Watts JL et al. (1994) Worm Breeder's Gazette "zu170 Defines a New Gene, par-6, and Can Act as a Suppressor of par-2"

Watts JL, Kemphues KJ

[Worm Breeder's Gazette, 1994]

zul70 defines a new gene, par-6, and can act as a suppressor of par-2 Jennifer Watts and Ken Kemphues Genetics and Development, Cornell University, Ithaca NY 14853

Nicole Toms et al. (2001) International C. elegans Meeting "DNA arrays and mutations that reverse the direction of the ALM cell migrations"

Brandi Patchen, Eric Aamodt, Nicole Toms, Jennifer Cooper

[International C. elegans Meeting, 2001]

We are interested in how different types of neurons are specified and are focusing on the cell divisions that give rise to ALM touch receptor neurons and BDU interneurons. Touch neuron-specific genes in the ALMs are activated by a MEC-3/UNC-86 heterodimer. UNC-86 is present in both the ALMs and the BDUs while MEC-3 is only present in the ALMs. PAG-3 represses mec-3 in the BDUs and activates mec-3 in the ALMs. After the ALM/BDU cell division, the ALMs migrate posteriorly and the BDUs migrate anteriorly. Mutations in pag-3 cause the BDUs to express touch neuron specific genes but the ALMs and BDUs still migrate to their normal positions, suggesting that the migration and differentiation are controlled by separate mechanisms. We are interested in how the ALM and BDU cell migrations are controlled. We have identified a 104 bp DNA sequence from the mec-3 upstream control region that, when present in the ALM touch receptor neurons in 50-100 copies, causes ALM migration defects. In animals transformed with high copy extrachromosomal arrays containing the mec-3 upstream sequence, the ALM touch receptor neurons failed to migrate to their normal positions and sometimes migrated anteriorly. Furthermore, the PLM touch receptor neurons showed a number of axonal defects. Their axons were often short, misplaced, and some ended in a bulge that may have been a stalled growth cone. The ALM migration defect did not result from RNA interference (RNAi) because double stranded RNA that matched the mec-3 upstream sequence did not induce ALM migration defects or PLM axonal defects. These defects did not result from nonspecific effects of carrying a transgenic array because most arrays did not cause either ALM migration defects or PLM axonal defects. In this study, the ALMs and PLMs were being visualized by GFP fluorescence. Arrays that did not contain the active sequence but had very bright GFP fluorescence did not cause the ALM or PLM defects, which shows that the defects did not result from GFP expression. Instead, the ALM migration and PLM axonal defects resulted from transgenic arrays containing many copies of a specific 104 bp DNA sequence. Transgenic arrays containing this sequence did not affect all cell migrations. The mec-3 upstream sequence appears to be sequestering (titrating out) a specific DNA-binding factor that is required for the ALMs to migrate correctly. Because titration of this factor reversed the direction of ALM migrations, it may be part of a program that specifies both the direction and extent of ALM migrations. The titrating sequence is from the mec-3 gene, which is a master regulator of touch receptor neuron genes, so the factor or factors that bind this sequence may also be involved in specifying the fate of the ALM touch receptor neurons. Perhaps the ALM and BDU migrations are controlled by a factor that is preferentially segregated into the ALMs and away from the BDUs during the ALM/BDU cell divisions. Cells receiving more of this factor, the ALMs, might migrate posteriorly while cells receiving less of this factor, the BDUs, might migrate anteriorly. If this hypothesis is correct, mutations in genes that produce this factor, or produce proteins that interact with this factor, should cause the ALMs to migrate anteriorly rather than posteriorly. We have isolated mutations that phenocopy the ALM migration defect and will use these mutations to identify genes and proteins involved in controlling the direction and extent of the ALM migrations.

Sato K et al. (2006) Mol Biol Cell "Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans."

Audhya A, Oegema K, Sato K, Grant BD, Schweinsberg P, Sato M

[Mol Biol Cell, 2006]

Monitoring Editor: Jennifer Lippincott-Schwartz Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, since fusion can proceed in spe-9 fertilization mutants but is blocked by RNAi-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

Jennifer Pilipiuk et al. (2006) European Worm Meeting "Analysis of Epithelial Genes during Postembryonic Development of C. elegans"

Olaf Bossinger, Jennifer Pilipiuk, Gisela Helbig

[European Worm Meeting, 2006]

Jennifer Pilipiuk, Gisela Helbig and Olaf Bossinger. We wish to understand how epithelial polarity and tissue integrity is maintained. Here, we consider the role of DLG-1 (Discs large), LET-413 (Scribble), ERM-1 (Ezrin-Radixin-Moesin), and the catenin-cadherin complex (HMP-1HMP-2HMR-1) during postembryonic development of C. elegans. In the course of embryogenesis these genes play an important role in the establishment and maintenance of epithelial polarity, the formation of the lumen, and cell-cell adhesion. In contrast, during larval and adult development only newly established epithelia (e.g. the spermatheka or the vulva) show severe defects after bacterial RNAi against DLG-1, LET-413 and ERM-1, while the depletion of the catenin-cadherin complex seems not to cause visible defects. Nevertheless, how polarity of already established epithelia (e.g. the intestine or the hypodermis) is maintained during postembryonic development remains elusive. In the case of DLG-1, our results suggest that a small amount of protein is sufficient, but cannot fall below a certain threshold without causing defects. Intestinal and hypodermal polarity during larval and adult development of C. elegans might also depend on other, so far unidentified proteins. A detailed analysis of DLG-1, LET-413 and ERM-1 phenotypes during postembryonic epithelial development in C. elegans will be presented and discussed.

Keith A Boroevich et al. (2004) West Coast Worm Meeting "Identification of Regulatory Elements in Caenorhabditis elegans using Orthology Biased Gibbs Sampling"

Steven JM Jones, David L Baillie, Keith A Boroevich

[West Coast Worm Meeting, 2004]

Much attention has recently been placed on the problem of identification of gene regulatory sequences. Two main computational approaches exist: 1) identification of sequences that share similarity to known regulatory elements, and 2) de novo identification of common motifs in a set of co-regulated genes. Orthology Biased Gibbs Sampling (OrBS) applies a modification of the Gibbs Sampler put forth by Lawrence, Altschult et al . 1 in a attempt to solve this problem. Additional features include analysis of negative strand, multiple (or no) occurrences of a motif in any given sequence and identification of multiple unique motifs, all of which have appeared in previous incarnations of the Gibbs Sampler. The unique feature of OrBS is the use of comparative genomics as an informative prior and in the calculation of motif probability. OrBS will be applied to the identification of regulatory elements C. elegans . Clustering of spatially co-regulated genes as defined by the C. elegans Gene Expression Project (see Johnsen et al . poster). This data is more amenable to regulatory element detection than the more common microarray data because the sequence in which a cis -acting element may reside is strictly defined. Interspecies sequence conservation will be identified through alignment of the C. elegans gene promoter region to the promoter region of the C. briggsae ortholog using LAGAN 2 . Predicted regulatory elements will be verified in vivo using site directed mutagenesis and promoter::GFP fusions. References: 1 Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. (1993). Science 262: 208-214. 2 Brudno M, Do C, Cooper G, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). Genome Research 13: 721-731.

Hicks T et al. (1992) Worm Breeder's Gazette "Two Approaches to Aligning the Genetic and Physical Maps on Chromosome I:I. Cosmid Rescue of Mapped Lethals."

Rose AM, Hicks T

[Worm Breeder's Gazette, 1992]

For the left portion of chromosome I, where we have recovered lethals using sDp2 ,we are using cosmid rescue to align the genetic and physical maps. We also hope to obtain an estimate of the density of identified essential genes per cosmid. We have been using established transgenics (kindly sent to us by Heidi Browning and Janet Paulsen in Susan Strome's lab). As well we are constructing our own transgenics in order to have a representative collection of (frozen) genetic strains which carry cosmids or YACs for the majority of the sDp2 region (Thomas Hicks, Jennifer McDowall and Sheldon McKay, unpublished). The transgenics will be used as duplication strains and crossed to lethals to test for rescue. As a beginning, a set of lethals very close to dpy-5 are being tested. This set consists of fifty-five EMS-induced lethal mutations (from screens using sDp2 )that had been positioned within 0.5 map unit of dpy-5 .In order to subdivide this set, we have used the duplications hDp22 and hDp24 described by McKim and Rose (1990 Genetics 124:115). Lethal mutations within the intervals are being complementation tested to define essential genes. So far, one interval which contains three mutations has been completed and the genes, let-576 ,let-577 and let-578 defined. Complementation testing for the other two intervals is not as yet complete, but one gene, let-575 ,has been identified which has at least three alleles. Lethals in the hDp24 to hDp22 interval are being crossed to strains constructed by Heidi Browning, which carry either the cosmid C24H1 or the YAC Y51G8 .The YAC rescues at least two of the lethals, h466 and h407 .The latter is also rescued by the cosmid C24H1 .

Michelle L. Tucci et al. (2007) International Worm Meeting "Rescue of dopaminergic behavioral deficits induced by alpha-synuclein overexpression via genetic and chemical effectors."

Shusei Hamamichi, Michelle L. Tucci, Cody J. Locke, Guy A. Caldwell, Kim A. Caldwell

[International Worm Meeting, 2007]

Alpha-synuclein is the key molecule involved in synucleinopathies since multiplication of the wildtype alpha-synuclein locus is sufficient for progressive loss of dopamine (DA) neurons and the development of Parkinsons disease. We have constructed two isogenic strains of C. elegans that co-express GFP and alpha-synuclein under the dat-1 promoter and have found that both lines exhibit DA neurodegeneration. Notably, the level of DA neurodegeneration corresponds to the level of alpha-synuclein expression as determined by semi-quantitative RT-PCR. Furthermore, both strains exhibit an age-related decline in DA neuron survival. For example, in worms expressing the higher level of alpha-synuclein, there is almost no degeneration when the animals are 3 days old, while 7 and 10 day-old animals have 25 and 36% degeneration, respectively. In mammalian systems, DA is implicated in the neurotoxicity of alpha-synuclein in DA neurons. We have discovered that the dorsal CEP neurons, which are post-synaptic to the ADE dopamine-producing neurons, degenerate significantly more often than the ventral CEP neurons, which do not synapse onto other dopamine producing neurons (p=0.02; Fisher Exact Test). DA neurodegeneration induced by alpha-synuclein overexpression was significantly rescued by overexpression of several genes implicated in Parkinsons disease (Cao et. al., J Neuroscience 25:3801; Cooper et al., Science 313:324; Hamamichi et al., 16th International Worm Meeting). Furthermore, several chemicals isolated from ongoing drug screening efforts significantly enhance DA neuron survival in our model. Alpha-synuclein-expressing worms were also tested for deficits in the DA-specific behavior of basal slowing in response to the presence of bacteria (Sawin et al., Neuron 26:619). We found that these worms displayed a locomotory defect for this phenotype that was often observed prior to the loss of DA neurons. Thus, it is possible that the behavioral deficit represents a more sensitive readout for DA neuron loss than visual inspection of the neurons. Furthermore, genetic and chemical enhancers of DA neuron survival often rescued this DA-specific behavioral phenotype.

Shu Hamamichi et al. (2007) International Worm Meeting "Hypothesis-based RNAi Screening to Identify Neuroprotective Genes in a Parkinson's Disease Model."

Kim A. Caldwell, Guy A. Caldwell, Adam L. Knight, Renee N. Rivas, Shu Hamamichi

[International Worm Meeting, 2007]

Genomic multiplication of the locus encoding human alpha-synuclein, a polypeptide with a propensity toward intracellular misfolding, results in Parkinson''s disease (PD). Using worms overexpressing human alpha-synuclein::GFP fusion in body wall muscles, we have established conditions that facilitate the rapid identification of factors that influence the misfolding of this protein over time. Here we report the results of a systematic RNAi screen of approximately 950 candidate genetic targets to identify factors that enhance misfolding of alpha-synuclein over the course of aging in C. elegans. The mature bioinformatics data associated with C. elegans biology provides an opportunity to more rapidly discern novel candidate effector proteins for subsequent functional analysis. In this context, our targets were primarily prioritized based on hypothetical bioinformatic associations to existing genes (parkin, Nurr-1, UCHL-1, DJ-1, PINK-1, LRRK2, and ATP13A2) and cellular pathways (ubiquitin-proteasome system, ER-associated degradation pathway, unfolded protein response, and autophagy) with implications for PD. This screen revealed 14 genes that when knocked-down, consistently resulted in increased alpha-synuclein misfolding; none of these genes were associated with a prior link to PD. We previously established the application of C. elegans toward functional validation of an evolutionarily conserved and neuroprotective protein, Rab1, using a neurodegenerative model of age- and dose-dependent alpha-synuclein-induced degeneration in the dopamine neurons of transgenic worms (Cooper et al., 2006, Science). Here we significantly expand our list of neuroprotective genes through subsequent analysis of the positive targets from our RNAi screen. We generated transgenic nematodes that overexpressed the cDNAs corresponding to a prioritized subset of these hits in dopamine neurons. Strikingly, these animals revealed that 5 out of 7 tested gene products significantly protect against dopamine neuron loss. These proteins include two trafficking proteins, two proteins of unknown function with high homology in humans, and one gene already reported to cause neurodegeneration in knockout mice. Our combined bioinformatic/genomic screening approach provides a valuable paradigm for the investigation of disease mechanism using C. elegans. Furthermore, the gene products identified from these functional analyses represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting 1% of the population over 65 years of age.

Suzan J Holt et al. (2003) International Worm Meeting "A dauer approach to survival: Modulate internal oxygen exposure and manufacture metabolites de novo."

Donald L Riddle, Suzan J Holt

[International Worm Meeting, 2003]

A lowered metabolic rate and availability of stored lipids are thought to contribute to the longevity of dauer larvae, which can remain non-feeding for months. At least 5% external oxygen is required for lipid utilization in starving Caenorhabditis (1). The small size of a C. elegans dauer larva, 20 m in diameter, might indicate that oxygen passage by cuticular diffusion is sufficient for maintenance of aerobic physiology. Nevertheless, recent evidence from gene expression profiles suggests that anaerobic metabolism is augmented in dauer larvae. Serial analysis of gene expression (SAGE) has revealed that the dauer larva, though developmentally arrested, has a surprisingly complex transcriptome (2). We compared transcript abundance between the dauer and mixed-stage profiles for 59 genes with confirmed or predicted involvement in carbohydrate metabolism. The most prominent transcript was for phosphoenolpyruvate carboxykinase (PEPCK). The pyruvate kinase:PEPCK SAGE tag ratio for either profile supports a glycolytic shunt such as that found in parasitic helminths which are characterized by succinate fermentation and anaerobic ATP formation. However, the transcript patterns for key enzymes in the glyoxylate cycle, gluconeogenesis, and for glycogen phosphorylase reflected higher potential involvement for PEPCK in carbohydrate synthesis in mixed stages compared to the dauer. Transcripts for additional proteins with predicted functions in oxygen-independent routes for energy generation were dauer-enriched. The morphology of dauer larvae together with apparent upregulation of anaerobic pathways indicates that the dauer interior could be hypoxic. These data may help explain the hypoxia resistance (Hyp) of N2 dauer larvae and may have implications regarding the adult Hyp phenotype of certain daf-2 mutants (3). The lowering of the aerobic metabolic rate, as well as oxygen level, may lower the rate of oxidative damage accumulation and contribute to dauer longevity, as long as the -oxidation of lipids is not seriously compromised. In addition, fermentation byproducts, used as alternative nutrient sources, could serve to slow the depletion of lipid stores and extend the survival period.1. Cooper and Van Gundy. 1970. J. Nematol. 2:305; 2. Jones, Riddle, Pouzyrev, Velculescu, Hillier, Eddy, Stricklin, Baillie, Waterston, and Marra. 2001. Genome Res. 11:1346; 3. Scott, Avidan, and Crowder. 2002. Science 296:2388.

Jennifer Schisa et al. (2003) International Worm Meeting "Investigation of chemotaxis mutants in an undergraduate genetics laboratory"

Jennifer Schisa, Katie M Morrison

[International Worm Meeting, 2003]

A three-week series of C. elegans-based laboratories has been implemented in an introductory college genetics course (JS) that is based upon those used successfully in a high school program (KMM) and others (Jennifer Vowels, Debbie Birnby, Wendy Schackwitz, Jim Thomas). These labs are performed near the end of the semester, after students have been introduced to basic molecular biology concepts and at the same time that the lecture discusses the use of genetics to study behavior. The first session is a computer-based laboratory to introduce students to C. elegans and the vast array of information available at the C. elegans WWW server, Wormbase, and NCBI. Students are guided through questions and prompts to learn how to find different types of information such as: the literature published regarding chemotaxis in C. elegans, the names of researchers working on this problem, and the proteins that have been implicated in regulating chemotaxis. The second lab introduces students to the worm via three activities: 1) distinguishing males, adult hermaphrodites, and L4 worms; 2) identifying basic anatomical parts of the worm; and 3) sorting unc, dpy, and rol worms from a plate containing all three mutants. The first and third activity give students the opportunity to learn how to pick worms correctly, a skill they will need in the third lab. At the end of the second lab, students are assigned three unknowns (that they will identify in the third lab via chemotaxis assays) and pick worms to fresh plates in preparation for lab 3. The unknowns include two osm mutants, 1 che mutant, 1 che mutant marked with dpy, and wild-type. The third lab includes an attractance assay (based on Bargmann et al., 1993) and an avoidance assay, each performed with the three unknowns and wild-type controls. Students calculate avoidance and attractance indexes and perform statistical analyses to determine the identity of their unknowns. This series of laboratories could easily be expanded to incorporate student-directed projects, investigating other possible effectors of C. elegans behavior. Bargmann, C.I., Hartweig, E., and Horvitz, H.R. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74: 515-527.