Shinya, Ryoji et al. (2015) International Worm Meeting "Unusual sex determination systems in Bursaphelenchus okinawaensis and B. xylophilus."

Chen, Anthony, Hasegawa, Koichi, Kikuchi, Taisei, Tsai, Isheng J., Sternberg, Paul W., Shinya, Ryoji

[International Worm Meeting, 2015]

Sex determination mechanisms are unexpectedly diverse in organisms and change rapidly during evolution. Among nematodes, molecular sex determination mechanisms have been well characterized in a few species of Caenorhabditis. Caenorhabditis species have an XO male/XX hermaphrodite or female sex determination system, and it is thought that the XX/XO sex determination system is predominant among nematodes. Although XX/XY or environmental sex determination systems have been also suggested for some nematodes, the molecular mechanisms of these systems are still unclear. Recently we have studied the sex determination mechanisms of the hermaphroditic fungal feeding nematode, Bursaphelenchus okinawaensis, and the gonochoristic plant-parasitic nematode, B. xylophilus. First, we observed B. okinawaensis and B. xylophilus chromosomes to constrain their sex determination mechanisms. The chromosome number is the same between males and hermaphrodites/females of B. okinawaensis and B. xylophilus, with all of the chromosomes being apparently identical, ruling out an XO/XX system. To investigate possible environmental effects on their sex determination, we scored the ratio of sex under the various types of culture conditions; however, no significant difference in sex ratio was observed. Although we have thoroughly investigated the possibility of other types of sex determinations that have been reported in other organisms, our results suggested that the sex determination of B. okinawaensis and B. xylophilus does not correspond to any of the currently known mechanisms. We thus performed a comparative genomic analysis between males and hermaphrodites/females using next-generation sequencing to investigate any genomic differences at higher resolution. Our results confirmed that males and hermaphrodites/females have the same number of chromosomes. We are still working on the analysis of the sequence data to determine whether there is any difference in the nuclear and mitochondrial genomes between males and females/hermaphrodites. In this presentation, we will discuss the unique sex determination mechanisms of B. okinawaensis and B. xylophilus based on all of these of experiments. The sex determination system of the nematodes seems to be more diverse than we thought. .

Jun-ichi Aikawa (2001) International C. elegans Meeting "Molecular characterization of heparan sulfate/heparin N-deacetylase/N-sulfotransferase in worms"

Jun-ichi Aikawa

[International C. elegans Meeting, 2001]

Heparan sulfate binds and activates a large variety of growth factors, enzymes and extracellular matrix proteins. These interactions largely depend on the specific arrangement of sulfated moieties and uronic acid epimers within the chains. These oligosaccharide sequences are generated in a step-wise manner, initiated by the formation of a linkage tetrasaccharide which is then extended by copolymerization of alternating a1,4GlcNAc and b1,4GlcA residues. As the chains polymerize, they undergo a series of sulfation and epimerization reactions. The first set of modifications involves the removal of acetyl units from subsets of GlcNAc residues, and the addition of sulfate groups to the resulting free amino groups. These reactions are catalyzed by a family of enzymes designated as GlcNAc N-deacetylase/N-sulfotransferases (NDST), since they simultaneously. Four members of the family have been identified in vertebrates, with single orthologs present in Drosophila and C. elegans. We have revealed tissue-specific expression pattern and unique enzymatic properties of these four isozymes1,2). In fly, loss of NDST (sulfateless) results in unsulfated chains and defective signaling by multiple growth factors and morphogens. I reconstituted cDNA for worm NDST from EST clones and 5' RACE products. Enzymatic activities will be discussed. 1) Aikawa, J. & Esko, J. D J. Biol. Chem. 274, 2690-2695 (1999) 2) Aikawa, J., Grobe, K., Tsujimoto, M. & Esko, J. D J. Biol. Chem. 276, 5876-5882 (2001)

Husson, Steven J. et al. (2011) International Worm Meeting "C. elegans mutants defective in neuropeptide amidation enzymes are abnormal in egg-laying behavior."

Landuyt, Bart, Schoofs, Liliane, Gottschalk, Alexander, Horvitz, H.Robert, Temmerman, Liesbet, Husson, Steven J., Ringstad, Niels, Meelkop, Ellen

[International Worm Meeting, 2011]

Egg laying has mainly been studied at the behavioral, neuronal and neurochemical levels, but little is known about the biochemical control of the relevant neuropeptidergic signaling systems. Biosynthesis of endogenous peptides requires processing enzymes, such as proprotein convertase 2, which is encoded by egl-3 (1, 2), and a carboxypeptidase encoded by egl-21 (3, 4). Mutants defective in these genes have egg-laying defects, consistent with the finding that FMRFamide-like peptides (FLPs) have been linked to egg laying behavior. C. elegans enzymes that carry out the last step in the production of biologically active peptides, the carboxy-terminal amidation reaction, have not been characterized. This multistep reaction involves hydroxylation of the glycine a-carbon by a peptidyl-a-hydroxylating monooxygenase (PHM), followed by a cleavage reaction performed by peptidyl a-hydroxyglycine a-amidating lyase (PAL) to generate a glyoxylate molecule and the a-amidated peptide. In vertebrates, both enzymatic activities responsible for the carboxyterminal amidation reaction are contained in one bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM). By contrast, invertebrates generally express two separate enzymes encoded by two different genes. Here we report the identification and characterization of C. elegans amidating enzymes using bioinformatics to identify candidate genes and mass spectrometry to compare the neuropeptides in wild-type and newly generated mutants. Mutants lacking a functional PHM displayed an altered neuropeptide profile, showed impaired egg laying behavior and had a decreased brood size. Interestingly, PHM mutants still displayed fully processed amidated neuropeptides, probably as a result of the presence of a bifunctional PAM, the main amidating enzyme in vertebrates. Our data indicate the existence of a robust complementation system for the amidation reaction of neuropeptides in nematodes and suggest the involvement of amidated neuropeptides in egg laying. (1) S. J. Husson et al., J. Neurochem. 98, 1999 (2006); (2) J. Kass et al., J. Neurosci. 21, 9265 (2001); (3) S. J. Husson et al., J. Neurochem. 102, 246 (2007); (4) T. C. Jacob, J. M. Kaplan, J. Neurosci. 23, 2122 (2003).

Crittenden SL et al. (1997) International C. elegans Meeting "TEMPERATURE-SENSITIVE REPRESSION OF TRANSGENES CARRYING A fem-3(gf) MUTANT 3'UTR"

Crittenden SL, Kimble JE

[International C. elegans Meeting, 1997]

We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant fem-3 3'UTR at the 3' end. The fem-3(gf) mutations map to the fem-3 3'UTR and release fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a lag-2 promoter::GFP construct that carries either a strong or a weak fem-3(gf) 3'UTR. The lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.

Von Stetina, Stephen E. et al. (2009) International Worm Meeting "Finding Regulators of Cadherin-Independent Epithelialization."

Mango, Susan E., Von Stetina, Stephen E.

[International Worm Meeting, 2009]

How are polarized epithelia established and maintained? This question is of critical importance, as the loss of epithelial polarity is associated with metastasis(1). There are many well-studied protein complexes that lie in specific membrane compartments with roles integral to the epithelial cell. The E-cadherin-containing adherens junction serves to link neighboring epithelial cells together while the more basal tight junction functions to separate the apical and basolateral surfaces. For some cells, E-cadherin is the major initiator of cell polarity and epithelium formation via cell-cell adhesion(2). However, recent studies have discovered E-cadherin independent polarity pathways(3-6). C. elegans offers a powerful system to study this cadherin-independent mechanism, as E-cadherin is dispensible for the initiation of epithelial polarity in nematodes(4). We study cadherin-independent epithelium formation during pharynx development. Nine pharyngeal arcade cells undergo a mesenchymal-to-epithelial transition to link the pharynx to the outer epidermis(7). Ablation of the arcade cells results in a Pharynx unattached (Pun) phenotype, in which the pharynx fails to connect to the epidermis(7). Pun animals die as they are unable to eat. Our lab has undertaken a genetic screen for Pun mutants that fail to form the arcade cell epithelium (Portereiko and Mango, unpublished). This screen revealed that loss of the central-spindlin component ZEN-4/MKLP1 induces a Pun phenotype because the arcade cells fail to polarize(8). We are currently studying where and when ZEN-4 is needed for arcade cell polarization. We have also undertaken a structure/function analysis of this mitotic kinesin in order to elucidate its role in epithelialization. In addition, we are in the process of cloning several mutants that were isolated in the Pun mutagenesis screen. (1). J. M. Lee, S. Dedhar, R. Kalluri, E. W. Thompson, J Cell Biol 172, 973 (Mar 27, 2006). (2). L. N. Nejsum, W. J. Nelson, J Cell Biol 178, 323 (Jul 16, 2007). (3). A. F. Baas et al., Cell 116, 457 (Feb 6, 2004). (4). M. Costa et al., J Cell Biol 141, 297 (Apr 6, 1998). (5). T. J. Harris, M. Peifer, J Cell Biol 167, 135 (Oct 11, 2004). (6). W. B. Raich, C. Agbunag, J. Hardin, Curr Biol 9, 1139 (Oct 21, 1999). (7). M. F. Portereiko, S. E. Mango, Dev Biol 233, 482 (May 15, 2001). (8). M. F. Portereiko, J. Saam, S. E. Mango, Curr Biol 14, 932 (Jun 8, 2004).

Dustin L Updike et al. (2005) International Worm Meeting "The TIP60/SWR1 Chromatin Remodeling Complex Functions with the Selector Gene PHA-4 to Endow Cells with Pharyngeal Competence"

Dustin L Updike, Susan E Mango

[International Worm Meeting, 2005]

A critical question in developmental biology is how complex programs of gene expression are orchestrated by a class of regulators known as selector genes. Selector genes code for transcription factors that autonomously govern the fates of groups of cells related to each other by virtue of their cell type, position or affiliation to an organ (1). For example, the FoxA transcription factor PHA-4 dictates the identity of cells within the C. elegans pharynx. Embryos that lack pha-4 fail to generate pharyngeal cells, and these cells acquire an alternative ectodermal fate instead (2,3). PHA-4 functions in combination with additional transcription factors to modulate genes at distinct stages and in different cell types during pharyngeal development (4,5). Here we explore the role of PHA-4 during transcriptional regulation. We identified components of the TIP60/SWR1 complex in a screen for loci that interact genetically with PHA-4. The predicted histone acetyltransferase mys-1 and chromatin remodeling factor ssl-1 each enhanced partial loss of pha-4 activity. In other organisms, the SSL-1 complex exchanges nucleosomes with histone H2A for those containing the variant H2Az (6). Nucleosomes containing H2Az appear less stable than those carrying H2A, which may facilitate the subsequent removal of nucleosomes for activation or repression (7). Consistent with this idea, C. elegans H2Az enhances the pharyngeal phenotype of pha-4 alleles, similar to ssl-1. Furthermore, we observe H2Az associated with PHA-4 target promoters in nascent pharyngeal cells. We propose that the MYS-1/SSL-1 chromatin remodeling complex functions with PHA-4 to endow cells with pharyngeal competence by restructuring the chromatin environment around PHA-4 target genes. Chromatin restructuring may render these promoters susceptible to binding by additional transcription factors that modulate pharyngeal gene expression in conjunction with PHA-4.1) R. S. Mann, S. B. Carroll, Curr. Opin. Genet. Dev. 12, 592 (2002). 2) S. E. Mango, E. J. Lambie, J. Kimble, Development 120, 3019 (1994). 3) M. A. Horner et al., Genes Dev. 12, 1947 (1998). 4) J. Gaudet, S. Muttumu, M. Horner, S. E. Mango, PLoS Biol. 2, e352 (2004). 5) W. Ao, J. Gaudet, W. J. Kent, S. Muttumu, S. E. Mango, Science 305, 1743 (2004). 6) G. Mizuguchi, X. Shen, J. Landry, W. H. Wu, S. Sen, C. Wu, Science 303, 343 (2004). 7) D. W. Abbott, V. S. Ivanova, X Wang, W. M. Bonner, J. Ausio, J. Biol. Chem. 276, 945 (2001).

Henderson ST et al. (1997) International C. elegans Meeting "REGULATION OF CELL FATE BY LAG-2 AND OTHER DSL PROTEINS"

Crittenden SL, Kimble JE, Henderson ST, Gao DL

[International C. elegans Meeting, 1997]

DSL (for DELTA, SERRATE, LAG-2) ligands act with LNG (for LIN-12, NOTCH, GLP-1) receptors to regulate cell fates in many organisms. To understand how DSL proteins shape metazoan development, we have investigated control by LAG-2 as well as two new DSL proteins. LAG-2, a predicted transmembrane protein, acts in many cell fate decisions (1,2,3). A systematic study of LAG-2 functional domains lead to four new conclusions. First, the non-conserved N-terminal region is critical for LAG-2 activity. Second, EGF-like repeats are not essential. Third, membrane association is required for rescue, but not signaling. Curiously, fusion to GFP can confer rescuing activity upon secreted LAG-2 forms, suggesting that GFP fusion proteins, when secreted, may stick in the membrane or aggregate outside the cell. Fourth, the intracellular domain normally lowers activity: LAG-2 mutants lacking this domain are hyperactive. In addition, a secreted LAG-2 placed under control of the ges-1 intestinal promoter can induce germline tumors, but membrane bound LAG-2 under the same control cannot. Therefore, the secreted form appears to retain activity across two basement membranes. How does LAG-2 control germline mitosis? One idea is that the length of distal tip cell processes may determine the extent of LAG-2 signaling (4). However, we have not been able to confirm this idea. We propose that the LAG-2 DTC signal is not limited to areas of cell-cell contact. Instead, we suggest that the signal is propagated in the germline by downstream factors. The sequencing project has identified two other genes that have the potential to encode DSL proteins, one on cosmid F15B9 and another on F58B3. We have constructed reporter constructs and begun antisense injections of both. Results on expression patterns of these DSL homologs and their antisense phenotypes will be reported. (1) Lambie, E. J., and Kimble, J. (1991). Development 112, 231-240. (2) Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924. (3) Tax, F. E., Yeargers, J. J., and Thomas, J. H. (1994). Nature 368, 150-154. (4) Fitzgerald, K., and Greenwald, I. (1995). Development 121, 275-4282.

Katja Ziegler et al. (2007) International Worm Meeting "A Sodium:Neurotransmitter symporter Family protein required for the induction of antimicrobial peptides in C.elegans."

Jonathan Ewbank, Nathalie Pujol, Katja Ziegler

[International Worm Meeting, 2007]

C. elegans responds to infection with the nematophagous fungus Drechmeria coniospora by up-regulating the expression of antimicrobial peptides including certain NLPs (neuropeptide-like proteins) and CNCs (Caenorhabditis bacteriocins) (1). We have generated a reporter strain containing the promoter of the antimicrobial peptide gene, nlp-29, fused to gfp. This results in a strain in which green fluorescence is highly induced after infection with D. coniospora. Coupled with the Union Biometrica BioSort, this provides us with a valuable tool to study infection-dependent induction in a qualitative and quantitative fashion. Using this strain and a direct visual screen following EMS mutagenesis, we identified 5 Nipi (no induction of peptide after Drechmeria infection) mutants (see abstracts by Pujol et al., Zugasti et al.). This study describes the isolation, characterization and SNP mapping of nipi-2. The mutant was found to carry a mutation in a conserved residue of a member of the sodium-dependent neurotransmitter transporter family in C. elegans. We are currently further characterizing this SNF protein and investigating its role in the induction of nlp-29 that follows infection. Progress will be reported at the meeting. 1.Couillault, C., Pujol, N., Reboul, J., Sabatier, L., Guichou, J. F., Kohara, Y. & Ewbank, J. J. (2004) Nat Immunol 5, 488-494. .

Garriga G et al. (2000) West Coast Worm Meeting "Role of the Caenorhabditis elegans homologs of cdk5 and p35 in migration and axon outgrowth"

Harbaugh TE, Garriga G

[West Coast Worm Meeting, 2000]

While cdk5 was originally identified in vertebrates based on its homology to the cell cycle regulated cdc2, further study has demonstrated a function not in the cell cycle but instead in post-mitotic neurons. Mouse knockouts of cdk5 and its activator, p35, result in defects in the pattern of neuronal migration in the cortex (1,2). Studies in cultured rat neurons demonstrated that the cdk5/p35 kinase activity can promote neurite outgrowth (3). The C. elegans homolog of cdk5 is 74% identical to mouse, and the p35 homolog contains a 159 AA region that is 54% identical. Translational GFP reporter constructs for cdk5 and p35 are expressed in the cytoplasm of most neurons beginning around the two-fold stage of embryogenesis and continuing into adulthood. Simultaneous overexpression of both cdk5 and p35 produces a strongly uncoordinated phenotype, with defects in fasciculation and axon pathfinding. Along with the expression pattern, these phenotypes suggest a role for cdk5 and p35 in neuronal development. The mig-18(k140) mutation was identified based on defects in distal tip cell migration (4) and may represent a Ce-cdk5 allele. Extrachromosomal arrays containing the Ce-cdk5 genomic region can partially rescue the distal tip cell migration defect of mig-18(k140). Furthermore, sequencing of the Ce-cdk5 genomic region revealed a point mutation predicted to change a conserved histidine into a tyrosine. To confirm that k140 is a Ce-cdk5 allele, we plan to isolate additional mig-18 mutants and determine whether they contain Ce-cdk5 molecular lesions. Also, we will screen a deletion library for alleles of Ce-cdk5 and its activator Ce-p35. (1) Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K, J Neurosci, 18:6370-7 (1998) (2) Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai LH, Neuron, 18:29-42 (1997) (3) Nikolic M, Dudek H, Kwon YT, Ramos YF, Tsai LH, Genes Dev, 10:816-25 (1996) (4) Nishiwaki K, Genetics, 152:985-97 (1999)

Schroeder, Frank et al. (2021) International Worm Meeting "Carboxyesterases and intestinal granules in the biosynthesis of novel families of nematode small molecule signals"

Wrobel, Chester, Schroeder, Frank, Sternberg, Paul, Cohen, Sarah

[International Worm Meeting, 2021]

Signaling molecules derived from attachment of diverse primary metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, the extent to which various inter- and intra-organismal signaling pathways are controlled by small molecules is unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of a large library of small molecules, including most modular ascarosides as well as previously undescribed modular glucosides (1). Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism. We further show that a family of carboxylesterases (CEST's) that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides, suggesting that modular glucosides, like the ascarosides, serve signaling functions. For example, biosynthesis of some modular glucosides is starkly upregulated in long-lived daf-2 mutants, suggesting that insulin signaling is modulated by small molecules produced in a lysosome-dependent manner. Parallel studies in C. briggsae and other nematode species indicate that assembly of modular metabolites via CEST enzymes is widely conserved in nematodes. Further exploration of LRO function and cest homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms. (1) H. H. Le, C. J. J. Wrobel, S. M. Cohen, J. Yu, H. Park, M. J. Helf, B. J. Curtis, P. R. Rodrigues, P. W. Sternberg, F. C. Schroeder. Modular metabolite assembly in C. elegans depends on carboxylesterases and formation of lysosome-related organelles. eLife, 9, e61886, 2020.