Krishna S et al. (1997) International C. elegans Meeting "SPECIFICITY IN TGF-b SIGNALING"

Krishna S, Padgett RW

[International C. elegans Meeting, 1997]

TGF-b signaling involves the formation of a heteromeric complex between type I and II serine threonine kinase receptors. This complex is iniated by ligand-binding, and upon phosphorylation of the type I receptor by the type II kinase, signal is transduced from the receptors to SMADs, which are downstream components that are phosphorylated by the receptor complex. In this manner, the extracellular signal can produce intracellular changes. In C. elegans, the type II receptor daf-4 is involved in two parallel TGF-b-like pathways. Because the phenotype of the previously known type I receptor at the time, daf-1, could not account for those exhibited by mutations in the sma [2,3,4] genes (which were shown to function downstream of daf-4, Savage et. al.,PNAS 93, 790 794.), we hypothesized that another type I receptor participates in the Small pathway. Therefore, we have cloned and characterized sma-6, which encodes a type I receptor kinase which, presumably through association with DAF-4, transduces signal to the downstream SMAD components sma-[2, 3, 4]. We are currently examining the developmental role of sma-6 and the Small pathway, and we are using a null allele of sma-6 to facilitate this. The two characterized TGF b-like systems in C. elegans, the Small and Dauer pathways, provide an opportunity to examine how signaling specificity is generated and maintained in general TGF-b-like signaling mechanisms.

Krishna S et al. (1998) East Coast Worm Meeting "sma-6 and the Issue of Specificity in TGF-beta Signaling"

Krishna S, Padgett RW

[East Coast Worm Meeting, 1998]

In C. elegans, the TGF-beta-like type II receptor daf-4 intersects two distinct pathways. It is involved in the regulation of the dauering process in association with the type I receptor daf-1. In addition, it is also required in body size determination and male tail patterning, roles that do not require daf-1. We have cloned and characterized a novel type I receptor, and show that it is encoded by sma 6. Previously, we have demonstrated that the sma-2, -3, and 4 genes, members of the SMAD family, are downstream signaling components involved in only the Sma/Mab pathways. This study provides developmental genetic evidence that the interaction of the type II receptor with different type I receptors and their corresponding SMADs confers specificity in TGF-beta-like signaling. We show that there is minimal cross-talk between the Sma/Mab and dauer pathways and that sma-6 regulates male tail morphogenesis only through the small genes.

Krishna S et al. (1996) East Coast Worm Meeting "TGF-beta Superfamily Signalling in C. elegans"

Padgett RW, Krishna S

[East Coast Worm Meeting, 1996]

The TGF-b superfamily comprises a group of secreted proteins which are involved in fundamental biological processes including, but not limited to, axis specification, mesoderm induction, osteogenic differentiation, tissue regeneration, and neoplastic growth. Though there has been intense interest in recent years to determine the pathways by which TGF-b signalling occurs, the mechanism by which the signal is transduced from the ligand is still relatively poorly understood. Previous work examining the transduction of signal at the level of the membrane has resulted in the formulation of a general model by which this may occur. Transmembrane receptors which interact specifically with TGF-b superfamily ligands have been isolated and have been demonstrated to be required for proper transduction in several organisms. A subset of these receptors contain a serine-threonine kinase domain, and can be categorized into two classes, type I and type II, which differ considerably with each other in their non-kinase regions. It is thought that the mature protease-cleaved dimeric ligand interacts with appropriate type II receptors followed by the formation of an oligomeric complex with specified type I receptors. This complex then conceivably transduces the appropriate signal to downstream molecules, eventually resulting in intracellular changes and/or altered expression patterns of specific gene targets. In the worm, the dauer and small pathways are examples of mechanisms controlled by TGF-b-like ligands. In the dauering pathway, the products of daf-1, daf-4 are receptors and serve to transduce the signal. Additionally, the dwarfins comprise a group of genes which encode products that have been implicated in the TGF-b transduction pathway in the nematode (Savage et al., PNAS 1996; Savage et al., WBG 1995) and in Drosophila (Sekelsky et al., Genetics 139: 1347-1358, 1995) . In the worm, the sma-2, sma-3, and sma-4 genes are dwarfins that have been characterized by our group. The mutant phenotypes of these genes are a subset of those exhibited by daf-4. However, the sma phenotypes are not observed in other dauer mutants. Thus, daf-4 intersects the dauering and small pathways. Since daf-1 does not exhibit the small phenotype, we hypothesized that there should be another type I receptor transducing a TGF-b-like signal to the sma gene products, that when mutated, results in small animals. Using molecular tools, we had identified tre-1 as a novel type I receptor in C. elegans. tre-1 demonstrates a large degree of homology with other type I receptors, particularly the thickveins gene in Drosophila. Searches of extant mutations in the region of tre-1 indicated that sma-6 was nearby. Given that four genes mutate to a small phenotype, other genes in the pathway may also yield the same phenotype. Based on these data, the possibility was raised that sma-6 may encode tre-1. Germline transformation experiments confirmed this hypothesis, whereby a 9kb genomic fragment containing the tre-1 gene and flanking regions extending, but not encompassing, adjacent predicted transcripts was sufficient to rescue sma-6 mutants and revert F1 transformed progeny to wild-type length. We are currently continuing to characterize the expression of tre-1 and analyze interactions with other components of the small pathway. These results helps us to define the specificity of TGF-b signalling by demonstrating that different type I receptors are able to elicit varying biological responses. Furthermore, we have completed a large genetic screen (see abstract by Savage et al.) in order to identify other mutations that yield small animals. Our results with sma-6 validate this screen, and we hope to isolate other novel components of the pathway.

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.

Shetgiri P et al. (1997) International C. elegans Meeting "MOLECULAR AND GENETIC CHARACTERIZATION OF LON-1"

Shetgiri P, Padgett RW, Krishna S, Savage C

[International C. elegans Meeting, 1997]

Our lab has characterized a TGF-b signaling pathway in C. elegans defined by genes that mutate to a Small phenotype. In our ongoing efforts to elucidate the TGF-b signaling pathway, we have become interested in the question of whether negative regulators of the pathway exist. Several lines of evidence have implicated lon-1 as a negative element of this pathway, although it is unclear how directly it is involved. First, lon-1 mutations suppress the Small phenotypes of the TGF-b pathway components daf-4, sma-6, sma-2, sma-3, and sma-4, but do not suppress mutations in sma-1 which is not part of this signaling pathway. Second, sma-6 and sma-4 rescuing constructs introduced into worms often confer a Lon phenotype, suggesting that overactivity of the pathway can result in this phenotype. Finally, we have now found that transgenic worms carrying lon-1 rescuing constructs (see below) are often Small. We are characterizing lon-1 molecularly and genetically to understand how it exerts its effect on body size and at what level it intersects with the Small pathway. We have identified rescuing cosmids and l clones from the lon-1 genomic region by transformation rescue. The smallest rescuing clone contains two predicted ORFs, and we are currently trying to narrow the rescuing activity further. We have also continued to analyze the interactions of lon-1 with Small mutants. In particular, lon-1 alleles are epistatic to sma-6 null alleles, suggesting that mutations in lon-1 bypass the TGF-b signaling pathway. Our data therefore favor a model in which lon-1 is not a negative regulator of the pathway, but potentially a negatively regulated target of the pathway.

Y Suzuki et al. (1999) International C. elegans Meeting "Mediators and Modulators of dbl-1 Functions in Body Size Control"

GA Morris, Y Suzuki, WB Wood

[International C. elegans Meeting, 1999]

Size regulation of body components is a poorly understood aspect of animal growth and development. We have previously shown that the dbl-1 gene, a C. elegans gene encoding a TGF- b ligand, is a dose-dependent positive regulator of cell size and body size increase during post-embryonic development of the worm (1). Identification of additional components that act upstream and downstream of dbl-1 could provide new insights into the mechanisms of size regulation. Studies of the epistatic relationships between known lon genes and components of the dbl-1 signal transduction pathway have placed lon-2 and lon-3 upstream of dbl-1 , and lon-1 downstream of the Smad genes of the pathway (2, 3). More careful analysis has shown, however, that dbl-1 lon-3 double mutants are small but slightly longer than dbl-1 single mutants, leaving open the possibility that lon-3 acts independently of the dbl-1 pathway. We are in the process of cloning lon-3 to allow further analysis of its function. To identify potential targets of the body size control pathway, we are carrying out screens for suppressors of the Sma and Lon phenotypes caused by dbl-1(lf) mutations and dbl-1 overexpression, respectively. In a screen of 14,000 haploid genomes for suppressors of the Sma phenotype, we have isolated a number of candidate suppressors. Some isolates exhibit phenotypes similar to the Lon-1 phenotype, and others exhibit phenotypes that resemble the semi-Sma phenotypes of dbl-1 lon-3 double mutants or dbl-1 heterozygotes. We plan genetic and molecular characterization of genes defined by these suppressors. 1. Suzuki, Y., Yandell, M. D., Roy, P. J., Krishna, S., Savage-Dunn, C., Ross, R. M., Padgett, R. W. and Wood, W. B. (1999). Development 126: 241-250. Similar results have been obtained by Morita, K., Chow, K. L., and Ueno, N. (1999). Development 126: 1337-1347. 2. Savage, S., Padgett, R., and Baird, S. (1994) WBG 13(2): 38 3. Suzuki, Y., Morris, G., and Wood, B. (1999) WBG 15(5): 44

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.

Colavita A et al. (1997) International C. elegans Meeting "Suppressors of Ectopic UNC-5 Expression Identify a Novel TGF-b Involved in Axon Guidance in C. elegans."

Zheng H, Culotti JG, Krishna S, Padgett RW, Colavita A

[International C. elegans Meeting, 1997]

UNC-5 encodes a transmembrane receptor that guides migrating cells and pioneer axons away from ventral sources of secreted UNC-6 netrin guidance molecules in C. elegans. When ectopically expressed in the touch receptor neurons, which don!t normally respond to UNC-6, UNC-5 is able to confer UNC-6 responsiveness to their growth cones. This result forms the basis of a screen to look for mutations in components of the UNC6/UNC-5 signaling cascade - defects in components of this pathway should suppress the ability of ectopic UNC-5 to steer growth cones in response to UNC-6. This screen yielded mutants of 8 genes, one of which, unc-129, is a new uncoordinated mutant with axon guidance defects like those of unc-5 and unc-6 mutants. unc-129 was found to encode a novel member of the TGF-b family of secreted signaling proteins. unc-129-reporter transgenes are expressed in motorneurons and in dorsal muscles. Promoter bashing results indicate that the muscle expression is required for motor axon guidance; we are not yet certain whether motorneuron expression is also required. Ectopic expression of this TGF-b indicates that its spatial patterning is important for guiding cell and growth cone migrations. We are examining whether unc-129 acts on the UNC-6/UNC-5 signaling cascade as anticipated by the rationale for the suppressor screen, or whether it acts by an independent mechanism.

Josefin Nystrom-Friberg et al. (2003) International Worm Meeting "Two new loci affecting body length in C. elegans."

Simon Tuck, Armand Leroi, Josefin Nystrom-Friberg, Richard W Padgett, Zai-Zhong Shen

[International Worm Meeting, 2003]

Mutations in genes encoding a signal transduction pathway activated by a ligand in the TGF- faimly, DBL-1 can profoundly affect body size in C. elegans. Loss of function mutations in genes in the pathway causes a Sma phenotype characterized by a marked reduction in body size whereas constitutive activation of the pathway increases body length (Lon). Worms displaying the Sma or Lon phenotypes appear to have the same numbers of cells as wild type suggesting that the pathway affects body size by affecting cell size rather than cell number. The pathway may excert its efffects in part at least by affecting endoreduplicaton of hypodermal nuclei, and in part by regulating expression of components of the cuticle. Two targets of the pathway have been identified, lon-1which encodes a PR-related protein and lon-3 which encodes a cuticle collagen1, 2, 3, 4. Mutations in lon-1 affect both endoreduplication and body length, whereas mutations in lon-3 affect length without affecting endoreduplication. In a screen for new mutations causing a Lon phenope we have isolated 2 alleles, sp10 and sp11, of a locus on X, lying between unc-9 and lin-15. The mutations are all recessive and 100% penetrant; homozygous mutants are approximately 23 % longer than wild type but display no other obvious defects. Morphometric analysis indicates that the phenotype caused by these mutations is very similar to that caused by overexpression of DBL-1. We have identified a cosmid that rescues the mutant phenotype. The screen also identified a single allele, sp9, of a locus between unc-3 and lin-15 on X. This mutation causes an incompletely penetrant Lon phenotype; homozygous mutants are also slightly Egl. 1. Morita K, Flemming AJ, Sugihara Y, Mochii M, Suzuki Y, Yoshida S, Wood WB, Kohara Y, Leroi AM, Ueno N, EMBO 21: 1063-1073 (2002); 2. Maduzia LL, Gumienny TL, Zimmerman CM, Wang H, Shetgiri P, Krishna S, Roberts AF, Padgett RW, Developmental Biology 246: 418-428 (2002); 3. Nystrom J, Shen ZZ, Aili M, Flemming AJ, Leroi A, Tuck S, Genetics 161: 83-97 (2002); 4. Suzuki Y, Morris GA, Han M, Wood WB, Genetics 162:1631-1639 (2002).

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.