Musset-Bilal F et al. (1994) Worm Breeder's Gazette "Characterization of the C. elegans Basement Membrane-Associated Protein SPARC"

Musset-Bilal F, Schwarzbauer JE, Ryan CS

[Worm Breeder's Gazette, 1994]

Characterization of the C. elegans Basement Membrane- Associated Frederique Musset-Bilal, Carol S. Ryan, and Jean E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544

Blumenthal TE et al. (1993) Worm Breeder's Gazette "The C. elegans Cleavage and Polyadenylation Signal"

Fields CA, White O, Blumenthal TE

[Worm Breeder's Gazette, 1993]

THE C. ELEGANS CLEAVAGE AND POLYADENYLATION SlGNAL Tom Blumenthal, Department of Biology, Indiana University. Bloomington, IN 47405; Owen White and Chris Fields, Institute For Genomic Research, Gaithersburg, MD, 20878

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

David Michaelson et al. (2006) Development & Evolution Meeting "Soma-germline Interactions Required for Robust Larval Germline Proliferation"

Rachael Lader, E. Jane Hubbard, David Michaelson, Dina Esposito, Chris Ryan, John Maciejowski, Jessica Dy-Johnson

[Development & Evolution Meeting, 2006]

Proper control of cell proliferation is critical for normal development. While the molecular basis for the proliferation/differentiation decision is relatively well-characterized in certain developmental contexts, less is known about cell-cell interactions that control of the extent of proliferation within a developing organ. The C. elegans germ line offers a tractable model system for exploring this problem. Previous work (McCarter et al., 1997; Killian and Hubbard, 2005), indicates that the distal pair of somatic gonadal sheath cells (Sh1) promote robust larval germline proliferation. We are taking several approaches to identify the molecular basis for this interaction. Forward genetic screens, molecular analysis and directed RNAi studies identified a critical role for optimal ribosome biogenesis in the sheath (Killian and Hubbard, 2004; Voutev et al., submitted) for its germline proliferation-promoting activity. To identify additional players, a genome-wide RNAi screen and candidate screens are underway. One screening strategy is based on the observation that reduced larval germline proliferation inhibits gonad arm extension, delays meiotic entry, and thereby uncouples the coordination between somatic gonad and germline development. As a result, under certain conditions, reducing larval germline proliferation can enhance proximal germline tumor formation (Pro phenotype; Killian and Hubbard, 2005; see also abstract by Maciejowski and Hubbard). Thus we are screening for soma-autonomous RNAi-induced enhancers of Pro. In addition, candidate screens suggest that the insulin pathway contributes to robust larval germline proliferation.

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.

S Golik et al. (1999) International C. elegans Meeting "FMRFamide-related peptides and egg-laying"

W Schafer, L Hardaker, S Golik

[International C. elegans Meeting, 1999]

The egg-laying motorneurons release multiple neurotransmitters, each of which appears to control a specific aspect of egg-laying behavior. Under favorable conditions, wild-type worms fluctuate between alternative behavioral states for egg-laying: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Switching between states, as well as egg-laying within the active state, are stochastic, Poisson-like processes. We showed previously that serotonin, released primarily from the HSNs, facilitates the switch from the inactive to the active state, while acetylcholine, released from both the HSNs and VCs, promotes egg-laying within the active state. Li and Chalfie have shown that both the HSNs and VCs contain one or more FMRFamide-related peptides, and provided pharmacological evidence that these neuropeptides may control of egg-laying behavior. To investigate the roles of these peptides in more detail, we have analyzed the egg-laying behavior of deletion mutants (kindly provided by Chris Li) that are defective in production of specific FaRP precursors. We have found that one of these mutants, defective in the gene flp-1, have dramatically lengthened inactive phases, but completely normal egg-laying within the active phase. Moreover, although these mutants are stimulated to lay eggs by serotonin with approximately normal dose responses, their rate and pattern or egg-laying on serotonin are abnormal. Thus, we hypothesize that the FLP-1 peptides and serotonin act in parallel to affect the same step in egg-laying behavior--inducing the switch between the inactive and active phases. Experiments to investigate the cellular and molecular mechanisms for the FLP-1 peptides' affects on egg-laying will be described. Thanks to Chris Li for discussions and for providing strains.

Zocher E et al. (2018) MicroPubl Biol "Dominant-negative VPS-4 disrupts ODR-10::GFP distribution but has limited effects on chemotaxis"

Ruth N, Dahlberg C, Zocher E

[MicroPubl Biol, 2018]

Accumulation of ODR-10 depends on the sex and fed state of C. elegans (Ryan et al. 2014), though the precise mechanism of its regulation is not known. We tested whether the ESCRT pathway is required to maintain normal accumulation and distribution of ODR-10::GFP in AWA neurons under well-fed and starved conditions. The conserved AAA ATPase VPS-4 is required as the final step of the ESCRT pathway, to dissociate the machinery as cargo is moved from endosomes to multivesicular bodies (Babst et al. 2011, Kim et al. 2011). Mutation of Glutamate 219 to Glutamine (E219Q) in VPS-4 results in a dominant negative enzyme (VPS-4 DN). VPS-4 DN disrupts normal accumulation of the glutamate receptor, GLR-1, in a cell autonomous manner (Chun et al. 2008). We investigated a potential role for VPS-4 in ODR-10 regulation using fluorescence microscopy and an attraction chemotaxis assay. We expressed VPS-4 DN under the odr-10 promoter in animals expressing ODR-10::GFP (Figure 1A). Using live-imaging (200X (total) magnification), ODR-10::GFP was not visible in over half of the wild type, well-fed animals. Using this baseline, we measured changes in ODR-10::GFP (abundance and localization) and found that VPS-4 DN increased the frequency with which we observed animals with aberrant ODR-10 accumulation (Figure 1B). In 8 animals (out of 58) there was observable fluorescence in the cell bodies of AWA neurons (1 with fluorescence exclusively in the cell body). Starvation exacerbated this effect: 26 out of 39 animals had fluorescence in the cell bodies of AWA neurons (7 with fluorescence exclusively in the cell body). In contrast to previously published data that relied on different imaging parameters (Ryan, et al., 2014) we also observed that starvation of animals led to increased frequency of visible fluorescence in the cilia of AWA in WT animals (Figure 1B). Expression of VPS-4 DN had no effect on the ability of animals to react to the chemoattractant diacetyl under well-fed conditions (Figure 1C). However, VPS-4 DN expression in the AWA neuron resulted in significantly lower chemotaxis indices, compared to wild type, fed animals (Figure 1C, asterisk). Starvation of animals has a subtle effect on the abundance of ODR-10 that accumulates in the cilia of AWA neurons, but that accumulation does not affect the efficiency of chemotaxis in wild type animals (Figure 1B and C). However, when food stress was paired with a disruption of the ESCRT pathway the aberrant accumulation of ODR-10::GFP correlated with a modest but significant decrease in chemotaxis index (Figure 1C). It is still unknown whether ODR-10 is a direct target of the ESCRT machinery or other ubiquitin-related pathways. These data could also represent indirect effects due to changes in availability or localization of cofactors that are required for normal receptor processing and/or localization within the AWA neuron.

Consortium TCeGS (1992) Worm Breeder's Gazette "Update on cDNA Sequencing"

Consortium TCeGS

[Worm Breeder's Gazette, 1992]

This is another interim report on the state of the cDNA sequencing and mapping project that we are carrying out alongside the genomic sequencing effort. We have now sequenced 1222 of the 1743 clones in Chris Martin's library. Of these 27 appear to be duplicates in the strong sense that their sequence looks identical and they come from neighboring micro-titre wells that were most likely cross-contaminated. The following results therefore come from the 1195 left after removing these duplicates. Remember that we are only producing a single gel reading from each clone (5' end of the cDNA insert). Similarities: There are 373 (31%) with BLASTX scores to the public protein databases greater than 100, which is a high enough threshold that these similarities are very likely to be meaningful. A list of these is given on the following pages. [Figures excluded due to low reproducability] Trans-spliced leaders: 166 sequences (14%) had trans-spliced leaders at the 5' end, 149 to Sl1 and 17 to SL2 . Repeat isolations: A number of genes clearly have been cloned more than once. We can unambiguously detect this only when the 5' ends of the inserts overlap, which happens for 334 sequences that fall into 130 overlap groups. This is not out of line with Chris Martin's estimate of repeat frequency. It leaves us with 991 distinct sequences of which 861 are represented only once. However it is likely that there are additional repeats that we don't see this way because we have different parts of the same gene. Hits to previously determined worm sequences: There were exact matches to 13 previously determined C. elegans sequences in the public databases, and to 3 of the 44 predicted genes on the first 4 genomic cosmids that we have sequenced. The latter figure allows us to generate an estimate of (44/3)*991 = 14,500 genes in the worm, but the 95% confidence limits for this are 6,000 to 50,000, so we need more data to make this a useful approach to estimating gene density. Mapping: we are now in the process of mapping all the clones using the YAC "polytene" filters. This data will be in ACEDB and will also be provided if you ask for a clone. All the sequences, similarity and mapping data will be in ACEDB, and any clone is available along with more detailed data from the St. Louis lab.

Crisp A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Age-related Cholinergic Neurodegeneration by Overexpression of apl-1 in C.elegans"

Pierce-Shimomura J, Crisp A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Alzheimer's disease (AD) is the most common cause of dementia. It is characterized by selective degeneration of cholinergic neurons involved in memory. Dying neurons are surrounded by dense plaques primarily composed of beta-amyloid peptide (Aβ). Aβ is just one cleavage product from the protein APP. Mutations in APP that affect the processing of Aβ result in early-onset AD; however, a single additional wild-type copy of APP can also lead to AD, as seen in all individuals with Down syndrome. Aβ plaques were originally thought to be the primary cause of neurodegeneration, but new research suggests the role of the APP in AD is more complex than originally appreciated. The labs of Dr. Chris Li and Dr. Chris Link have pioneered the use of C. elegans to study APP function and dysfunction. For instance, overexpression of human Aβ in C. elegans muscle leads to the formation of Aβ aggregates(1). Overexpression of multiple copies of the APP-related gene, apl-1, in C. elegans leads to gross phenotypes, including partial lethality, arrested development, and vacuolization(2). We are testing whether expression of only one additional wild-type copy of apl-1 results in AD-related phenotypes. DIC and fluorescence microscopy were used to evaluate worms for signs of neurodegeneration. We have discovered that overexpression of apl-1 in a single copy results in the age-related degeneration of a specific subset of cholinergic neurons in C. elegans. To quantify the impact of degeneration, we track the deterioration of two natural behaviors (swimming and egg-laying) that rely on these neurons. These worms displayed no obvious defects in early adulthood but began to display defects in egg-laying and swimming by the third day of adulthood ('middle age'). In addition, ablation of these specific neurons in wild-type individuals recapitulates the behavioral defects observed in egg-laying and swimming. These results suggest that the behavioral defects observed in the overexpression strain are primarily due to the selective degeneration of these neurons. The quantifiable link between these behaviors and neurodegeneration allows us to test strategies to recover the function mediated by the degenerated cholinergic neurons. We are now studying the mechanisms by which apl-1 overexpression causes death of cholinergic neurons in the worm and in the process, generate novel hypotheses about the mechanism of Alzheimer's disease. 1. Link CD. (1995) PNAS 92, 9368-9372. 2. Hornsten A. et al. (2007) PNAS 104, 1971-1976.

Estevez M et al. (1990) Worm Breeder's Gazette "daf-4, a Gene Controlling Dauer Larva Development, Encodes a Serine/Threonine Kinase"

Estevez M, Albert PS, Riddle DL

[Worm Breeder's Gazette, 1990]

Loss-of-function mutations in daf-4 confer a dauer-constitutive phenotype. Two transposon-tagged alleles of daf-4 have been isolated, and Southern blots probed with the labeled transposon sequence revealed a novel restriction fragment in the mutant DNA that was absent in non-mutant DNA. A transposon-containing fragment was cloned, and sequences flanking the transposon were used to screen Bob Barstead's cDNA library and Chris Link's genomic library. Four overlapping genomic clones and five cDNA clones were isolated. Microinjection of one of the genomic clones into the germline of daf-4 mutants transforms their progeny to wild type. Approximately 1 kb of contiguous cDNA sequence has been determined, and the deduced amino acid sequence shows 30% identity to the C. elegans brane receptor kinase (Georgi et al., 1990, Cell 61: 635) over a 222 amino acid region in the catalytic domain. The deduced amino acid sequence also shares 37% identity with PSK-J3 ( from HeLa cells), a member of the cdc2/CDC28 kinase family over a 107 amino acid region in the catalytic domain. Since genetic interactions suggest that daf-4 acts downstream of the daf-1 receptor, we propose that it mediates the second step in a kinase cascade that normally acts to prevent dauer larva development, and permit growth.