complete the late step of cytokinesis . In addition, according to the report of Jantsch-Plunger V et al., ZEN-4 and CYK-4 are thought to cooperate to complete the late step of cytokinesis, because their locations are similar and interdependent. Thus, through our deficiency screening, these two cytokinesis genes were identified as similar PCD-defective mutations. Our screening covered ~80% of the genome, and no other Deficiency embryos exhibited large corpses similar to those of tDf6 and nDf41 . Therefore, we speculate that at most one or two more genes are required zygotically for the late step of cytokinesis during embryogenesis.

Nakano Y et al. (2000) Japanese Worm Meeting "Analysis of large body size mutants."

Nakano Y, Ohshima YM

[Japanese Worm Meeting, 2000]

In order to analyze the mechanisms for body size determination, at least ten larger size mutants were isolated after EMS mutagenesis. Some of them had up to twice as much volume as that of the wild type in adults. Four larger size mutants, FK16(ks16;daf-6), FK204(ks60), FK205(ks61) and FK206(ks62) were back crossed two or three times with N2. The original and back crossed mutants were examined as to their body size and other phenotypes. There are at least two types of growth curve in these large size mutants. For g-6(ks61), the volume of the mutant is larger than that of N2 by 50~100% at all points, including larval developmental stages. In contrast, the difference in volume between the mutant and wild type begins to be apparent after becoming adults for g-3(ks60) and g-8(ks62). Major organs of the large mutants seem to be larger than those of the wild type roughly in proportion to the body size. The nuclei of the intestine, the largest organ, were counted as a measure of the cell number, and the results suggest that cell size but not cell number is increased in the intestine of the large mutants. Males of the four large size mutants occasionally have additional rays in the region anterior to ray1. All the four large size mutants seem to have elongated lifespan. Especially, g-3(ks60) had 92% longer mean life span than wildtype (23 vs 12 days) and 195 % longer maximum life span (56 vs 19 days). The back crossed mutants were also subjected to STS genetic mapping. The four mutations for enlarged body size are distributed on two linkage groups (g-3(ks60) and g-6(ks61) on LGIII, gig-1(ks16) and g-8(ks62) on LGIV). Further mapping will be done for cloning of the corresponding genes. We are also trying to isolate more large size mutants.