Ludewig, A.H. et al. (2017) International Worm Meeting "Pheromones control lifespan via sirtuins by generating oxidative stress."

Pulido, D.C., Schroeder, F.C., Thornton, S.N., Ludewig, A.H.

[International Worm Meeting, 2017]

Previous work showed that chemosensation of hermaphrodite produced pheromones ascr#2 and ascr#3 increases C. elegans adult lifespan and stress resistance (Ludewig 2013), whereas male-produced pheromones of then unknown identity shorten hermaphrodite lifespan (Gems 2000, Maures 2014). We show that the male-produced ascaroside ascr#10 underlies male-induced hermaphrodite progeria, and that pheromone-mediated lifespan increase and decrease depend on several different sirtuins (Ludewig, 2013; Ludewig, unpublished). Sirtuins, a family of NAD+ dependent histone deacetylases, have been implied as conserved regulators of lifespan and stress resistance in various species ranging from yeast to vertebrates. However, the mechanisms by which sirtuins influence lifespan are purely understood. We show that sensing of lifespan-increasing ascarosides transiently increases reactive oxygen species (ROS) levels in the mitochondria and requires the transcription factor skn-1/Nrf, a general regulator of stress responses. Antioxidants abolish the effects of ascarosides on lifespan, demonstrating that ROS production is necessary for sirtuin-dependent longevity. Mitochondrial ROS production is sirtuin-dependent and requires an intact nicotinamide catabolic pathway, indicating that ascaroside-mediated effects on lifespan depend on conversion of NAD+ into nicotinamide by sirtuins, similar to previously reported effects of sir-2.1 over expression (Schmeisser 2013). Our research uncovered an endogenous lifespan regulatory system based on antagonism of longevity-promoting and progeria-inducing ascarosides that act by modulating sirtuin-dependent ROS production. Furthermore, our work suggests that the intensely discussed variability of sirtuin dependent lifespan phenotypes in C. elegans may be due to the confounding effects of worm-produced ascarosides accumulating on experimental plates, in conjunction with other environmental stimuli activating skn-1/Nrf.

Ludewig, A.H. et al. (2017) International Worm Meeting "Larval crowding accelerates C. elegans development and reduces lifespan."

Ludewig, A.H., Schroeder, F.C., Panda, O., Thornton, S.N., Pulido, D.C., Artyukhin, A.B.

[International Worm Meeting, 2017]

Environmental conditions experienced during animal development have sustained impact on maturation and adult lifespan. We recently showed that in C. elegans developmental rate and adult lifespan depend on larval population density and that this effect is mediated by excreted small molecules. We established the time point of first egg laying as ultimate and easy accessible marker of developmental pace and for full maturity (= egg laying assay). We found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Furthermore, larval population density but not population density during adulthood, strongly affected C. elegans lifespan. Population-density dependent developmental acceleration (Pdda) was dramatically enhanced in fatty acid beta -oxidation mutants that are defective in the production of dauer-inducing ascarosides. In contrast, Pdda is abolished by synthetic ascarosides or steroid hormones that act on the nuclear hormone receptor DAF-12. However neither ascarosides nor any of the known steroid hormones are required for Pdda, instead it is mediated by another larval population density-dependent signal (Ludewig 2017, in press) that antagonizes the effects of the dauer pheromone. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.

Church DL et al. (1991) International C. elegans Meeting "SPECIFICITY OF TRANS-SPLICING IN C. ELEGANS"

Church DL, Wickens MP

[International C. elegans Meeting, 1991]

Trans-splicing attaches a 22-nt leader to the 5' end of 1020% of mRNAs in the nematode, C. elegans. We are studying the specificity of the reaction, by which a given pre-mRNA receives, predominantly, only one of two spliced leaders (SLl or SL2). Is the specificity for SLl vs. SL2 conferred by the sequence immediately upstrearn of the trans-splice acceptor site? We have constructed a chimeric gene (tra-2/unc-54) comprising 70 nt upstream of the first exon of a trans-spliced gene (tra-2, which predominantly receives SL2) followed by the 'body' of a nontrans-spliced gene (unc- 54). We have analyzed trans-splicing in a stable transgenic strain carrying the tra2/uncS4 construction. PCR analysis suggests that both the endogenous tra2 message and the tra2/unc54 RNA receive SLl and SL2. Primer extension studies indicate that most of the tra2/unc54 RNA is not trans-spliced.To study the 5' ends of the .trans-spliced tra2/unc54 RNAs, we have used the RACE ('rapid amplification of DNA ends') method. By cloning, screening and sequencing RACE products, we have verified that SLl is trans-spliced to the tra2 acceptor site of at least some tra2/unc54 RNAs. Taken together, the data suggest that the tra2 sequence contained in the tra2/unc54 RNA does not direct the preferential trans-splicing of SL2. (D.C. is supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research.)

Alicea, Bradly et al. (2015) International Worm Meeting "An experimental evolution model of C. Elegans adaptability."

Schroeder, Nathan, Androwski, Rebecca, Alicea, Bradly

[International Worm Meeting, 2015]

While the genetic architecture of C. elegans is well-studied, the adaptability of various strains and their reproductive fitness is less understood. To address this, an experimental-based model will allow for characterization of C. elegans adaptability amongst strains which may exhibit low reproductive potential and/or survivability under selection. Two experimental approaches representing within- and between-generation survivability (preconditioning and experimental evolution, respectively) will allow survivability to be assessed. Preconditioning [1] assesses adaptability within a generation using environmental selection, while experimental evolution [2] assesses adaptability via cross-generational artificial selection. For a given strain, populations grown from single founder individuals are repeatedly subject to genetic drift and then selected for fecundity (largest brood size). These results demonstrate how specific genotypes exhibit robustness in the face of adaptive constraints.Biological adaptability involves both survivability and reproductive fitness. Survivability is the degree to which a single founder worm can survive and produce viable clones (e.g. the number of generations constituting a single genealogy). Reproductive fitness is the mean population size for a given genealogy. In strains with high survivability, founder populations with high reproductive fitness should also generate subsequent founder populations with a high degree of survivability. Strains with high reproductive fitness but low overall progeny sizes may also exhibit high adaptability if selection results enhances adaptation to varying environmental conditions. While this model does not control for stochastic effects and demographic constraints [3], it does allow for genealogies with consistently high survivability [4].1. Calabrese, E.J. et.al, Tox App Pharm, 222(1), 122-128 (2007).2. Kawecki, T.J. et.al, Trends Ecol Evol, 27(10), 547-560 (2012).3. Szendro, I.G. et.al, PNAS, 110(2), 571-576 (2012).4. Wahl, L.M., Krakauer, D.C., Genetics, 156(3), 1437-1448 (2000).

Song, Myung Chul et al. (2015) International Worm Meeting "C09F5.1, the BRICHOS containing protein functions in stress resistance."

Han, Ching Tack, Song, Kyung Hee, Song, Myung Chul, Kim, Sung Hee

[International Worm Meeting, 2015]

C09F5.1, one of several genes expressed differentially in heat sensitive C. elegans mutants was isolated. To confirm whether C09F5.1 is involved in heat stress response, the expression pattern of C09F5.1 in heat stress condition was analyzed, showing the induction of expression of mRNA and protein by 33degC heat treatment in wild type N2. The induction of C09F5.1 protein by heat treatment delayed in heat shock transcription factor-1(HSF-1) mutants (sy441) compared to N2 implying that the HSF regulates the transcription of C09F5.1 as HSPs. C09F5.1 RNAi-treated worms became more sensitive to heat stress than control worms. These results indicate that C09F5.1 transcribed by HSF-1 may function in heat stress response. To find the putative functional domain of C09F5.1, the computational analysis of C09F5.1 protein was done. The results showed that C09F5.1 consists of an N-terminal region, a type II transmembrane domain and a BRICHOS domain. The various BRICHOS proteins are known related with familial dementia (BRI2), chondrosarcoma (Chondromodulin-1) and interstitial lung disease (SP-C). BRICHOS domain containing proteins function as molecular chaperone(Sanchez-Pulido, Devos et al. 2002). Furthermore recent studies revealed that BRICHOS domain directly interacts with Abeta42 peptide and delays amyloid-beta fibril formation(Willander, Presto et al. 2012, Hermansson, Schultz et al. 2014). When the C09F5.1 was expressed in animal cells (293T), the C09F5.1 protein directly interacted with Abeta42. In order to identify the chaperone activity of C09F5.1 on abnormal protein aggregation, we generated transgenic worms expressing each of the C09F5.1 protein with full length, BRICHOS deletion and N-terminus deletion in Abeta42 expressing worm. The transgenic worms expressing the BRICHOS domain deleted mutant showed the increased survival rate compared to worms expressing Abeta42 solely. These results suggest C09F5.1 BRICHOS containing protein has chaperonin functions under stress but not through the BRICHOS domain only. Currently we are studying the mechanism of protection from amyloid toxicity by the N-terminus of C09F5.1.

Cheung, V.H.W. et al. (2013) International Worm Meeting "Characterizing the individual roles of CSR-1 isoforms across development in Caenorhabditis elegans."

Cheung, V.H.W., Claycomb, J.M.

[International Worm Meeting, 2013]

The nematode C. elegans expresses 26 Argonaute (AGO) proteins which interact with different small RNAs in the worm to regulate of gene expression1. Interestingly, only one AGO has been shown to be essential: CSR-1 (chromosome segregation and RNAi deficient)1. Loss of CSR-1 causes multiple defects in the animal: impaired ability to raise an RNAi response, abnormal germline morphology, chromosome mis-segregation, and embryonic lethality1,2,3. Various studies have explored roles for CSR-1 in modulating centromeric chromatin2, RNAi1,2, histone mRNA maturation4, small RNA-independent translational regulation5 and meiotic silencing of unpaired chromsomes3. However, these studies have neglected to address the contributions of two distinct isoforms of CSR-1 to overall CSR-1 function. Initially identified by Claycomb et al., the two CSR-1 isoforms differ by presence/absence of a 5'exon encoding an RG-rich motif and have differential expression across developmental stages2. We are currently using an integrated approach to examine isoform-specific functions across C. elegans tissues and development. Epitope tagged transgenes for each isoform will be used to characterize each form of CSR-1. Preliminary immunolocalization studies have revealed a differential localization pattern for the long isoform of CSR-1 in mitotic embryos distinct from the overall pattern of CSR-1 localization which was previously not appreciated. These observations will be further explored and supplemented with analysis of small RNA and protein binding partners of each isoform to determine the subset of isoform-specific cofactors functioning across C. elegans development. 1. Yigit, E. et al. Cell 127, 747-757 (2006). 2. Claycomb, J.M. et al. Cell 139, 123-134. (2009). 3. She, X., Xu, X., Fedotov, A., Kelly, W.G., & Maine, E.M. PLoS Genet. 5, e1000624. (2009). 4. Avgousti, D.C., Palani, S., Sherman, U., & Grishok, A. EMBO J. 31, 3821-3832 (2012). 5. Friend, K. et al. Nat. Struct. Mol. Biol. 19, 176-184 (2012).

Wesley C Lindsey et al. (1999) International C. elegans Meeting

Wesley C Lindsey, Steven W L'Hernault

[International C. elegans Meeting, 1999]

During spermatogenesis, the ER/Golgi derived, fibrous body-membranous organelles (FB-MOs) play a central role in the proper segregation of cytoplasmic components into the spermatids. Normally, FB-MO's arise in the developing spermatocyte. The FB first appears as short fibers of Major Sperm Protein (MSP) in the body of the MO. The FB-MO complexes increase in size as the spermatocyte develops and then segregate to the budding spermatid, transporting material required for these spermatids to undergo spermiogenesis. After the spermatids bud off of the residual body, the FB-MO complex disassociates. MSP fibers depolymerize to reassemble in the pseudopod, and the MO's fuse with the plasma membrane, releasing its contents onto the cell surface and forming a permanent fusion pore. Mutation of spe-10 prevents the proper function of the FB-MOs and spermatogenesis is disrupted. The FB-MO complexes break down prematurely, the fibrous bodies are left behind in the residual body and do not disassemble as in wild type spermatids. The membranous organelles segregate to spermatids, but then become swollen and vacuolated. Associated with these defects is improper localization of the nucleus in spermatids. So, resulting spermatozoa have vacuolated MO's, short pseudopods, and they fail to translocate. Previous work positioned spe-10 on chromosome V, covered by deficiency sDf35. Three factor mapping places it between unc-42 and sma-1 . (Shakes, D.C., and Ward, S. 1989. Dev. Biol . 134, 307-316.) On the physical map, this region is well represented by overlapping YACs and cosmids. Therefore, we are attempting to clone spe-10 by transgenic rescue. We have successfully rescued the spe-10 mutant phenotype by YACs containing wild-type DNA that covers the region between unc-42 and sma-1 . We are now attempting cosmid rescue, in order to narrow down this region. Progress towards the cloning of spe-10 will be reported.

Darryl Conte Jr et al. (2004) East Coast Worm Meeting "The germ granule protein PGL-1 is required for efficient RNA interference"

Darryl Conte Jr, Craig C Mello, Yingdee Unhavathaya

[East Coast Worm Meeting, 2004]

In C. elegans , very small quantities of dsRNA are sufficient to generate RNA interference not only in the animal that is exposed to dsRNA but also in subsequent generations (1, 2). These findings indicate that amplification and inheritance of a silencing agent(s) (such as siRNA) are important to the mechanism of RNAi. P-granules, the worm equivalent of germ granules, are expressed in the maternal germline, deposited into early embryos and are implicated in translational regulation of maternal mRNAs. Therefore, we hypothesized that the germline P-granules of C. elegans may be important for the transmission of a silencing factor and hence for RNAi. An examination of mutations in known P-granule components revealed that the PGL-1 protein is required for RNAi. siRNAs failed to accumulate in pgl-1 mutant worms exposed to dsRNA targeting a maternal mRNA, while siRNAs appeared to accumulate in the embryos of wild-type worms. Remarkably, animals challenged with dsRNA targeting zygotic mRNAs expressed in somatic tissues produced progeny with a reduced penetrance of RNAi phenotypes. These data are consistent with the hypothesis that PGL-1 may be important for efficient transmission of a silencing agent to the progeny of animals exposed to dsRNA. PGL-1 encodes a protein with an RGG-box found in RNA-binding proteins and is required for fertility in C. elegans (3). An attractive scenario is that PGL-1 interacts with and transmits siRNA to the progeny of animals exposed to dsRNA. Alternatively, PGL-1 may regulate the activity of the RNAi pathway by modulating the accessibility, localization or expression of RNAi pathway components or intermediates. We will present our recent efforts to elucidate the function of PGL-1 in RNAi and the relationship to the role of PGL-1 in development. 1) Fire et al. (1998) Nature 391:806-811 2) Grishok, Tabara and Mello (2000) Science 287:2494-2497 3) Kawasaki et al. (1998) Cell 94:635-645 This work is funded by National Institutes of Health grants GM058800 (to C.C.M.) and GM063348 (to D.C.)

L'Hernault SW et al. (2000) East Coast Worm Meeting "The spe-10 and spe-21 genes encode sperm transmembrane proteins that contain a DHHC-CRD zinc finger motif"

L'Hernault SW, Lindsey WC

[East Coast Worm Meeting, 2000]

Spermatogenesis in C. elegans is a good model for studying asymmetric partitioning of organelles during cellular differentiation. This one-way differentiation pathway begins with a stem cell and ends with a terminally differentiated cell, the spermatozoa. Like most organisms, C. elegans spermatids are compact cells that eliminate unnecessary components during their differentiation. This occurs because certain components are actively retained within spermatids while others are eliminated by placement within the acellular cytoplast (the residual body). We are interested in how asymmetric partitioning participates in the development of this specialized cell and how this process is genetically controlled. The C. elegans spe-10 and spe-21 genes affect spermatogenesis so that abnormally small nonfunctional spermatozoa are found in these mutants. Mutations in spe-10 disrupt the proper function of ER/Golgi derived, fibrous body-membranous organelles (FB-MOs) that are known to play a central role in the proper segregation of cytoplasmic components into spermatids (Shakes, D.C., and Ward, S. 1989. Dev. Biol. 134, 307-316.). The details of the mutant spe-21 phenotype are currently being investigated by electron microscopy. We have positionally cloned both of these genes, and they both encode 3-4 pass transmembrane proteins that contain a cysteine-rich domain (CRD) with a DHHC zinc finger motif (DHHC-CRD), and mutantations in either gene appear sperm specific in their phenotype. The DHHC-CRD zinc finger motif is a recently identified motif that is present in organisms from yeast to man, and this motif is thought to be involved in protein-protein interactions. This is the first known instance of two genes encoding a DHHC-CRD zinc finger motif that affect the same cellular pathway. An intriguing speculation is that Spe-10 and Spe-21 interact via this motif to form a heterodimer. Presently, we are continuing our analysis of spe-10 and spe-21 by generating allelic series, electron microscopy, and raising peptide-specific antibodies to look at the subcellular localization of the proteins. Such analysis will broaden our understanding of asymmetric partitioning and allow us to further elucidate the spermatogenesis pathway.

Weaver DC et al. (1996) East Coast Worm Meeting "Unc-62 is (likely to be) a regulator of hypodermal differentiation"

Wood WB, Weaver DC, Edgar LG

[East Coast Worm Meeting, 1996]

The unc-62 gene is currently defined by the embryonic lethal allele, s472, two alleles that show partial maternal effect embryonic or Ll lethality, ct344 and t2012 (kindly provided by Richard Feichtinger and Ralf Schnabel), and a weaker allele that causes an Unc phenotype, e644. The dead animals always display severely disorganized body morphology, most notably in the posterior. See ref. 1 and our poster for a more complete discussion of the genetics. We used the anti-LIN26 antibody (which stains all hypodermal nuclei) and the MH27 antibody (which outlines tight junctions between cells) to examine the distribution and number of hypodermal cells at various stages of embryogenesis, particularly at the start of morphogenesis. Self progeny embryos from mothers homozygous for either unc-62(ct344) or unc-62(ct2012) have about 30 fewer hypodermal cells than wild type and mislocalize the P, V, and C derived hypodermal cells. Other hypodermal cells appear to be largely normal, consistent with correct formation and positioning of the buccal opening, rectal opening, and tail spike. Using 4D Nomarski microscopy, we are attempting to identify alterations in cell lineage that could be responsible for the observed defects. Preliminary results of this analysis will be presented. unc-62(ct344) has been rescued by two overlapping subclones defining a region of 15 kb. This region has been sequenced by the Sequencing Consortium, and GeneFinder analysis predicts 5 transcripts in the rescuing region. One appears to be a novel transposon and is being excluded from consideration. The other four transcripts comprise two similar pairs of nearly identical genes. At least one member of each gene pair is transcribed based on the presence of the corresponding cDNA in the Okema embryonic library. The chromosomal organization of these two predicted gene pairs suggests that an ancient inverted duplication generated the original members of each pair and a second, more recent, inverted duplication arose to produce the current four copies. All of these genes display significant similarity to a growing family of proteins that have members in yeast, slime mold, guinea pig, and human. The human homolog (pl9Skp1) is directly involved in regulating the activity of CycACDK2 kinase and thereby controls entry into S phase (ref. 2). Northern analysis shows that these genes are highly expressed in early embryogenesis and expressed in adults (though not in the germ line). We are sequencing the mutant alleles to identify the molecular lesions and determine which of these genes actually encodes the unc-62 activity. 1. Weaver, D.C., et al., WBG 13(5):68. 2. Zhang, H. et al. Cell 82: 915925 (1995).