Andrew Singson et al. (2002) West Coast Worm Meeting "Mutants that affect gamete function at fertilization"

Emily Putiri, Indrani Chatterjee, Pavan Kadandale, Brian Geldziler, Marty Nemeroff, Andrew Singson

[West Coast Worm Meeting, 2002]

The main goal of our lab is to understand the molecular mechanisms of fertilization. Initially, we have focused on the characterization of a group spe and fer genes required by sperm for fertilization. Although sterile, worms with these mutations produce morphologically wild-type sperm with normal motility that can come in contact with oocytes. Therefore mutations in these genes disrupt gamete recognition, adhesion, signaling and/or fusion. We have expanded our studies to include gene products required by the oocyte for fertilization. We are using RNAi to test candidate egg cell surface components for possible functions in fertilization. Additionally, we are conducting a genetic screen to identify conditional "egg sterile" mutants. We will report our progress on the genetic, phenotypic and molecular analysis of these genes. This work will lead to a better understanding of the specialized cell-cell interactions that occur at fertilization.

Mendel, Jane et al. (2015) International Worm Meeting "WormBook News."

Harris, Todd, Mendel, Jane, Chalfie, Martin, Sternberg, Paul, Wang, Qinghua, Hobert, Oliver

[International Worm Meeting, 2015]

WormBook (http://www.wormbook.org/) is a comprehensive, open-access collection of original peer-reviewed chapters covering the biology of C. elegans and other nematodes. WormBook also includes WormMethods, which contains protocols ranging from the basics of C. elegans culture to advanced imaging techniques, WormHistory, which contains personal recollections of the early days of nematode research, and the Worm Breeder's Gazette, an informal newsletter. We are continually updating the older chapters in WormBook and WormMethods, as well as adding chapters on new topics. For example, we will shortly add several WormMethods chapters describing aspects and functions of WormBase.We are very happy to announce the addition of an Introduction to WormBook: "A Transparent Window into Biology: A Primer on Caenorhabditis elegans", by Ann Corsi, Bruce Wightman, and WormBook editor-in-chief, Marty Chalfie, is scheduled to be copublished by Genetics and WormBook in June.Additional developments include listing WormBook chapters in PubMed Central and upgrading the search feature of WormBook, WormMethods, and The Worm Breeder's Gazette.We welcome your feedback and comments on how we can improve WormBook, WormMethods, and The Worm Breeder's Gazette to make them more useful to you, the worm community.

Avery L et al. (1985) Worm Breeder's Gazette "mutants that pump when wild-type worms don't."

Avery L, Horvitz HR

[Worm Breeder's Gazette, 1985]

We are studying pharyngeal pumping in order to understand the development and function of the nervous system that controls pumping. At the last worm meeting, we described a method for isolating mutants that pump under conditions in which wild-type worms don't. Worms are suspended in liquid with microscopic iron particles; then the worms that have pumped iron are pulled out with a small magnet. Using this selection we have isolated two different sorts of pumping mutants and are now characterizing them. Well-fed wild-type worms pump very little in the absence of bacteria. Three of our mutants do pump under these conditions, so we call them pumping constitutive. One mutation, n1304, is a new allele of unc-31 IV. n1304 has a recessive Unc phenotype similar to that of unc-31( e169), and e169 pumps constitutively. The two alleles fail to complement for both phenotypes. The remaining two mutants look normal in the dissecting microscope. We also have one mutant that pumps in the presence of ivermectin, a drug that normally inhibits pumping and also prevents worms from growing. (We thank Marty Chalfie for telling us about ivermectin.) Although the mutant was selected only for ability to pump in the presence of ivermectin, it also grows on plates containing ivermectin: perhaps the lethal effect of the drug is due entirely to its inhibition of pumping. None of our pumping-constitutive mutants grows on ivermectin plates.

Gu GQ et al. (1994) Worm Breeder's Gazette "mec-5 Encodes a Collagen Needed For Touch Sensitivity."

William CM, Chalfie M, Gu GQ

[Worm Breeder's Gazette, 1994]

mec-5 Encodes a Collagen Needed for Touch Sensitivity Guoqiang Gu, Chris William, and Marty Chalfie, Department of Biological Sciences, Columbia University in the City of New York, NY 10027 mec-5 is one of the genes required for mechanosensitivity in C. elegans. Although the touch receptor neurons do not function, they appear morphologically normal in mec-5 mutants. Peanut-lectin binding to the touch cell mantle, however, is absent (M. Chalfie and E. Hedgecock, unpublished data). By transformation rescue, we identified a 6.5 kb BamHI-Kpnl fragment in cosmid E03G2 which completely rescues the mec-5 phenotype. We isolated a 1.2 kb full length cDNA (Northern blots show a 1.2 kb mRNA) from the Chris Martin cDNA library. The cDNA sequence encodes a collagen. The protein (329 aa) has a 20 amino acid putative signal sequence at the N- terminus and 69 Gly-X-Y repeats starting at aa 63. These collagen repeats are followed by 60 additional amino acids. Because collagens are often glycosylated on hydroxyproline residues, the loss of MEC-5 may explain the lack of peanut- lectin binding. To verify that this collagen gene is mec-S, we probed genomic Southern blots with the 6.5 kb fragment and found that the 6.5 kb fragment was deleted in two mec-5 strains, the mut-2 derived strain TU828 and the Y-ray strain TU1031. We are now sequencing other mec-5 mutations and characterizing the expression pattern.

Ando H et al. (1989) Worm Breeder's Gazette "pharmacological characterization and cloning of kra-1 mutant."

Ando H, Kagawa H

[Worm Breeder's Gazette, 1989]

We have previously reported on isolation of ketamine response abnormal (KRA) mutants by temporary convulsive phenotypes (WBG vol10, No.3, 1988 and CSH 1989). Ketamine, a general anesthetic, is one of the non-competitive antagonist of N-methyl-D-aspartate (NMDA). Genetic and molecular study on genes that have influence on normal pharmacological response to such drugs must be an adequate approach to understanding the NMDA class of glutamate receptor. As reported previously, we identified a gene, kra-1 on LGV by genetic study on a strain kh-30. Genetic locus of kh-30 mutation site was determined between right side breakpoints of nDf32 and sDf20 (0.08mu in span). This mutation expresses a semidominant convulsive phenotype in 30mM ketamine solution or other NMDA antagonists, a strong inhibition of postembryonic development of 10mM ketamine containing NGA, a recessive cold sensitive Unc phenotype, and variable motility in usual condition. As suggested by John White, we also tested previously isolated strains including 26 unc loci derived from over 30 genes and some levamisole resistant strains appeared to be blocked their postembryonic development by ketamine. In addition, some mutant that have abnormal neuron networks showed ketamine resistance in postembryonic development. On the other hand, in immediate response to ketamine, hypersensitive paralyzing strains (whole body and head region restricted) could be found. We are observing pharmacological responses of KRA and other strains to acetylcholine antagonists or other reagents. Pharmacological and anatomical data will give us any suggestions about the ketamine functional site on neuron networks in the worm. Tc1 tagging of mec-1 gene is in progress with generous supply of Tc1 clones by Marty Chalfie. We do hope mec-1 linked fragment will be cloned for matching physical map and genetic map very soon. [See Figure 1]

Ma C et al. (1994) Worm Breeder's Gazette "mec-6 May Encode a Degenerin."

Chalfie M, Ma C, Gu GQ

[Worm Breeder's Gazette, 1994]

Mec-6 may encode a degenerin Charles Ma, Guoquiang Gu and Marty Chalfie. Dept. of Biological Sciences, Columbia University, NY, NY 10027 The gene mec-6 is needed for the function of a set of touch receptor neurons (ALMs, PLMs, AVM and PVM). In addition, its was found that recessive mutations in mec-6 are able to suppress neuronal degenerations caused by dominant mutations in the genes deg-1, mec-4, and mec-10. These three genes all belong to a family of genes encoding degenerin protein. Based on the phenotype of the dominant mutations and the similarities of the degenerins to the components of the vertebrate amiloride-sensitive sodium channel, it is likely that the degenerins form membrane channels that can mutate to cause neuronal degenerations. One attractive model is that MEC-4 and MEC-10 help to form the mechanosensory channel in the touch receptor cells. In rat, the amiloride-sensitive sodium channel is made of three different degenerin-like subunits. We have investigated whether a third degenerin subunit is found in the touch cells. The best candidate for such a component is the mec-6 protein product. We have found an apparent degenerin homologue that maps to the mec-6 region on chromosome 1. Using degenerative oligos that encode highly conserved regions of the degenerin gene family, we were able to PCR amplify a small fragment from the cosmid W01 A8 that shares sequence homology with the degenerins. In addition, we cloned a 1 .3kb genomic fragment that includes this region and found that this fragment detects a deletion in animals with the mec-6(u450) gamma-ray mutation. Several transcripts were detected on a northern blot using this genomic fragment as probe. We are testing whether these transcripts are absent in the mec-6(u450) animals. It seems likely that this degenerin is the mec-6 gene.

Freyd G et al. (1988) Worm Breeder's Gazette "identification of a transposon insertion in lon-2."

Freyd G, Horvitz HR

[Worm Breeder's Gazette, 1988]

Mutants of the gene lon-2 cause worms to grow to one-and-a-half times their normal length. Since cell lineages appear normal, we hypothesize that the gene either could be directly lengthening all cells in the animal, or that it could be affecting the structure of the cuticle, which is known to be important in maintaining the worm's body shape. A powerful approach to characterizing this gene would be its molecular cloning. We isolated a lon-2 allele (n1630) in TR679, a mut- 2 strain with a high frequency of transposon transposition. The unbackcrossed strain reverts at a high frequency, whereas the 10X backcrossed strain does not, leading us to believe a transposon has inserted into lon-2. We have subsequently identified an approximately 20kb HindIII restriction fragment that is likely to include at least part of the lon-2 gene. We used three-factor crosses with nearby markers, unc-78 and dpy-8, and subsequent Southern blot analysis with Tc1 probes to identify the band. We have analyzed six recombinants in the interval between unc- 78 and lon-2 (approximately 4.5 map units) and six recombinants between dpy-8 and lon-2 (approximately 0.5 map units). A band of about 20 kb is present in 6/6 recombinants that show the lon-2 phenotype and in 0/6 recombinants that are wild-type, mapping the band to within 0.2 map units on one side and 1.8 map units on the other side of lon-2.In addition, we have analyzed a spontaneous dominant revertant of this lon-2 allele. The mutation causing the reversion is tightly linked to lon-2 and is lacking the 20kb Tc1-hybridizing band present in the lon-2(n1630) mutant strains. These results suggest that a transposon excision event caused the reversion. Two other lon- 2 alleles (cgl1 and u383) have been isolated in mutator backgrounds in the laboratories of Jim Kramer and Marty Chalfie that we have not analyzed.

Jean-Claude Labbe et al. (2006) European Worm Meeting "A Novel Role for Nanos in PAR Protein-Dependent Cell Polarity"

Monica Gotta, Thomas Marty, Jean-Claude Labbe, Anne Pacquelet, Esther Zanin

[European Worm Meeting, 2006]

Jean-Claude Labb1, Anne Pacquelet, Esther Zanin, Thomas Marty and Monica Gotta. Asymmetric cell division is a fundamental process for the generation of cell diversity during the development of metazoans. We are interested in understanding how cells become polarized and divide asymmetrically. In the early C. elegans embryo PAR proteins are responsible for the establishment and maintenance of cell polarity. PAR-3, PAR-6 and PKC-3 are members of a protein complex localized at the anterior cortex of the embryo (referred to as the PAR-3/6/3 complex); the PAR-2 and PAR-1 proteins are present at the posterior cortex of the embryo. Mutations that disrupt PAR-2 function result in embryos in which the PAR-3/6/3 complex is mislocalized, leading to polarity defects and embryonic lethality. It has been previously shown that the lethality of par-2 mutants can be suppressed by reducing PAR-6 levels or by depleting CDC-42. This indicates that the lethality due to the absence of PAR-2 can be suppressed by conditions that affect the levels, the localization and/or the activity of the PAR-3/6/3 complex. . To identify new genes that either regulate or are regulated by the PAR-3/6/3 complex, we have performed a genome-wide RNAi screen looking for suppressors of par-2 lethality. One of the identified suppressors is NOS-3, the homologue of Drosophila Nanos. We find that nos-3 suppresses most of the phenotypes associated with loss of par-2 function, including early cell division defects, maternal effect sterility and mislocalization of the anterior PAR-3/6/3 complex. Strikingly, we find that asymmetric localization of PAR-1 at the posterior cortex is not restored. However, PAR-1 activity is essential in nos-3; par-2 double mutants, suggesting that the function of PAR-1 is independent of its cortical localization. Interestingly, nos-3 can also suppress the phenotypes associated with a null allele of par-2, indicating that NOS-3 impinges on the PAR pathway independently of the PAR-2 protein. Consistent with this, we find that NOS-3 regulates PAR-6 levels in the embryo. We are currently investigating the molecular mechanisms by which NOS-3 controls PAR-6 levels.

Marisa L Foehr et al. (2005) International Worm Meeting "SMA-9 antagonizes the TGF-beta signaling pathway in patterning the C. elegans postembryonic mesoderm"

Rachel Fairbank, Jun Kelly Liu, Amanda S Lindy, Nirav M Amin, Marisa L Foehr

[International Worm Meeting, 2005]

Our lab is studying mesodermal development using the C. elegans postembryonic lineage, the M lineage, as a model system. The M lineage gives rise to a variety of mesodermal cell types. In particular, the dorsal daughter of M (Md) produces two coelomocytes, while the ventral daughter Mv produces two sex myoblasts. The mechanisms regulating this dorsal-ventral asymmetry are not well understood. We have identified SMA-9 as one of the factors regulating this asymmetry. Mutations in sma-9 resulted in the transformation of the dorsal fates to the ventral fates in the M lineage. sma-9 encodes a nuclear localized C2H2 zinc finger protein that is homologous to the Drosophila protein Schnurri. Mutations in sma-9 also lead to small body sizes and male tail patterning defects (Liang et al., 2003). In order to understand how SMA-9 functions in the M lineage, we carried out a mutagenesis screen and isolated 7 suppressor mutations that specifically suppressed the M lineage defects of sma-9(cc604). One of our suppressor mutations jj3 is an allele of sma-3, which encodes a regulatory Smad of the DBL-1/TGF-beta signaling pathway. sma-3 mutations caused no M lineage defects on their own, yet they completely suppressed the M lineage phenotypes, but not the small body size, of sma-9 mutants. We further showed that mutations in genes encoding the DBL-1 ligand, the DAF-4 and SMA-6 receptors and the SMA-2 and SMA-4 Smads proteins all suppressed the M lineage defects of sma-9 mutants without suppressing their small body size phenotype. Since mutations in all the above TGF-beta pathway components caused no M lineage defects, their interactions with sma-9 have allowed us to uncover a novel role of the TGF-beta signaling pathway in patterning the postembryonic mesoderm. We showed that this genetic interaction between sma-9 and the TGF-beta pathway is specific, since mutations in the Wnt signaling pathway did not suppress the M lineage phenotypes of sma-9 mutants. The Drosophila SMA-9 homolog Schnurri has been shown to act as a transcriptional repressor (Marty et al., 2000). We therefore propose that in the C. elegans postembryonic mesoderm SMA-9 functions in parallel to the TGF-beta signaling pathway to antagonize its function. We are currently further testing this hypothesis and addressing possible targets of sma-9 in the M lineage.

Treinin M et al. (1994) Worm Breeder's Gazette "Neuronal Degeneration Caused by Mutations of an Acetylcholine Receptor in C. elegans."

Chalfie M, Treinin M

[Worm Breeder's Gazette, 1994]

NEURONAL DEGENERATION CAUSED BY MUTATION OF AN ACETYLCHOLINE RECEPTOR IN C. elegans Millet Treinin and Marty Chalfie Department of Biological Sciences Columbia University Dominant mutation in the genes deg-1. mec-4, and mec-10 result in the degeneration of specific sets of neurons. These three genes encode for membrane proteins which belong to the degenerin gene family. Degenerin homologues in rat (a,,B,yrENaC) are components of an epithelial, amiloride- sensitive, sodium channel. mec-4 and mec-10 probably form a similar channel which is needed for the function of the touch receptor neurons. Cell degeneration, which is caused by these mutations, is probably a result of changes in the ion permeability of these channels, leading to osmotic imbalance and cell death. Thus, we believe that mutations that cause neuronal degeneration can lead to the identification of channel genes. We have been analyzing a number of degeneration-causing mutation. One of them deg-3(u662J causes an uncoordinated phenotype and the degeneration of a small set of neurons. Because loss-of-function mutations in deg-3 produce no mutant phenotype, the gene may be non-essential. deg-3 encodes a homolgue of the a-7 nicotinic receptor, a vertebrate neuronal acetylcholine receptor which is highly permeable to Ca++. The degeneration-causing mutation in deg-3 affects the equivalent amino acid (in the second transmembrane domain) as is altered in the chick a-7 mutant (Galzi et al., Nature 359: 500-505 (1992)). Interestingly, the chick mutant receptor desensitizes slowly, a result consistent with the deg-3(u662) phenotype. Thus, we believe that the deg-3(u662)-caused cell death is a result of increased activity of the mutant channel. Support for this suggestion is provided by the partial suppression of the deg-3(u662) phenotype which is seen in the presence of d-tuborcurarine. Although the vertebrate a-7 proteins can form homooligomer receptors in Xenopus oocytes, the nature of the receptor in vivo is unknown. We have identified two extragenic suppressor genes (des-2(11) and des-3(1MJ) that appear to be important for deg-3 function. Analysis of these suppressors may shed some light on the regulation of deg-3 function. The deg-3(u662) mutation affects sensory neurons (touch receptor neurons and IL1 neurons) and interneurons (PVC, AVG), no motor neuron degeneration is seen. Expression of GFP, fused to the deg-3 upstream regions, is seen in the same postion as the degenerating cells. Although we do not know the function of deg-3, the extensive overlap between cells that express deg-3 and cells that express the degenerins mec- 4, mec-10, and deg-1 is intriguing, and suggest an interaction between these two types of channels.