Wendy B Iser et al. (2005) International Worm Meeting "Analysis of age-1/PI3K activity in subsets of neurons and in the intestine"

Wendy B Iser, Andrea Wilson, Catherine A Wolkow

[International Worm Meeting, 2005]

In C. elegans, lifespan, stress resistance and developmental arrest are controlled by the daf-2/insulin-like signaling pathway. Mutations in daf-2, encoding a homolog of mammalian insulin/IGF-I receptors, or age-1, encoding a homolog of the mammalian p110 catalytic subunit of PI 3-kinase (PI3K), can extend adult lifespan and cause constitutive dauer arrest. Previous analyses have indicated that age-1 activity in the nervous system can rescue adult lifespan (Wolkow et al. 2000). Longevity was also correlated with daf-2 activity in the nervous system, as well as in the intestine (Apfeld & Kenyon, 1998; Wolkow et al. 2000). Interestingly, daf-16, encoding a FOXO transcription factor which is the ultimate output of the daf-2/age-1 pathway, did not appear to function in neurons, but did function in the intestine, to regulate lifespan (Libina et al, 2003). We have recently expressed age-1 from two additional intestine-specific promoters and found that these transgenes can rescue adult lifespan. These results suggested two possibilities. First, daf-2 and age-1 may act in either the nervous system or the intestine to control lifespan. Alternatively, promiscuity of nervous system-specific transgenes may have provided sufficient intestinal age-1 activity to rescue lifespan. To further examine where age-1/PI3K acts to control adult lifespan, we have constructed additional transgenes expressing age-1 from promoters restricted to subsets of neuron. In addition, we have developed an assay to monitor the level of intestinal daf-2 pathway activity. During starvation or oxidative stress, intestinal ACE-2 acetylcholinesterase activity relocalizes from cytoplasmic vesicles to the nuclear or perinuclear compartment. ACE-2 relocalization requires intestinal daf-2/age-1 pathway activity. We found that transgenes expressing age-1 from neuron-specific promoters could partially rescue adult lifespan without rescuing intestinal ACE-2 localization. This rescue was not restricted to specific neurons, as lifespan was partially rescued by age-1 expression from either motorneuron- or interneuron-specific promoters. These results appear to support the conclusion that age-1 activity in the nervous system affects adult lifespan. To further test this hypothesis, we are conducting genetic screens to identify additional genes necessary for cell non-autonomous control of lifespan by age-1/PI3K signaling.

Sambongi Y et al. (1998) European Worm Meeting "Physiological roles of C. elegans copper transport ATPase"

Wakabayashi T, Sambongi Y, Yoshimizu T, Futai M

[European Worm Meeting, 1998]

Copper is essential for cuproenzymes, but its excess causes cell damage through oxidizing proteins and lipids. Therefore, intracellular copper ion concentration must be tightly regulated. Two human genetic disorders, Menkes and Wilson diseases, are defective in copper metabolism. Genes responsible for both disorders encode homologous heavy metal P-type ATPases. To understand the physiological roles of the ATPase, we cloned a homologous cDNA from C. elegans (CUA-1, a 1,238 amino acid protein) that has sequence identities of 46% and 43% to Menkes and Wilson disease proteins, respectively. We showed that the CUA-1 cDNA could rescue the yeast Dccc2 mutant (copper ATPase gene disruptant), suggesting that CUA-1 functions as a copper transporter. The upstream region of cua-1 was fused with the reporter genes (b-galactosidase or GFP) and introduced into the N2 worms. High level expression of the reporter gene was observed specifically in pharyngeal terminal bulb muscle pm6 and most anterior intestinal cells in the transgenic adult worms, suggesting that CUA-1 functions primarily in the digestive organs. In addition to these cells, prominent expression was found in hypodermal cells in larval stages. We also report subcellular localization of CUA-1 and discuss physiological role(s) of this ATPase in C. elegans. We thank Yuji Kohara for providing yk29a9 clone and Andy Fire for GFP and lacZ vectors.

Chris R Gissendanner et al. (2002) Mid-west Worm Meeting "NHR-6: a nuclear receptor transcription factor required for ovulation."

Tri Q Nguyen, Claude V Maina, Chris R Gissendanner, Marius Hoener, Jianping Xiao, Ann E Sluder

[Mid-west Worm Meeting, 2002]

The C. elegans genome encodes greater than 270 nuclear receptor (NR) genes (Sluder et al, 1999). Among these are fifteen genes that, based on comparative sequence analysis, encode nuclear receptor proteins conserved across metazoan phyla (Sluder et al, 1999). One of these conserved NR genes is nhr-6, which encodes the only C. elegans member of the NGFI-B NR sub-family (Wilson et al, 1992; Kostrouch et al, 1995; Sluder et al, 1999). We have initiated a functional analysis of nhr-6. Data from RNAi experiments and analysis of the deletion mutant lg6001 have established a role for nhr-6 in gonad development and/or function. Specifically, nhr-6(RNAi) and nhr-6(lg6001) animals have extremely low brood sizes and lay abnormally shaped eggs. Further analysis demonstrated that these phenotypes were due to an ovulation defect. Oocytes undergoing ovulation in lg6001 hermaphrodites frequently fragment entering the spermatheca. Only a fraction of the oocyte fragments become fertilized and many of these fail to develop properly. Oocyte fragments that remain in the gonad can become endomitotic (Emo phenotype). An nhr-6::GFP transgene is expressed in the spermatheca, suggesting a role for nhr-6 in spermathecal development and/or function. Currently, our analysis of nhr-6 is focused on the following: 1) a detailed characterization of nhr-6 expression and the ovulation defect observed in lg6001 mutants; 2) the in vivo significance of a conserved Akt/PKB phosphorylation site within the DNA binding domain of NHR-6; and 3) a comparative biochemical analysis of NHR-6 and an NGFI-B homolog (DiNHR-2) from the filarial nematode Dirofilaria immitis. The results of these investigations will be presented. Wilson, T.E., Paulsen, R.E., Padgett, K.A., and Milbrandt, J. (1992). Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors. Science, Vol. 256(5053), 107-10.

John Peloquin et al. (2007) International Worm Meeting "Worms and Filthy Lucre: Applying C. elegans to natural product development in industry."

Stuart Reeves, John Peloquin

[International Worm Meeting, 2007]

Diamond V Mills is a medium sized Agricultural Biotechnology company with more than 63 years of success in producing dietary supplements and ingredients. Recently the company has moved into human nutrition through its formation of Embria Health Sciences. Thus, the product repertoire has increased and is increasing. Our products arise naturally as the result of subtle alterations of fermentative processes, which are still not completely understood. Furthermore, their effects on animal biology are pleotropic. This presents some serious economic challenges in that far more product prototypes can be produced than economically tested in vertebrate models to demonstrate efficacy. Because many biochemical and signal transduction pathways are conserved between C. elegans and other animals, we have begun to develop C. elegans-based medium through-put screens to identify new product prototypes. We present preliminary data on anti-aging effects of an Embria product as an example of our future goals. (this work was initiated with technical assistance from Mark A. Wilson and Cathy Wolkow of the NIA).

G Aspoeck et al. (1999) International C. elegans Meeting "Complete closure of the anterior and posterior hypodermis needs the C. elegans homeobox gene ceh-43."

G Aspoeck, TR Buerglin

[International C. elegans Meeting, 1999]

The genome sequencing project sequenced a close ortholog of the Drosophila Distalless (Dll) and the vertebrate Dlx genes with 73% amino acid identity and a conserved intron position in the homeodomain (Stock et al.,1996; Wilson et al., 1994). Distalless/Dlx genes function in nervous system development and the distal outgrowth of appendages (antennae, limbs, legs etc.) in many species (Panganiban et al., 1997 and references therein). ceh-43::gfp reporter constructs are first expressed in some (not all) ABxxxxx cells and later in neuronal precursors and neurons of the head and tail, occasionally also in syncytial hypodermis. The expression matches with the staining of the antibody against the Drosophila DLL protein (Panganiban et al., 1997). RNAi using a cDNA clone kindly provided by Yuji Kohara is 100% embryonic or L1 lethal. Embryos develop normally until the 1.5x stage; subsequently, cells emerge from the buccal cavity and sometimes the tail, resulting in rupture of the embryo. In the few embryos that manage to hatch the pharynx and intestine are completely detached from the hypodermis. ceh-43::gfp expressing neurons and axons look normal in these animals. We believe that the RNAi phenotype is specific because two deficiencies that cover the gene show the described phenotypes. We think that the embryonic phenotype may either be due to incomplete closure of the anterior and posterior hypodermis, or because these points are too weak to withstand the internal pressure exerted during morphogenesis. jam-1::gfp (kindly provided by Jeff Hardin) expression shows that the hypodermal cell sheet looks normal before embryos rupture. The larval attachment problem might arise from a lack or weakness of the same cell-cell or cell-matrix adhesion molecules or mechanisms. References: Panganiban et al.(1997). Proc Natl Acad Sci USA 94:5162-6 Stock et al.(1996). Proc Natl Acad Sci USA 93:10858-63 Wilson et al.(1994). Nature 368:32-38

Martin Gutternigg et al. (2006) European Worm Meeting "Glycosidases of Caenorhabditis elegans involved in N-glycan processing"

Martin Gutternigg, Matthias Hackl, Ute Stemmer, Iain B. H. Wilson, Petra Geier, Gunter Lochnit, Ramona Ranftl, Katharina Paschinger, Verena Jantsch, Dorothea Lubich

[European Worm Meeting, 2006]

Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.

John Peloquin (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Screening natural product prototypes for activity using C. elegans"

John Peloquin

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Diamond V Mills has produced innovative and effective natural fermentation products for more than 64 years with our complex proprietary fermentation processes. Recently, Diamond V''s Innovation Program has generated a number of new product prototypes we must evaluate for biological activity and efficacy. Discovery and development of novel health and nutrition products for animal and human markets involves many economic and logistical challenges. Perhaps most importantly, common vertebrate models (in vitro and in vivo) are less than optimal when screening multiple prototypes in the early stages of product development. Because of these challenges, Diamond V developed a medium-throughput screen using Caenorhabditis elegans to help identify promising new product prototypes. Previously, C. elegans was shown to be an excellent whole-animal model for host-pathogen interactions (Moy et al. 2006) and to show the influence of natural products on aging (Wilson et al. 2005). As in these two studies, we presently rely on lifespan alteration as an indicator of biological activity when comparing our prototypes to appropriate controls. We further investigate the most promising of these prototypes in targeted vertebrate models (in vitro and in vivo) to better understand their biological and health effects. Our C. elegans model has helped Diamond V increase the efficiency and cost-effectiveness of the early stages of new product development. With our worm-based assay, we can reduce our use of vertebrate animals, ensure efficacy of our products, and increase the efficiency and cost-effectiveness of our new product development process.

Chris R Gissendanner et al. (2002) East Coast Worm Meeting "NHR-6: a nuclear receptor transcription factor required for ovulation."

Claude V Maina, Jianping Xiao, Marius Hoener, Tri Q Nguyen, Ann E Sluder, Chris R Gissendanner

[East Coast Worm Meeting, 2002]

The C. elegans genome encodes greater than 270 nuclear receptor (NR) genes (Sluder et al, 1999). Among these are fifteen genes that, based on comparative sequence analysis, encode nuclear receptor proteins conserved across metazoan phyla (Sluder et al, 1999). One of these conserved NR genes is nhr-6, which encodes the only C. elegans member of the NGFI-B NR sub-family (Wilson et al, 1992; Kostrouch et al, 1995; Sluder et al, 1999). We have initiated a functional analysis of nhr-6. Data from RNAi experiments and analysis of the deletion mutant lg6001 have established a role for nhr-6 in gonad development and/or function. Specifically, nhr-6(RNAi) and nhr-6(lg6001) animals have extremely low brood sizes and lay abnormally shaped eggs. Further analysis demonstrated that these phenotypes were due to an ovulation defect. Oocytes undergoing ovulation in lg6001 hermaphrodites frequently fragment entering the spermatheca. Only a fraction of the oocyte fragments become fertilized and many of these fail to develop properly. Oocyte fragments that remain in the gonad can become endomitotic (Emo phenotype). An nhr-6::GFP transgene is expressed in the spermatheca, suggesting a role for nhr-6 in spermathecal development and/or function. Currently, our analysis of nhr-6 is focused on the following: 1) a detailed characterization of nhr-6 expression and the ovulation defect observed in lg6001 mutants; 2) the in vivo significance of a conserved Akt/PKB phosphorylation site within the DNA binding domain of NHR-6; and 3) a comparative biochemical analysis of NHR-6 and an NGFI-B homolog (DiNHR-2) from the filarial nematode Dirofilaria immitis. The results of these investigations will be presented.

M Afaq Shakir et al. (2001) International C. elegans Meeting "Expression of C. elegans"

Bob Campbell, M Afaq Shakir

[International C. elegans Meeting, 2001]

Vertebrate reproduction and metabolism are tightly controlled by glycoprotein hormones that interact with leucine-rich repeat G-protein coupled receptors (LGRs). A C. elegans homolog of the vertebrate hormone receptors ( Ce LGR) was identified by the C. elegans Genome Sequencing Consortium (1). When expressed in mammalian cells Ce LGR constitutively activates production of the second messenger cAMP, but does not respond to vertebrate hormones (2). We have taken advantage of available tools to study the possible function of Ce LGR in growth and development of C. elegans . Ce LGR:: gfp reporter constructs were made using 4.5kb of the 5' promoter sequence and 12 of the 13 exons (including the predicted transmembrane regions). Following co-injection with the rol-6 transformation marker, GFP expression patterns were determined from the roller progeny. Strong expression was observed in many tissues associated with sensory projections to the external environment including nose sensillia, inner labial sheath and socket cells, CEP neurons, amphid sheath and socket cells, ADE and PDE neurons, vulva and male hook, spicule sensillum and ray sensory neurons. In addition, bright staining was also observed in lumenal cells of intestine and CAN neurons in both hermaphrodites and males. RNAi using a full-length coding sequence in N2 animals did not result in a detectable effect on development, general behavior, or reproduction. RNAi in the roller animals of Ce LGR::GFP strain resulted in marked suppression of intestinal GFP signal, but little or no change in expression at other sites. Studies are in progress to observe the possible phenotype using heat shock promoter fused to Ce LGR cDNA. Also, there are a number of mutants available in the vicinity of the Ce LGR locus that affect one or more cell types that expressed the Ce LGR::GFP reporter. Results from rescue studies of some of these mutants will be presented. 1. Wilson R., et al. Nature 368:32-38. (1994). 2. Kudo M., et al. Molecular Endocrinology 14:272-284. (2000).

Manser J (1996) West Coast Worm Meeting "CELL MIGRATION GENE mig-10 CAN ENCODE TWO PROTEINS WITH SIMILARITIES TO A FAMILY OF SH2 DOMAIN PROTEINS"

Manser J

[West Coast Worm Meeting, 1996]

Mutations in mig-10 cause incomplete embryonic migration of neurons CAN, ALM and HSN as well as shortened posterior excretory canals. To define more precisely the role of mig-10 in C. elegans development, we have begun a molecular characterization. We have identified mig-10 coding sequence by transformation rescue of mutant phenotypes and DNA sequencing of ct41 (amber) and e2527 (TTTCAA splice acceptor) mutant alleles. Genomic sequencing by us and the genome consortium (1) initially predicted a MIG-10 product of 650aa's containing a large region (~300 aa's) of similarity with a recently identified family of mammalian SH2 domain proteins, Grb7 and Grb10 (2,3). We call this region of similarity the GM domain (for Grb and Mig). The precise functions of Grb7 and Grb10 are not known, but current evidence supports a role in signal transduction: Grb7 is overexpressed in certain breast cancers where it is bound to the growth factor receptor HER2 (4), while Grb10 can interact with the insulin receptor (5). By sequencing cDNA clones and RT-PCR products, we have identified two SL1-trans-spliced mig-10 transcripts which contain different first exons. One transcript can encode the predicted 650aa MIG-10 product, the other a 667aa isoform. Neither predicted MIG-10 protein contains an SH2 domain, but both share with Grb7 and Grb10 the GM domain, a pleckstrin homology (PH) domain (within the GM domain) and proline-rich sequences. The PH domain is commonly found in signal transduction and cytoskeletal proteins, although its function remains controversial (6). Pro-rich sequences like those found in MIG-10 can bind SH3 domains found in many signaling and cytoskeletal proteins (6). Our mosaic analysis reveals that mig-10 acts cell nonautonomously during development of the excretory canals and suggests a possible focus within descendants of the AB lineage, possibly epidermis. We hypothesize that mig-10 may function in signal transduction and/or cytoskeletal organization outside of motile cells. We are currently undertaking additional experiments to clarify the role of mig-10, including immunolocalization of MIG-10 proteins. 1. Wilson, R. et al. 1994. Nature 368: 32-38. 2. Margolis, B. et al. 1992. Proc. Natl. Acad. Sci. 89: 8894-8898. 3. Ooi, J. et al. 1995. Oncogene 10: 1621-1630. 4. Stein, D. et al. 1994. EMBO J. 13: 1331-1340. 5. Hansen, H. et al. 1996. J. Biol. Chem. 271: 8882-8886. 6. Pawson, T. 1995. Nature 373: 573-580.