Elaine R Mardis et al. (2003) International Worm Meeting "Long oligomer microarray resource for the C. elegans community"

Jon Armstrong, John D McPherson, Wesley Warren, Darin Blasiar, John Spieth, Elaine R Mardis

[International Worm Meeting, 2003]

We report our efforts to develop and test a spotted microarray resource for distribution to the C. elegans community containing 1-2 long oligos (nominally 65mers) per predicted gene, along with appropriate control elements. Working with Wormbase curators (D. Blasiar and J. Spieth), we have obtained the most recent annotation to create the gene set from which the array oligos will be designed. The gene set is subdivided into three subsets: fully confirmed genes, partially confirmed genes and unconfirmed genes. Of the latter subset, we have determined whether any other type of supporting evidence exists (microarray, RNAi, BLAST, C. briggsae) to further subdivide the genes. We then propose to design a single unique oligo for each gene that is fully or partially confirmed, or is unconfirmed but with other supporting evidence of expression. For the entirely unconfirmed gene predictions, we will design two oligos. Our oligo design algorithm (J. McPherson) selects the optimal array oligo based on several criteria including; uniqueness with respect to the entire genome, Tm, low secondary structure potential, low homopolymer content, and with a 3-most bias, when possible. In addition, for partially confirmed genes, we will attempt to limit the oligo placement to within the confirmed portion(s) of the gene. A similar attempt will be made of unconfirmed genes with supporting evidence. Test arrays have been printed and distributed to several C. elegans labs with prior microarray experience and data sets. We will present the results of this testing, details of our Web-based support for this resource, and details on planned distribution of the full genome microarrays. We acknowledge NHGRI for funding this activity.

Yuichi Iino et al. (2008) C.elegans Neuronal Development Meeting "Behavioral Strategy for Salt Chemotaxis and Underlying Neurons"

Kazushi Yoshida, Yuichi Iino, Takanobu Tagawa

[C.elegans Neuronal Development Meeting, 2008]

C. elegans shows chemotaxis to water-soluble chemoattractants such as sodium chloride. How this behavior is achieved was previously addressed by several investigators. Among them, Jon Pierce-Shimomura analyzed the behavior of animals during chemotaxis using computerized worm-tracking system and proposed a pirouette strategy (1). Worms often change the direction of locomotion by a stereotyped behavior called pirouette, a bout of reversals coupled to sharp turns. It was proposed that worms show pirouettes more often when the concentration of salt decreases, biasing their movement towards salt concentration peak. This response was directly tested and confirmed by applying salt concentration change to restricted worms, making this strategy widely accepted (2-4). We reexamined the chemotaxis behavior by using a worm-tracking system, and found another strategy called weather-vane strategy. We focused on the behavior during run, the period in which worms move forward without displaying pirouette. Quantitative analysis reveals that during run, worms tend to curve towards the side with higher concentration of salt. The average curving rate is roughly proportional to the geometrical gradient of salt concentration in the direction perpendicular to the orientation of locomotion. This strategy in principle drives the worm to orient towards the peak of salt concentration. We are further analyzing the statistical nature of the behavior. By computer simulation, neither pirouette strategy alone nor weather-vane strategy alone generates chemotaxis as efficient as real worms, but combination of the two leads to chemotaxis quantitatively similar to real worms, suggesting that worms employ the two strategies in parallel during chemotaxis. To understand the neural basis of the behavior, we laser-ablated several candidate neurons and performed worm-tracking analysis on treated animals. So far, some neurons important for both pirouette and weather-vane strategies have been identified. The neural and behavioral mechanism of chemotaxis will be discussed.

Hideaki Hiraki et al. (2007) International Worm Meeting "Semi-automatic system for the creation of cell shape models in C. elegans embryogenesis."

Yuji KOHARA, Hideaki Hiraki

[International Worm Meeting, 2007]

Cell to cell interactions play critical roles in early embryogenesis, therefore, it is very important to have information about the arrangements of cells, cell shapes and the contact among them. We have been developing a computer system to create cell shape models from a time series of confocal microscopic images of the embryo whose plasma membrane is labeled with a vital fluorescent dye or a fluorescent protein. In this system, cell shapes are automatically calculated by a seeded region growing algorithm from a 3D image and a set of seed point coordinates. Manual editing of the seed coordinates is required and is assisted by its graphical user interface. We applied this system on the "dub" data set in WORM 4D CDs kindly provided by Bill Mohler, and obtained cell shape models of 24-200 cell stages successfully for the most part. This time, in collaboration with Jon Audhya, we performed live recording of OD58, the strain expressing the fusion of GFP with pleckstrin homology domain which is targeted to the plasma membrane. We successfully obtained data sets of 1-24 cell stages. We also developed a post-processing system to remove the bumps of cell shapes derived from image noises by implementing an active balloon model under gradient vector flow. As a result, the phenotype of par-2 RNAi embryos could be evaluated in terms of cell volumes and cell-to-cell contacting areas. We are improving the system more, and plan to apply this system to compare the cellular arrangements and the cell-to-cell contacts among mutant embryos and the embryos from other species closely related to C. elegans.

Dasgupta, Krishnakali et al. (2009) International Worm Meeting "Understanding the role of PKC-1 signaling pathway in regulating Dense Core Vesicle secretion."

Sieburth, Derek, Dasgupta, Krishnakali

[International Worm Meeting, 2009]

Synaptic vesicles (SV) and dense core vesicles (DCV) both exhibit calcium dependent neurotransmitter release. However, differences in their biogenesis, release properties, and recycling suggest that each type of organelle may use some unique proteins to carry out their functions. PKC-1, a protein kinase C ortholog in worms, is one such candidate for a unique DCV regulating molecule, since it has been shown to regulate neuropeptide secretion, while possibly not affecting SV release.(1) PKC-1 is most similar to the human eta and epsilon isoforms which are activated by the second messenger DAG via their C1 domains. In order to identify additional components that act in the PKC-1 pathway to regulate DCV secretion, we have used a constitutively active PKC-1 mutant which lacks the autoinhibitory domain and is predicted to be active in the absence of DAG. Expression of this transgene specifically in motor neurons causes hypersensitivity to an acetylcholinesterase inhibitor, aldicarb (Hic) and loopy locomotion. We screened for suppressors of either the loopy or Hic phenotype of animals expressing the constitutive PCK-1, and identified ~30 suppressors that restore movement or aldicarb response. We are in the process of mapping them and characterizing their effects on neuropeptide secretion. Presently, we have identified one aldciarb resistant candidate allele, vj3, which shows decreased uptake of NLP-21::YFP by coelomocytes and increased accumulation of DCVs in axons. However, vj3 mutants do not cause an accumulation of the SV marker at synapses, suggesting that vj3 mutants specifically reduce DCV secretion. vj3 maps to a small interval on LG I that does not contain any known neurotransmitter secretion genes. We are currently in the process of fine mapping, and tesing candidate genes for aldicarb resistant phenotypes by RNAi. 1.PKC-1 regulates secretion of neuropeptides; Derek Sieburth1, Jon M Madison & Joshua M Kaplan; Nature Neuroscience 10, 49 - 57 (2007).

Squiban, Barbara et al. (2009) International Worm Meeting "In vivo monitoring of gene transcription in the innate immune response."

Squiban, Barbara, Ewbank, Jonathan

[International Worm Meeting, 2009]

Infection of C. elegans with the fungus Drechmeria coniospora provokes the rapid induction of the antimicrobial peptide gene nlp-29. Transgenic worms with a pnlp-29::GFP reporter gene exhibit green fluorescence after infection. A needle prick also results in a very rapid increase in GFP expression. Initially, the fluorescence is restricted to the site of injury and then extends to the entire major epidermal cell syncytium, hyp7. This result is intriguing since all the >100 nuclei in hyp7 share the same plasma membrane and cytoplasm. The spread of fluorescence might reflect the diffusion of GFP following gene activation in the nucleus that is closest to the wound site, or an increase in the number of nuclei expressing the reporter gene. To characterize better this response, one needs to be able to assay specific gene transcription, at the single nucleus level. N. Broude''s group has described a method that could be adapted to achieve this. It is based on GFP complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In it, eIF4A is split into two, and each fragment fused to split fragments of GFP. Coexpression of the two protein fusions (A-F1 and B-F2) in the presence of a transcript containing the specific RNA aptamer results in GFP fluorescence. Thus, the spatial and temporal changes in fluorescence seen within cells should reflect the dynamics of transcript production, localization and degradation. Unfortunately, when the A-F1 and B-F2 proteins were expressed together in worms, a strong fluorescence was observed, both in the cytoplasm and in the nucleus. The fluorescence appeared static in its distribution and was independent of the presence of the aptamer. One explanation is that, in C. elegans, the A-F1 and B-F2 molecules aggregate irreversibly. Attempts to circumvent the problem by diluting the injected transgenes were unsuccessful. We have therefore turned to a less sophisticated, indirect approach, based on the observation that a MAB-9::GFP fusion protein is localized to a very limited number of epidermal cell nuclei. Jon Audhya kindly provided us with a mCherry-histone reporter construct. We have placed this under the control of certain nlp promoters, and have generated transgenic lines that show no fluorescence in the absence of injury, but have red nuclei after wounding. We are therefore now in a position to follow the dynamics of gene expression at the single nucleus level within the epidermal syncytium. Thanks to J. Hodgkin and all members of his lab, who hosted JE during early stages of the project. Pujol et al (2008) Curr Biol 18:481 Valencia-Burton et al (2007) Nature Methods 4:421. Wollard & Hodgkin (2000) Genes & Dev 14:596.