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lab has undertaken a genetic screen for Pun mutants that fail to form the arcade cell epithelium (Portereiko and Mango, unpublished).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).

Wirshing, A. C. E. et al. (2017) International Worm Meeting "The C. elegans spermatheca is a model contractile tube for elucidating the roles of actin interacting proteins in tissue contractility."

Cram, E. J., Wirshing, A. C. E.

[International Worm Meeting, 2017]

Actin cytoskeleton organization and composition determine the mechanical properties of cells and tissues. There is evidence that disruption of the actin cytoskeleton underlies the pathophysiology of contractile tissue diseases including asthma and hypertension. Elucidating the mechanisms for organization of the actin cytoskeleton in contractile tissue is challenging due to the presence of numerous actin interacting proteins and the dynamic nature of actin regulation. Much of our current understanding comes from in vitro and ex vivo experiments. In this work, we use the C. elegans spermatheca as a novel in vivo model for identifying and characterizing genes involved in actin organization and tissue contractility. The spermatheca is a sack-like organ of the reproductive system that houses the sperm. During ovulation, the oocyte is propelled from the proximal gonad, through the spermatheca, where it is fertilized, and into the uterus. This process occurs approximately 150 times per gonad arm requiring robust regulation of contraction. To identify actin interacting and regulatory genes required for gonad contractility, we used a candidate RNAi screen of more than 200 genes. We have identified actin nucleators, crosslinkers, and motor proteins required for spermathecal contractility. Here we highlight the role of actin motor protein non-muscle myosin II, nmy-1, in not only spermathecal contractility, but also organization of the spermathecal actin network. Using 4-D confocal microscopy to observe spermathecal actin in live animals during ovulation, we show that prominent parallel actomyosin bundles develop from a loose meshwork during the first ovulation. Bundle development requires active myosin and bundle periodicity, tortuosity, connectivity, and orientation are regulated by myosin activity. We conclude that tight spatiotemporal control of myosin activity is required for formation and maintenance of the functional spermathecal actin network.

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.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

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.

Yuen, Grace J. et al. (2011) International Worm Meeting "Enterococcus infection of Caenorhabditis elegans as a model of innate immunity."

Ausubel, Frederick M., Pukkila-Worley, Read, Yuen, Grace J.

[International Worm Meeting, 2011]

Enterococcus is a Gram-positive commensal that is found in the gastrointestinal and biliary tracts of all healthy humans. It is also an important opportunistic pathogen, causing nosocomial urinary tract and wound infections that are often complicated by antibiotic drug resistance. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both strains. We have previously shown that infection with either strain leads to gut distention, but only E. faecalis is able to establish a persistent infection and kill the nematode. We now provide evidence that at least two canonical C. elegans immune signaling pathways are important for survival during infection with both E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by an E. faecium infection, mutations in the PMK-1 and FSHR-1 immune signaling pathways lead to an immunocompromised phenotype, where pmk-1 and fshr-1 mutants die rapidly upon E. faecium feeding. This new finding suggests that an active immune response is required to keep E. faecium infection "in check" in the worm intestine, and that E. faecium is indeed pathogenic to the nematode. We are now using microscopy and genetic studies to understand the biology of the Enterococcus infection in C. elegans and to identify novel Enterococcus-activated immune signaling pathways.

Fuentes, A. et al. (2015) International Worm Meeting "Diet affects neurodegenerative processes in C. elegans."

Fuentes, A., Calixto, A., Garcia, V., Palominos, MF.

[International Worm Meeting, 2015]

C. elegans has been used to understand the dietary impact on development and behavior (Macneil & Walhout., 2013), but very little is known about the role of diet on neurodegenerative processes. To study the effect of diet on neuronal degeneration, we use animals expressing the hyperactivated degenerins mec-4d expressed in the touch receptor neurons (TRNs) and deg-1 expressed in the PVC neurons. We measured the impact of different bacterial diets on neuronal integrity in the respective degeneration models. mec-4d animals with GFP marked TRNs were fed with E. coli OP50, B, HT115 and K12; Commamonas aquatica, Commamonas testosteronii and Bacillus megaterium and their TRN integrity assessed both morphologically (by visual inspection) and functionally (by the touch response) at 72 hours where most axons are degenerated (Calixto et al., 2012). The maximum protection was elicited by E. coli HT115 diet, with up to 50% wild type axons while the control strain E. coli OP50 and its precursor B only protects 3% at 72 hours post hatching. Like in the mec-4d animals, deg-1 animals PVC show a higher percent of functional response when animals are fed E. coli HT115. E. coli HT115 is capable of protecting neurons during large periods of time after adulthood since animals at 168 hours post hatching still showed 16.5 % of wild type axons. E. coli K12, Comammonas aquatica, testosteronii and Bacillus megaterium protected less (between 10 and 17% of wild type axons) than E. coli HT115 and more than E. coli OP50. E. coli HT115 is still capable of generating neuroprotection when diluted 1:100 in E. coli OP50, showing that E. coli OP50 does not produce neurodegeneration. Additionally, this shows that a very small amount of the protective food is necessary to elicit neuronal protection. E. coli HT115 does not produce dietary restriction and supports well animal growth, with 100% animals reaching adulthood at 72 hours vs 100% in E. coli OP50. Dead bacteria produce the same levels of neuronal protection than live bacteria, indicating that the interaction between bacteria and intestine is not required. Our data suggest that E. coli HT115 is capable of triggering pathways involved in neuroprotective processes early in animal development (first 12 hrs after hatching), highlighting the relevance of developmental time when the protective diet is ingested. We are currently performing transcriptomics analysis to identify the bacterial components from E. coli HT115 that mediate neuroprotection.

Knight JK et al. (1998) West Coast Worm Meeting "GAD-1 may interact with POP-1"

Knight JK, Wood WB

[West Coast Worm Meeting, 1998]

We have previously reported that the maternal-effect gene gad-1, encoding a protein with WD repeats, is required for gastrulation initiation (1). In gad-1(ct226ts) mutants at 25C, the E daughter cells divide early, with the timing and division plane of MS daughters, and fail to migrate into the embryo. However, in most mutant embryos as well as in gad-1(RNAi) embryos, the E cell progeny correctly express endoderm differentiation markers, and we have shown by laser ablation that only E progeny can do so. Given that members of the Wnt pathway have been shown to be involved in endoderm specification, we investigated the potential interaction of gad-1 with one member of this pathway, pop-1. In pop-1 mutant embryos, both the MS and E blastomeres differentiate into gut. In about 10% of gad-1 mutant embryos (n=333) and 8% of gad-1(RNAi) defective embryos (n=198), no gut is produced. To ask whether E adopts another fate in these embryos, we recorded five mutant embryos through the 6th cell cycle, but we could find no lineage differences between embryos that do and do not make gut. When we constructed a pop-1;gad-1 double mutant, we found that 64 % of the animals make no gut (compared to 10% for gad-1 embryos). This result suggests that in the pop-1 mutant background, gad-1 affects the ability of both the E-like MS cell and the true E cell to make gut. In support of this view, laser ablations of the true E cell in pop-1;gad-1embryos showed that in 11%, the E-like MS cell was capable of making gut; ablations of the E-like MS cell showed that in 33%, the true E cell was capable of making gut. Interestingly, MS-derived pharynx is also reduced in gad-1 mutants, suggesting that both daughters of the EMS blastomere are compromised by the gad-1 mutation. Whereas the above interactions between gad-1 and pop-1 appear to be complex, the cell lineage of pop-1;gad-1 embryos looks indistinguishable from that of gad-1 embryos. Whether or not gut is made in pop-1;gad-1 embryos, the E cells divide early and on the surface of the embryo, with a timing characteristic of the MS lineage (6/6 embryos, analyzed through the first 6 cell cycles). All other lineages are indistinguishable from wild type. In summary, we have found that: 1) gad-1 affects MS as well as E cell fates; 2) gad-1 and pop-1 interact in a complex manner in determining E cell fates; but 3) gad-1 appears completely epistatic to pop-1with regard to effects on the cell lineage, suggesting that cell behavior and subsequent cell fates in the E lineage can be separated. 1) Knight, J. K. and W. B. Wood (1998), Devel. Biol., in press