Loewen, R.A. et al. (2015) International Worm Meeting "Pharyngeal gland development in the root-knot nematode Meloidogyne hapla."

Kormish, J, Tenuta, M, Williamson, V.W., Loewen, R.A.

[International Worm Meeting, 2015]

Root-knot nematodes (RKN) are plant parasitic nematodes that cause massive crop loss in North America and worldwide. RKN are adept at invading and feeding on plant tissue with secretions they produce and release largely from their gland cells [1]. The collection of proteins within these secretions, called the "secretome," have been studied intensely using transcriptomics [2] and proteomics [3]. In our laboratory the highly derived gland cells of RKN are being studied by comparing glandular development in the model RKN Meloidogyne hapla to the non-parasitic nematode Caenorhabditis elegans. The pharyngeal and gland important transcription factors, PHA-4 and HLH-6 respectively [4], are being examined for their role in transcriptional regulation of parasite-specific genes. Putative homologues of these transcription factors have been identified in M. hapla: MhPHA-4 and MhHLH-6. Conservation of the DNA-binding motifs of the MhHLH-6 and CeHLH-6 proteins suggests a conserved function in binding of transcriptional regulatory elements. This conservation has led to the hypothesis that MhHLH-6 will play an important role in gland cell development and function in M. hapla and other RKN. Studies are ongoing using RT-qPCR to verify life-cycle expression levels of Mhpha-4 and Mhhlh-6. Isolates of M. hapla from Canada are being used in the analysis, and are currently being inbred for development of isogenic strains for sequencing [5]. Identification of genes controlled by MhPHA-4 and MhHLH-6 is underway, and progress on this study will be presented. It is predicted that identification of genes unique to M. hapla, an obligate plant parasite, when compared to the free-living C. elegans, will reveal genes that have been uniquely acquired to pharyngeal function during evolution of RKN. Our goal is to identify genes essential for parasitism, such as those regulating the function of the gland cells and their secretions. This approach will provide new targets for knockdown during RKN infection. The overlying goal of this research is to develop effective control mechanisms against parasite specific targets to inhibit this pest in the agricultural setting. References: [1] Rutter W et al. 2014. Mol Plant-Microbe Interact. 27: 965-974.; [2] Huang G et al. 2003. Mol Plant-Microbe Interact. 16: 376-381.; [3] Bellafiore S et al. 2008. Plos Pathog. 4: 1-12.; [4] Mango SE. 2009. Annu Rev Cell Dev Biol. 2009;25:597-628.; [5] Liu QL, Williamson VM. J Nematol. 2006. 38:158-64.

Tang, Lois et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Asymmetric regulation of the homeobox gene ceh-5 in early embryogenesis of C. elegans"

Abou-Zied, Akram, Hench, Jurgen, Tang, Lois, Shlyueva, Daria, Cesnulevicius, Konstantin, Burglin, Thomas

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

Homeobox genes are highly conserved transcription factors that play key roles in the development of humans and animals (1). From a 4D microscopy expression screen performed in the Brglin lab, a homeobox gene ceh-5 displaying a unique asymmetrical expression was revealed. This ortholog of the human VAX genes is expressed in three different groups of cells during gastrulation. An intriguing aspect of ceh-5 expression in the two bilaterally symmetric c ell groups is a left-right asymmetry in their expression levels. Little is understood about cell fate specification after the beginning of gastrulation, and also how left-right asymmetries are generated; ceh-5 , therefore, provides an excellent entry point to study these phenomena. We are dissecting the promoter region of ceh-5 to understand how the distinct spatio-temporal expression is generated. We created a series of deletions in the promoter region of ceh-5 fused to GFP that were used to make transgenic animals. The GFP expression patterns in the transgenic worms were monitored using 4D-imaging. This investigation revealed a 21bp in the promoter region containing an E-box motif essential for the regulation of ceh-5 . This motif is highly conserved between different Caenorhabditis species and is known to be a binding site for basic helix-loop-helix (bHLH) transcription factors (2). In order to look for transcription factors involved in regulating the expression of ceh-5 , we screen via RNAi knockdown, searching for candidates that would create an altered ceh-5 expression pattern. Although the screen is not yet saturated, putative bHLH transcriptional regulators of ceh-5 have been identified. 1) Burglin, T.R. 2005. Encyclopedia of Molecular Cell Biology and Molecular Medicine (ed. R.A. Meyers). 2) Yamada, K. and Miyamoto, K. 2005. Front Biosci.

Erich M Schwarz et al. (2003) International Worm Meeting "Comparative analysis of cis-regulatory sequences using four Caenorhabditis species."

Nora Mullaney, Erich M Schwarz, Tristan De Buysscher, Paul W Sternberg, Barbara J Wold, John A DeModena, Eunpyo Moon, Hiroaki Shizuya

[International Worm Meeting, 2003]

Comparing homologous cis-regulatory DNA sequences from three or more genomes has advantages over pairwise comparison of only two. Cis-regulatory sequences are short (6-20 bp) and tolerate substantial variation. Purely random pairing of unrelated 100-bp DNA segments is expected to yield two perfect 6 bp matches. Alignment of a third or fourth sequence should greatly lower the frequency of false positive regions, allowing small but real cis-regulatory sequences to be efficiently detected. This increased resolution should also allow direct comparison between phylogenetically conserved sequences and statistically overrepresented sequences, which may yield complementary views of regulatory elements. In the Caenorhabditis genus, C. remanei appears to be most closely related to C. briggsae; two other species, CB5161 and PS1010, comprise the two closest known and culturable Caenorhabditis species outside the elegans-briggsae group (Fitch, 2000). CB5161 is closest to C. elegans, and PS1010 the next most divergent; these two species thus provide an evolutionarily graded series. We have constructed fosmid libraries from CB5161 and PS1010, and begun sequencing individual fosmids for comparative analysis of genes involved in vulval or sensory neuron development. At the same time, we have devised the Mussa software package to adapt the algorithms of Davidson and coworkers (Brown et al., 2002) to multiple sequence analysis. At this writing, we have sequence data from the egl-30, lin-11, and mab-5 loci of both CB5161 and PS1010. Initial results of sequencing and comparative sequence analysis will be presented. References: Brown, C.T., Rust, A.G., Clarke, P.J., Pan, Z., Schilstra, M.J., De Buysscher, T., Griffin, G., Wold, B.J., Cameron, R.A., Davidson, E.H., and Bolouri, H. (2002). New computational approaches for analysis of cis-regulatory networks. Dev. Biol. 246, 86-102; Fitch, D.H.A. (2000). Evolution of Rhabditidae and the male tail. J. Nematol. 32, 235-244.

Ellis HM et al. (1998) East Coast Worm Meeting "C. elegans era, homolog of an E. coli cell cycle gene?"

Golden A, Powell BS, Court DL, Ellis HM

[East Coast Worm Meeting, 1998]

Members of the GTPase superfamily are found in organisms ranging from bacteria to humans. These proteins function as molecular switches, regulating a variety of cellular processes including signal transduction, cell cycle regulation and protein translocation. The E. coli GTPase, ERA (E.coli ras-like) is an essential protein required for normal progression through the cell cycle1. Reduction of era function results in cell cycle arrest after chromosome partitioning, but prior to cytokinesis. Sequence comparisons suggest that ERA and its homologs in other bacteria, C.elegans, mouse and human form a new subfamily of GTPases. These proteins show extensive homology both in the GTPase domain and in a C-terminal region of unknown function. In searches of the C.elegans database with the conserved carboxy-terminal region of E. coli ERA we identified a single homolog, E02H1.2, which we will refer to as Ce-ERA-1. Ce-ERA-1, is 31% identical (49% similar) to E.coli ERA in the GTPase domain and 19% identical (43% similar) to E.coli ERA in the C-terminal region. RT-PCR experiments generated an SL2 spliced cDNA with an exon structure matching that predicted by the genome project. The function of Ce-era-1 is unknown. Attempts to disrupt Ce -era-1 function by RNA-mediated interference did not generate any obvious phenotype. We are currently taking two approaches to the analysis of Ce-era-1 function: 1) Expression of transgenes carrying mutations predicted to produce dominant activated or dominant negative phenotypes. 2) Isolation of null mutations in a PCR based screen for deletions. In addition we have generated polyclonal antibodies against Ce-ERA peptides and will examine the temporal and spatial patterns of Ce-ERA expression. 1. Britton, R.A., Powell, B.S., Dasgupta, S., Sun, Q., Margolin, W., Lupski, J.R., and Court, D.L.(1998) Cell cycle arrest in Era GTPase mutants: a potential growth regulated checkpoint in Escherichia coli. Molecular Microbiology 27(4), 739-750. This work was sponsored by the National Cancer Institute, DHHS, under contract to ABL.

Evan Z Macosko et al. (2007) International Worm Meeting "Regulation of C. elegans roaming behavior by a multi-component pheromone."

Jon Clardy, Rebecca Butcher, Jim Thomas, Evan Z Macosko, Cori Bargmann

[International Worm Meeting, 2007]

Overall movement during animal foraging can take two general strategies: exploration of a wide area (roaming) and restriction to a local area (dwelling). C. elegans, when feeding on a bacterial lawn, has been shown to alternate between roaming and dwelling states.i Recordings of worm movements on food indicate that the behavioral state changes are arrhythmic and can be modified by food quality. We found that a multi-component pheromone released from incised or lysed worms dramatically inhibits C. elegans roaming behavior. The same worm lysate also acts as a potent repellent, suggesting that this mixture could be analogous to an alarm pheromone. Our chemical analysis indicates that there are at least two important components of the worm lysate that inhibit roaming: (1) a derivative of the sugar ascarylose, which is also a component of dauer pheromoneii and (2) a small indole-containing compound. We are currently asking if the same compounds also induce acute avoidance behavior. To determine the neuronal circuitry that regulates roaming and dwelling, we killed individual cells by laser ablation. Loss of either ADL, ASK, or AWC increased roaming in the presence of pheromone. In contrast, ablation of ASI resulted in a constitutive dwelling phenotype. The interneurons AIZ and AIY are important for proper roaming and dwelling behavior, while AIB and AIA are less important. Finally, though AVA is a crucial command interneuron for many locomotory behaviors, we found that AVA-ablated animals roam relatively normally. We also tested candidate mutants for sensitivity to pheromone. Mutations in egl-4, a gene previously shown to antagonize roaming,i reduce pheromone sensitivity. Rescue of egl-4 using heterologous promoters indicates that the EGL-4 gene product functions in chemosensory neurons to promote dwelling. iFujiwara M., P. Sengupta and S.L. McIntire, 2002 Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-Dependent Protein Kinase. Neuron 36: 1091-1102. iiButcher R.A., M. Fujita, F.C. Schroeder, J. Clardy, Small Molecule Signaling of Dauer Formation in C. elegans, 2007 International Worm Meeting.

Powell BS et al. (2000) East Coast Worm Meeting "C. elegans era, homolog of an E. coli cell cycle gene?"

Golden A, Ellis HM, Powell BS, Court DL

[East Coast Worm Meeting, 2000]

Members of the GTPase superfamily are found in organisms ranging from bacteria to humans. These proteins function as molecular switches, regulating a variety of cellular processes including signal transduction, cell cycle regulation and protein translocation. The E. coli GTPase, ERA is an essential protein required for normal progression through the cell cycle1. Reduction of era function results in cell cycle arrest after chromosome partitioning, but prior to cytokinesis. Sequence comparisons suggest that ERA and its homologs in other bacteria, C.elegans, mouse and human form a new subfamily of GTPases. These proteins show extensive homology both in the GTPase domain and in a C-terminal RNA binding domain. In searches of the C.elegans database with the conserved carboxy-terminal region of E. coli ERA we identified a single homolog, E02H1.2, which we will refer to as Ce-ERA-1. Ce-ERA-1, is 31% identical (49% similar) to E.coli ERA in the GTPase domain and 19% identical (43% similar) to E.coli ERA in the C-terminal region. RT-PCR experiments generated an SL2 spliced cDNA with an exon structure matching that predicted by the genome project. The first gene in this operon E02H1.1 is homologous to the bacterial rRNA methyltransferase ksgA, which interacts genetically with E. coli era. Thus there might be a functional relationship between the two genes in the C. elegans operon. RNA-mediated interference of E02H1.1 results in a severe developmental delay and larval lethality. The function of Ce-era-1 is unknown. Attempts to disrupt Ce-era-1 function by RNA-mediated interference did not generate any obvious phenotype. A null allele of Ce-era-1 was recently identified in a PCR screen (R. Ranganathan and H.R. Horvitz, personal communication). Characterization of this allele will be presented. 1. Britton, R.A., Powell, B.S., Dasgupta, S., Sun, Q., Margolin, W., Lupski, J.R., and Court, D.L.(1998) Cell cycle arrest in Era GTPase mutants: a potential growth regulated checkpoint in Escherichia coli. Molecular Microbiology 27(4), 739-750.

Szentgyorgyi, Erik et al. (2015) International Worm Meeting "Polar body extrusion in the early C. elegans embryo."

Muller-Reichert, Thomas, Koenig, Julia, Szentgyorgyi, Erik

[International Worm Meeting, 2015]

Polar body extrusion (PBE) plays an essential role in genome haploidization during female meiosis. The underlying mechanism, however, is poorly understood. The process of extruding a small polar body out of the large oocyte can be considered as an extreme case of asymmetric cytokinesis. In mammalian cytokinesis, actomyosin-driven membrane constriction leads to a compression of the central spindle. ESCRT-III components then cause a secondary constriction at the intercellular bridge, which is accompanied with a severing of microtubules caused by spastin. Importantly, microtubule severing is the rate-limiting step in this process [1].We aim to unravel the mechanism of PBE in the early C. elegans embryo. How is the polar body extruded, and how are the constriction of the contractile ring and the disassembly of the central spindle coordinated in this system?To answer these questions we have started to combine live-cell imaging and electron tomography. Live-cell imaging of wild-type embryos indicated a difference in spindle disassembly during extrusion of the first and second polar body. In the first meiotic division, the central spindle appears to be disassembled in the middle between the separating homologous chromosomes. In contrast, spindle disassembly in meiosis II appears to take place adjacent to the chromatin involved in the formation of the female pronucleus. In addition, we aim to deplete components of the chromosomal passenger complex (CPC) and the centralspindlin complex by RNAi [2]. Since both complexes are likely to be involved in contractile ring formation and constriction during PBE we want to observe the influence of contractile ring constriction on central spindle disassembly. Furthermore, we have started to analyze taxol- and nocodazole-treated embryos to investigate the influence of microtubule stability on polar body extrusion. In parallel, we compare the ultrastructure of spindles in late anaphase I and II by electron tomography.Interestingly, we found no indication for the formation of helical structures involved in membrane constriction, indicating that PBE in C. elegans is most likely achieved through an ESCRT filament-independent mechanism.[1] Guizetti, J. et al.; Cortical Constriction During Abscission Involves Helices of ESCRT-III-Dependent Filaments; Science 331, 1616 (2011)[2] Green, R.A. et al.; Cytokinesis in Animal Cells; Annu. Rev. Cell Dev. Biol. 28:29-58 (2012).

Steven J Husson et al. (2005) International Worm Meeting "Caenorhabditis elegans: in search of neuropeptides by mass spectrometry"

Steven J Husson, Geert Baggerman, Liliane Schoofs, Elke Clynen, Arnold De Loof, Tom Janssen

[International Worm Meeting, 2005]

Peptides are the original signalling molecules in metazoan nervous systems. They are also ubiquitous in nervous systems that employ classical neurotransmitters where they serve as modulators, hormones and transmitters. Understanding the role of neuropeptides is however unfortunately hindered by the absence of primary sequence information and knowledge about their post-translational modifications. This sequence information has arrived very slowly, due to the huge efforts required in tissue collection and purification to ultimately isolate and functionally characterize a peptide. In total, only 12 C. elegans peptides have been biochemical isolated and identified to date, all of which are FMRFamide-related peptides (FaRPs). The major advance of using C. elegans as a model organism in neuropeptide research is the availability of its genomic sequence [1] from which 23 FMRFamide like peptide (flp) genes [2,3] and 32 neuropeptide-like protein (nlp) genes [4] were predicted. The genomic database also represents an indispensable foundation to perform a high throughput peptidomic study. By using two-dimensional nanoscale liquid chromatography, tandem mass spectrometry and database mining, we analysed a mixed stage C. elegans extract in which we identified 28 FaRPs and 20 NLP peptides. In addition, we were able to identify 15 entirely novel peptides derived from 10 precursors that were not identified or predicted from the genome in any way previously. Some of the peptides display profound sequence similarities with neuropeptides from other animals, suggesting that they have a long evolutionary history. The present identification of novel endogenous peptides offers opportunities for a greater understanding of neuropeptide biology in C. elegans. Since this nanoscale approach worked very well for an organism as tiny as C. elegans, we are convinced that virtually any organism of which the genome has been sequenced can be analyzed by this peptidomics technology. [1] The C. elegans Sequencing Consortium (1998) Science, 282: 2012-2018. [2] Li, C, Nelson, L.S., Kim, K, Nathoo, A, Hart, A.C. (1999) Ann. N.Y. Acad. Sci., 897: 239-252. [3] Kim, K., Li, C. (2004) J. Comp. Biol., 475: 540-550. [4] Nathoo, A.N., Moeller, R.A., Westlund, B.A., Hart, A.C. (2001) PNAS, 98: 14000-14005.