Chan S et al. (1991) International C. elegans Meeting "SEEKING UNC-40."

Culotti JG, Su M-W, Chan S, Hedgecock EM

[International C. elegans Meeting, 1991]

unc-40 is required with unc-6 to guide ventral migrations on the nematode epidermis (Hedgecock et al., Neuron 4, 61-85, 1990). We have positioned unc-40 genetically by 3-factor crosses with dpy-5 and bli-4 as flanking markers and we have isolated 5 new alleles of unc-40 (2 spontaneous, 2 gamma, and 1 EMS) making a total of 12. DNAs from cosmids covering this region (obtained from Alan Coulson and John Sulston) are being used to probe DNA and RNA from the mutants and DNA from strains carrying duplications that break to either side of unc-40 (obtained from Anne Rose's laboratory). We are also injecting cosmid DNAs into gonads to look for rescue by germline transformation. The brood sizes of various unc-40 alleles are very small, so we have resorted to injecting the wild type in order to create a series of transformed lines, each carrying an extrachromosomal array of a different cosmid. Each array will be passed into appropriately marked unc-40 animals to ask if any can rescue the mutant phenotype. Mosaic experiments are also underway to determine whether unc-40 acts in migrating cells.

Chan S et al. (1993) International C. elegans Meeting "unc-40 rescued."

Su M-W, Chan S, Jahromi A, Culotti JG, Hedgecock EM

[International C. elegans Meeting, 1993]

Chan S et al. (1995) International C. elegans Meeting "unc-40, CLONED, SEQUENCED, EXPRESSED"

Su M-W, Zheng H, Chan S, Culotti JG, Hedgecock EM

[International C. elegans Meeting, 1995]

unc-40 is required for guided circumferential migrations of pioneer axon growth cones and mesodermal cells on the basal surface of the epidermis of C. elegans. Although unc-40 mutations primarily affect migrations oriented towards the ventral side, some dorsally-oriented cell migrations, eg., distal tip cells, head mesodermal cells, are also affected. The unc-40 gene was cloned by mapping it relative to strain specific polymorphisms followed by transformation rescue using a cosmid from the region covering the unc-40 gene. By sequencing cDNA, genomic DNA and using RT PCR, we have characterized the entire coding region of this gene. From amino to carboxy terminus, the UNC-40 protein has 4 immunoglobulin domains, 6 fibronectin type III domains, a single transmembrane domain and an intracellular domain of 309 amino acids. This protein structure is very similar to two others, the human DCC (deleted colorectal cancer) protein and the chicken Neogenin. Both are expressed in the developing C.N.S., Neogenin is expressed in neurons as they extend axons, but not in neurons that have terminated neurite growth. Developmental Northerns have shown that unc-40 is most highly expressed during embryogenesis, when most axon outgrowth occurs. We are constructing unc-40-lacZ transgenes to examine the temporal and spatial expression of lacZ and GFP reporters from the unc-40 promoter..

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Saleel Raut et al. (2008) C.elegans Neuronal Development Meeting "HLH-3, a C. elegans Achaete/Scute protein, has a role in the axonal pathfinding of hermaphrodite-specific motor neuron (HSN) that is independent of UNC-40."

Aixa Alfonso, Saleel Raut

[C.elegans Neuronal Development Meeting, 2008]

The basic helix loop helix protein, HLH-3, is a member of the achaete/scute family in C. elegans hlh-3 mutants have an egg-laying defective (Egl) phenotype. We have shown (Doonan et al., 2008 submitted) that the mutant phenotype results from abnormal axonal pathfinding in hermaphrodite-specific motor neurons (HSNs) that fail to innervate the vulval muscles. The NETRIN/UNC-6 receptor UNC-40/DCC is necessary for the guidance of the HSN axons (Garriga et al.,1993; Chan et al., 1996; Gitai et al., 2003; Adler et al., 2006). We hypothesized that mutant animals are not uncoordinated (Unc). Currently we are looking at SYG-1, a receptor on HSN''s that binds with the guidepost signal SYG-2 for proper synapse formation (Shen et al., 2004), as a possible target of HLH-3.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Oppenheimer AJ et al. (1996) East Coast Worm Meeting "G-alpha-s signaling in C. elegans"

Kaplan JM, Oppenheimer AJ

[East Coast Worm Meeting, 1996]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate phenotypes dependent on G-protein signaling, we expressed mammalian G-proteins under the control of the glr-1 promoter, which drives expression in a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Expression of wild-type rat G-alpha-s impairs backward locomotion, while consitutively active rat G-alpha-s causes paralysis and neuronal degeneration in a subset of G-alpha-s-expressing cells, primarily the PVC and AVD neurons. Our model for the neurodegeneration is that activated G-alpha-s is misregulating an ion channel in AVD and PVC, causing osmotic imbalance and consequent swelling. Degenerating neurons appear enlarged and vacuolated, similar to those in worms carrying gain-of-function mutations in deg-1 and other degenerins. We tested whether activated G-alpha-s is killing cells by misregulating known ion channels. Loss-of-function mutations in deg-1, mec-6, unc-36, unc-2, and egl-19 were unable to suppress the degenerations, suggesting that G-alpha-s is not acting through these degenerins or calcium channels. Mutations in ced-3 and ced-4 do not prevent G-alpha-s-induced degenerations, indicating that G-alpha-s is not killing through apoptosis. To identify the components of the signaling pathway downstream of G-alpha-s, we screened for mutations that suppress the paralysis caused by activated G-alpha-s. In a screen of 7,760 haploid genomes, we identified 8 mutations that suppress paralysis and neurodegeneration and 8 mutations that suppress only paralysis. Five of the degeneration suppressors map to chromosome III and may be allelic. These mutations are semidominant, and several cause a hyperactive phenotype in the absence of activated G-alpha-s. Because hyperactivity also results from mutations in the Go signaling pathway, our suppressor mutations may identify a gene invovled in both the Gs and Go signaling pathways. We are continuing to map and characterize the suppressor genes.

Costi D. Sifri et al. (2007) International Worm Meeting "Immune evasion by staphylococcal exopolysaccharide during C. elegans infection."

Jakob Begun, Dietrich Mack, Costi D. Sifri, Jessica M. Gaiani, Frederick M. Ausubel, Holger Rohde, Stephen B. Calderwood

[International Worm Meeting, 2007]

We have previously shown that C. elegans can serve as a model host for the study of S. aureus pathogenesis and host innate immunity. Here we characterize nematode killing by S. epidermidis. C. elegans fed S. epidermidis survive 3-5 days, which is ~1 day longer than their lifespan on S. aureus. Inactivation of the ica operon (the biofilm exopolysaccharide [PIA] biosynthetic operon and Yersinia hms homolog) in S. epidermidis strain M10 greatly reduces nematode killing compared to the isogenic parental strain 9142 and a plasmid complemented mutant. In S. aureus, overproduction of PIA due to derepression of ica augments nematocidal activity. FITC-WGA labeling confirmed the presence of PIA within the digestive tracts of worms fed 9142 but not M10. Attachment of bacteria or bacterial matrix to the nematode cuticle was not observed. Nematodes briefly exposed to 9142 could not be rescued by transfer to M10, and worms fed dilutions of 9142 in M10 at ratios as low as 1:10e4, respectively, still die. Pulse/chase experiments using 9142, M10, and a second PIA-deficient strain, ATCC 12228, showed that durable colonization requires an intact ica operon. Disruption of N2 worms fed 1:100 9142/M10 mixtures for 20h confirmed significant enrichment of 9142 within the digestive tract of N2 but not sek-1 animals. Moreover, sek-1 and pmk-1 animals were found to be equally sensitive to M10- and 9142-mediated killing, in contrast to wild-type N2 worms. Several conclusions can be drawn from these studies. (a) S. epidermidis can infect and kill worms. (b) Disruption of PIA biosynthesis abrogates S. epidermidis virulence, while overproduction of PIA in S. aureus augments virulence. (c) PIA-producing S. epidermidis has a competitive advantage over PIA-deficient S. epidermidis within the nematode digestive tract. (d) Genetic analysis suggests that PIA may protect luminal staphylococci from SEK-1 p38 MAPK-regulated immune responses.

Park J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "A Novel Role for UNC-40/DCC and UNC-6/Netrin in Synaptic Partner Choice"

Park J, Wung W, Raman M, Knezevich P, Shen K, O'Hanlon S, Goyal A, VanHoven M

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

Neural circuits are required for nervous system functions including perception, thought and behavior. Much is known about critical early steps in circuit formation including cell migration, axon guidance, and localizing presynaptic components to required subcellular compartments. However, less is known about how individual neurons, after achieving these steps, choose the correct synaptic partners within dense target regions. To understand how correct synaptic partner choice is mediated, we developed a fluorescent trans-synaptic marker to label synapses between neurons of interest called Neuroligin-1 GFP Reconstitution Across Synaptic Partners, and labeled pre- and postsynaptic neurites with the mCherry fluorophore. These markers enable us to instantly assess correct synaptic partner choice by visualizing neurite contact and synaptogenesis between pre- and postsynaptic neurons. We have found that two proteins previously studied for their role in earlier steps in neural circuit formation, the UNC-40/DCC (Deleted in Colorectal Cancer) receptor and its secreted ligand UNC-6/Netrin, have a novel role in mediating synaptic partner choice between PHB sensory neurons and AVA interneurons. unc-40/DCC and unc-6/Netrin animals with normal cell-migration, axon guidance to the ventral nerve cord, and localization of presynaptic components, display synaptic partner choice defects: they have reduced PHB-AVA neurite contact (70% of wild-type) and synapse formation (50% of wild-type). Previous studies indicate that UNC-40/DCC is expressed in PHB, and UNC-6/Netrin is expressed in AVA neurons1,2. Cell-specific rescue experiments indicate that UNC-40/DCC and UNC-6/Netrin can function in the pre- and postsynaptic cells, respectively, and overexpression of UNC-6/Netrin is sufficient to increase PHB-AVA synaptogenesis, supporting a model in which UNC-40/DCC and UNC-6/Netrin mediate a novel direct interaction between PHB and AVA neurons. In addition, a membrane-tethered UNC-6/Netrin expressed in AVA rescues synaptogenesis, but not neurite contact, distinguishing a long-range role of netrin necessary for complete neurite contact, from a short-range signal sufficient for PHB-AVA synaptogenesis. Finally, we have developed a behavioral assay to test the function of PHB-AVA synapses that correlates the observed visual phenotypes in mutant and transgenic animals with synaptic function. 1. Wadsworth, WG, et. al, Neuron 16, 35-46 (1996)2. Chan, S. S. et al. Cell 87, 187-95 (1996)