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).

Yueyi I Liu et al. (2004) West Coast Worm Meeting "Global Identification of C. elegans Regulatory Motifs"

Stuart K Kim, Yueyi I Liu

[West Coast Worm Meeting, 2004]

Regulatory motifs are short sequences of DNA that regulate the level, timing, and location of gene expression. Identifying these motifs and their functions is crucial in our understanding of gene regulation and disease processes. We developed CompareProspector, a motif-finding program that takes advantage of cross-species sequence comparison to identify putative regulatory motifs from sets of co-regulated genes [1] . We applied CompareProspector to 30 sets of genes with very similar patterns of expression, identified from the C. elegans topomap [2] and individual DNA microarray experiments. The statistical significance of each candidate motif identified was evaluated using criteria such as motif enrichment-the ratio of prevalence of the motif in a given set of promoters to its prevalence elsewhere in the genome, and the expression coherence of genes with the motif. We identified twelve significant regulatory motifs, three of which have literature evidence confirming they are true regulatory motifs. Overall, these twelve motifs are found in the upstream regulatory regions of 2970 different genes, and may be involved in gene regulation in 24 clusters of co-expressed genes. The first known motif, with the consensus TGATAA, matches the consensus of known binding sites for GATA factors. As GATA factors are known to be involved in worm intestine development [3] and hyperdermis development, it is not surprising that the GATA motif is identified from a set intestine-specific genes (F. Pauli, unpublished), mount08 of the topomap, which is enriched in genes from the intestine, and several collagen-related datasets (mount14, 17, and 35 of the topomap). We correctly identified GATA sites in the promoters of genes known to be regulatory by GATA factors. Interestingly, the GATA motif is also identified from several data sets involved in the aging process. This result parallels that of Murphy and colleagues, who independently identified this motif from their data set of DAF-16 target genes [4] . Both our result and the result from Murphy suggest that GATA factors may be involved in worm aging. Motif 2, which is identified in the two heat shock-related data sets, matches the consensus of known binding sites for heat shock factors [5] . Motif 3 matches the consensus of heat shock associated sites (HSAS), a motif that was first predicted computationally to be involved in the heat shock process [6] and later experimentally validated to be involved in ethanol stress response (14 th International C. elegans Conference abstract 1113C). We are currently in the process of validating the rest of the motifs and their individual binding sites using mutagenesis studies of promoters with predicted motifs. 1. Liu, Y., Liu, X.S., Wei, L., Altman, R.B. and Batzoglou, S. (2004) Eukaryotic regulatory element conservation analysis and identification using comparative genomics . Genome Res. 14 , 451-8. 2. Kim, S.K., Lund, J., Kiraly, M., Duke, K., Jiang, M., Stuart, J.M., Eizinger, A., Wylie, B.N. and Davidson, G.S. (2001) A gene expression map for Caenorhabditis elegans . Science. 293 , 2087-92. 3. Maduro, M.F. and Rothman, J.H. (2002) Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm . Dev Biol. 246 , 68-85. 4. Murphy, C.T., McCarroll, S.A., Bargmann, C.I., Fraser, A., Kamath, R.S., Ahringer, J., Li, H. and Kenyon, C. (2003) Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans . Nature. 424 , 277-83. 5. Amin, J., Ananthan, J. and Voellmy, R. (1988) Key features of heat shock regulatory elements . Mol Cell Biol. 8 , 3761-9. 6. GuhaThakurta, D., Palomar, L., Stormo, G.D., Tedesco, P., Johnson, T.E., Walker, D.W., Lithgow, G., Kim, S. and Link, C.D. (2002) Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods . Genome Res. 12 , 701-12 .

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).

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Yukiko Yonezawa et al. (2007) International Worm Meeting "Role of the type-b insulin-like genes on larval diapause and adult lifespan in Caenorhabditis elegans."

Yukiko Yonezawa, Yasuo Kimura, Kenji Gouda, Tsuyoshi Kawano

[International Worm Meeting, 2007]

In Caenorhabditis elegans, the insulin/insulin-like growth factor-I signaling (IIS) pathway regulates larval diapause, adult lifespan, fat metabolism, and stress-resistance. C. elegans has 38 putative insulin-like peptides which are grouped into type-<font face=symbol>a</font> , -<font face=symbol>b</font> , and -<font face=symbol>g</font> according to their predicted disulfide bond pattern.1) The type-<font face=symbol>g</font> peptides have the same pattern as mammalian insulin family peptides. On the other hand, the type-<font face=symbol>b</font> peptides have one additional disulfide bond to the canonical three. In 2003, Murphy and co-workers reported that ins-7 categorized into the type-<font face=symbol>b</font> has relevancy on regulation of dauer larva formation and adult lifespan.2) This fact suggested that other type-<font face=symbol>b</font> genes also have physiological functions. Therefore, we attempted to investigate all of the type-<font face=symbol>b</font> genes by RNAi knockdown and/or gene-disruption. In this meeting, we will show positive/nagative regulation of larval diapause and/or adult lifespan by the type-<font face=symbol>b</font> genes. 1) Pierce, S. B., et al. (2001) Genes Dev. 15:672. 2) Murphy, C. T. et al. (2003) Nature 424:277.

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. .