Masami Shiimori et al. (2010) East Asia Worm Meeting "Role of Y14 in C. elegans germline sexual differentiation"

Hiroshi Sakamoto, Masami Shiimori, Kunio Inoue

[East Asia Worm Meeting, 2010]

The exon junction complex (EJC) deposited after splicing on mRNA is thought to be important for the mRNA fate. Of the EJC components, mammalian Y14 is retained on mRNA after export to the cytoplasm, implying that Y14 may have crucial roles in determining the mRNA fate in the cytoplasm. We reported previously that C. elegans Y14 is required for switching the germline sexual differentiation to the female mode in adult hermaphrodites. However, little is known so far about the mode of action of Y14 in the germline sexual differentiation. Using RNA interference (RNAi), we found that Y14 gene functions downstream of her-1 and upstream of fem-3 in the cascade of genes involved in the germline differentiation. Since tra-2 is known to function in the same genetic position and promote oogenesis, we examined tra-2 expression under the Y14(RNAi) background. In situ hybridization showed that tra-2 mRNA was accumulated in the nuclei of Y14(RNAi) animals, whereas a control mRNA was normally exported to the cytoplasm. We also observed accumulation of intron-containing mRNAs and abnormally processed mRNAs in Y14(RNAi) animals. In addition, intron-containing mRNAs are exported to cytoplasm in Y14(RNAi) animals. However, several lines of evidence indicate that Y14(RNAi) phenotype is not caused by the defect of the nonsense mediated mRNA decay (NMD) pathway, suggesting that Y14 is involved in the different aspect of mRNA metabolism. Intriguingly, immunostaining showed that TRA-2 intracellular domain was normally localized in the nuclei of somatic cells, but was retained on the cytoplasmic membrane in the germline in Y14(RNAi) animals. Molecular mechanisms underlying the tra-2 regulation by Y14 will be discussed.

Yasuaki Habara et al. (2004) East Asia Worm Meeting "Isolation of deletion mutants for the mrg-1 gene which is required for germline development in C. elegans"

Teruaki Takasaki, Toshinobu Fujiwara, Kiyoji Nishiwaki, Hiroshi Sakamoto, Kunio Inoue, Yasuaki Habara

[East Asia Worm Meeting, 2004]

Teruaki Takasaki et al. (2005) International Worm Meeting "MRG-1, a C.elegans chromodomain protein is essential for proper gene expression in the primordial germ cells"

Kunio Inoue, Kiyoji Nishiwaki, Hiroshi Sakamoto, Toshinobu Fujiwara, Yasuaki Habara, Teruaki Takasaki, Jun-ichi Nakayama

[International Worm Meeting, 2005]

During metazoan development, cell-type-specific gene expression is essential for cellular differentiation. In many animals, establishment of the primordial germ cell (PGC) depends on several maternal factors. However, how they regulate the germline gene expression is not fully understood. We previously reported that a novel maternal protein, MRG-1, is required for germline development in C.elegans (1). MRG-1 contains a chromodomain, which is often found in many chromatin-associated proteins, suggesting that MRG-1 is involved in chromatin remodeling in the germline. Here we show that MRG-1 is localized to nuclei and associated with chromosomes. In early embryos, MRG-1 is observed in all nuclei. In late embryos, MRG-1 staining is restricted to the nuclei of two PGCs. These observations suggest that MRG-1 has a function in chromatin remodeling in PGCs. To investigate whether MRG-1 is involved in germline gene expression in PGCs, we examined the expression of PGL-1, a component of P-glanules, which is known to be constitutively expressed in the germline. We found that mrg-1(RNAi) larvae have defects in zygotic expression of PGL-1 in PGCs. Our results suggest that MRG-1 plays an important role to ensure the proper gene expression in PGCs through chromatin remodeling. We also attempted to isolate the mrg-1 gene disruption mutants. Using a TMP/UV-based deletion mutant library, we screened 2,400,000 haploid genomes and found one candidate for the mrg-1 mutation containing 450bp deletion between exon 1 and exon 2, which may cause frameshift in the coding region. National BioResource Project (NBRP) also isolated another mrg-1 deletion mutant (2). The mutation (tm1227) deletes the 401bp region spanning the promoter region and a half of exon 1 of mrg-1. Characterization of these deletion mutants is currently in progress.(1) Fujita et al., Mech. Dev. 114, 61-69, 2002.(2) NBRP homepage, http://shigen.lab.nig.ac.jp/shigen/nbrp/nbrp.jsp

Teruaki Takasaki et al. (2007) International Worm Meeting "MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in C. elegans."

Zheng Liu, Yasuaki Habara, Kunio Inoue, Susan Strome, Jun-ichi Nakayama, Teruaki Takasaki, Kiyoji Nishiwaki, Hiroshi Sakamoto

[International Worm Meeting, 2007]

In most sexually reproducing animals, germ cells are the only cells that can give rise to the next generation, and thus are responsible for perpetuation of species. To serve this role, germ cells must preserve the properties of totipotency and immortality. Understanding the molecular mechanisms that ensure the special properties of germ cells remains a central issue in biology. Mammalian mortality-factor-related MRG15 is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the C. elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). In contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ vs. soma distinction.

William C Chen et al. (2003) International Worm Meeting "A screen for suppressors of the enhanced susceptibility to pathogen mutant, sek-1."

William C Chen, Man-Wah Tan

[International Worm Meeting, 2003]

MAPK signaling pathways are activated under various stress conditions and have been shown to be required for innate immunity in Caenorhabditis elegans and Arabidopsis thaliana (Asai et al. 2002). In C. elegans, mutants for nsy-1, a MAPKKK, and sek-1, a MAPKK, are highly susceptible to the pathogen, Pseudomonas aeruginosa PA14 (Kim et al. 2002). In addition, RNAi of the p38 MAPK, pmk-1, also led to enhanced susceptibility, but mutants for unc-43, which activates NSY-1 to regulate AWC cell fate, are not susceptible (Sagasti et al. 2001). Arsenic has also been shown to activate p38 MAPK signaling in mammalian cultured cells, and exposure of C. elegans to sodium arsenite induces pmk-1. Furthermore, sek-1 mutants are hypersensitive to sodium arsenite, and nsy-1 mutants are partially susceptible to arsenite treatment. However, unc-43 mutants are not sensitive to arsenite (Inoue et al. 2002). These findings mirror those of the susceptibility to PA14. In order to identify downstream targets of SEK-1 MAPK signaling which may be involved in the host response to PA14, sek-1 worms were EMS mutanenized and screened for suppressors of the sodium arsenite susceptibility. Mutants identified from the screen were then tested for possible resistance to PA14 in order to determine the degree of overlap between the MAPK-mediated response to arsenic and to pathogen. Three mutants resistant to arsenic and PA14 have been identified thus far and further studies will include molecular identification and characterization of these mutants. Asai, T., et al. (2002). "MAP kinase signaling cascade in Arabidposis innate immunity." Nature 415(6875): 977-83. Inoue, H., et al. (2002). "C. elegans p38 MAPK cascade mediates arsenical stress response." Worm Breeder's Gazette 17(2): 31; Kim, D. H., et al. (2002). "A Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Innate Immunity." Science 297(5581): 623-626; Sagasti, A., et al. (2001). "The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates." Cell 105(2): 221-32.

Patterson GI et al. (1996) East Coast Worm Meeting "Identification of new genes in dauer/TGF-beta signalling"

Ruvkun GB, Patterson GI

[East Coast Worm Meeting, 1996]

We are interested in understanding the mechanism by which food and pheromone signals are transduced to signal dauer arrest or non-dauer development. Don Riddle's group has identified a TGF-beta homologous pathway that is involved in this signal transduction process. daf-7 encodes a peptide ligand, and daf-1 and daf-4 encode transmembrane serine/threonine kinases that may be receptors for the daf-7 protein. Recent work by Takao Inoue, Annette Estevez and our laboratory (see abstract by A. Koweek and G. Patterson) has identified a family of genes that are likely to be involved in the signal transduction from these receptors; daf-8, daf-14, and daf-3 are all homologues of the drosophila gene Mad, which transduces signals from serine/threonine kinase receptors. The mechanism by which these receptors and Mad homologues transduce signal is unknown; we are identifying new genes that act in this pathway. In previous screens for suppressors of daf-7, the genes daf-3, daf-5 and daf-12 were identified by D. Riddle. We have identified four new genes that suppress daf-7. Two of the genes were identified by mutations that strongly suppress daf-7; the other two show weak suppression. We are trying to clone a strong suppressor called scd-1 (Suppressor of Constitutive Dauer formation--pronounced "scud"). We isolated two alleles as suppressors of daf-7; Takao Inoue provided two alleles he isolated as suppressors or daf-14 and daf-8. We mapped the gene between lin-2 and unc-9 on X, and we are doing finer mapping as well as injecting candidate cosmids for rescue. We have also begun a screen for suppressors of daf-3. We are mutagenizing a daf-7 daf-3 strain (daf-7 is dauer constitutive, daf-3 is dauer defective, and the double mutant is dauer defective). We will screen for dauers at 25 degrees C. daf-2, daf-19 and daf-23 are the known dauer constitutive mutants epistatic to daf-3. We hope to get new mutations that, while not dauer constitutive on their own, suppress the daf-3 mutation and allow the daf-7 mutation to force dauer formation. We will screen with both a null daf-3 allele and a partial loss-of function daf-3 allele.

H Qadota et al. (2004) European Worm Meeting "MUSCLE SPECIFIC FUNCTIONS OF EARLY EXPRESSED LETHAL GENES"

T Hikita, H Qadota, K Kaibuchi

[European Worm Meeting, 2004]

Muscle cells are well differentiated and have highly designed architecture. Many genes contribute to muscle cell differentiation and morphogenesis. To gain an insight into regulatory mechanism of muscle differentiation and morphogenesis, functional analysis of genes expressed in muscle cells using chromosomal deletion or RNAi knock down is a powerful approach. However, since some genes are also expressed in other tissue cells, it is hard to know defects in muscle cell caused by primary and cell autonomous effects or by secondary and non-cell autonomous effects. Furthermore, RNAi of some early expressed genes caused embryonic lethal phenotype, so it is impossible to investigate effect of RNAi knock down of such genes on muscle differentiation and morphogenesis. To avoid these problems, mosaic analysis or tissue specific rescue experiment is required. But, it takes time and effort to construct mosaic animals or tissue specific rescue lines. Previously we reported tissue specific RNAi system and established hypodermis specific RNAi system (Inoue et al., 2000 Japanese Worm Meeting) using tissue specific rescue of rde-1, RNAi resistant, mutant worms. To examine effect of gene knock down on muscle morphogenesis efficiently, we established muscle specific RNAi system and applied this system on early expressed and embryonic lethal genes.

Honnen, Sebastian et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "vang-1 modulates life span and stress resistance via insulin/IGF-1-like signaling"

Honnen, Sebastian, Kampkotter, Andreas, Bossinger, Olaf, Buchter, Christian, Schroder, Verena

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in C. elegans [1-3]. The developmental function of this pathway is to mediate the coordinated orientation of cells or structures within the plane of an epithelium or to regulate the organization of cell-cell intercalation that is required for correct morphogenesis [4, 5]. Here, we describe a novel role of VANG-1, the only ortholog of Strabismus/Van Gogh in C. elegans. We show that two alleles of vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. In addition, vang-1 shows enhanced resistance to thermal-, oxidative-stress and decreased lipofuscin accumulation. Life span extension in vang-1 depends on the insulin/IGF-1 like receptor DAF-2 and DAF-16/Foxo transcription factor. In addition to the modulation of life span, we also observed an extension of reproductive span but a decreased number of progeny, suggesting that VANG-1 links these crucial processes during nematode development. 1.Green, J., Inoue, T., and Sternberg, P. (2008). Opposing Wnt pathways orient cell polarity during organogenesis. Cell 134, 646-656. 2.Wu, M., and Herman, M.A. (2006). A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans. Dev Biol 293, 316-329. 3.Hoffmann, M., Segbert, C., Helbig, G., and Bossinger, O. (2009). Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling. Dev Biol. 4.Wang, Y., and Nathans, J. (2007). Tissue/planar cell polarity in vertebrates: new insights and new questions. Development 134, 647-658. 5.Wu, J., and Mlodzik, M. (2009). A quest for the mechanism regulating global planar cell polarity of tissues. Trends Cell Biol 19, 295-305.

Koweek AR et al. (1996) East Coast Worm Meeting "DAF-3 ENCODES A DOT GENE THAT ACTS OPPOSITELY TO TGF-BETA"

Koweek AR, Ruvkun GB, Patterson GI

[East Coast Worm Meeting, 1996]

We are using the dauer larva as a model to investigate how animals couple environmental cues to a specific developmental program. Several of the genes involved in the dauer decision are homologs of transforming growth factor beta (TGF-beta) signal transduction proteins. These genes may act during the formation and wiring of the neurons involved in dauer formation or may function in the actual transduction of the pheromone signal. We are interested in the downstream effectors of the TGF-beta signal transduction pathway, so we cloned daf-3, which suppresses the phenotype of mutations in daf-1 and daf-4, the TGF-beta receptors. We mapped daf-3 to the region between aex-3 and unc-1 and identified B0217 as a rescuing cosmid. This region had been sequenced by the Genome Sequencing Project. This sequence allowed us to locate on B0217 a member of the DOT family (downstream of TGF-beta). The first DOT gene identified was the Drosophila gene Mad, which is required for signaling by dpp, a TGF-beta homolog. The DOT family is a family of proteins consisting of two conserved domains ("domains I and II"), separated by a proline-rich domain. Nothing is known about the function of these domains. We have identified two daf-3 alleles with mutations in the DOT gene: mg90 is a homozygous viable deletion that removes all of daf-3, and mg125 is a missense mutation in a conserved residue of domain I. In contrast, eleven out of twelve mutations identified in the other DOT family mutants are located in domain II. Eight of these mutations lie in a small hotspot of 45 amino acids. We have sequenced this hotspot in nine daf-3 alleles, and none of them has a mutation in this hotspot. We have constructed a 15 kb rescuing subclone predicted to contain only the DOT gene. We have also characterized full-length cDNAs of daf-3, and we have found evidence of alternative splicing at the 5' end. Analysis of a daf-3 expression construct indicates that daf-3 is expressed in most neurons and in the intestine, but not elsewhere in the animal; thus daf-3 may be required for proper sensation of pheromone signal or proper transduction of the signal through the nervous system. We are planning to examine the localization and expression of daf-3 in various mutant backgrounds, as well as in the presence or absence of pheromone to try to understand the mechanism of signal transduction. The fact that daf-3 suppresses daf-1 and daf-4 indicates that daf-3 acts oppositely to all of the known members of the DOT family, which act as positive transducers of the TGF-beta signal. These positively acting DOTs include daf-8 and daf-14, which have a phenotype like that of daf-1 and act to positively transduce a daf-7 signal (personal communication, A. Estevez and D. Riddle, T. Inoue and J. Thomas). The prevalence of mutations in domain II in many DOT members, particularly in the hotspot, suggests that this region is important for the function of these positively acting DOT genes. Consistent with this model, daf-8 mutations affect domain II (personal communication, A. Estevez and D. Riddle), and daf-14 mutations affect the hotspot in domain II (personal communication, T. Inoue and J. Thomas). daf-3 mutations, on the other hand, do not cluster in the hotspot, and the location of the point mutation in daf-3 indicates that domain I is important for the function of daf-3. The combination of positively and negatively acting DOT genes in a single genetic system will allow us to investigate the biochemical function of the DOTs.

Honnen, S. et al. (2011) International Worm Meeting "C. elegans vang-1 modulates life span and stress resistance via insulin/IGF-1-like signaling."

Kampkotter, A., Buchter, C., Schroder, V., Bossinger, O., Honnen, S.

[International Worm Meeting, 2011]

The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in the nematode Caenorhabditis elegans (1-3). The developmental function of this pathway is to coordinate the orientation of cells or structures within the plane of an epithelium or to organize cell-cell intercalation required for correct morphogenesis (4-5). Here, we describe a novel role of VANG-1, the only C. elegans ortholog of the conserved PCP component Strabismus/Van Gogh. We show that two alleles of vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. In addition, vang-1 mutants show enhanced resistance to thermal and oxidative stress and decreased lipofuscin accumulation. Life span extension in vang-1 mutants depends on the insulin/IGF-1 like receptor DAF-2 and DAF-16/Foxo transcription factor. This is the first time that a correlation between a key player of the PCP pathway and the modulation of life span and stress resistance has been established. references: 1.Green, J., Inoue, T., and Sternberg, P. (2008). Opposing Wnt pathways orient cell polarity during organogenesis. Cell 134, 646-656. 2.Wu, M., and Herman, M.A. (2006). A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans. Dev Biol 293, 316-329. 3.Hoffmann, M., Segbert, C., Helbig, G., and Bossinger, O. (2010). Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling. Dev Biol. 4.Wang, Y., and Nathans, J. (2007). Tissue planar cell polarity in vertebrates: new insights and new questions. Development 134, 647-658. 5.Wu, J., and Mlodzik, M. (2009). A quest for the mechanism regulating global planar cell polarity of tissues. Trends Cell Biol 19, 295-305.