Eric Miska et al. (2002) European Worm Meeting "Functional analysis of the micro RNA genes of C. elegans"

Ezequiel Alvarez-Saavedra, David P Bartel, Eric Miska, Nelson C Lau, Bob Horvitz, Victor Ambros, Allison Abbott

[European Worm Meeting, 2002]

* HHMI and Dept. Biology, MIT, Cambridge, MA 02139, USA Dept. Genetics, Dartmouth Medical School, Hanover, NH, 03755, USA # Whitehead Institute for Biomedical Research and Dept. Biology, MIT, Cambridge, MA 02142, USA

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

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.

PR Stancombe et al. (1999) International C. elegans Meeting "Characterisation of neprilysin (NEP) and endothelin-converting enzyme (ECE) from C. elegans"

PR Stancombe, D Coates, M Sajid, RE Isaac

[International C. elegans Meeting, 1999]

Cell-surface peptidases participate in the postsecretory processing and metabolism of neuropeptides and peptide hormones. Neutral endopeptidase-24.11 (NEP) is the prototype of a mammalian subfamily of zinc metallopeptidases that also includes endothelin-converting enzyme (ECE), the product of the PEX gene and KELL, an erythrocyte cell-surface antigen. These enzymes have central roles in a variety of physiological and disease processes, including cardiovascular function, cartilage and bone metabolism, inflammation and embryogenesis. Human zinc metalloproteinases belonging to this subfamily are important targets for therapeutic drugs. C. elegans has in the region of 90 genes with the zinc-binding consensus motif HEXXH of the superfamily of zinc metalloproteinases. Cluster analysis of predicted amino acid sequences reveals a group of 8 genes with closer similarity to mammalian NEPs/ECEs than the others. Hydrophobicity plots of the predicted protein sequences for these 8 genes revealed 5 that gave the expected domain structure seen in mammalian homologues; a short NH 2 -terminus intracellular region, a single transmembrane domain and a large extracellular region with a COOH-terminus active site. Expression patterns of beta-galactosidase and GFP have been obtained for 4 of these genes. T16A9.4 has a largely neuronal expression pattern with a pharyngeal and vulva muscle component; ZK970.1 and T05A8.4 are expressed in body wall muscle cells; ZK20.6 is pharynx specific. RNA interference experiments are being conducted with these genes to determine any null phenotypes.

Gert Jansen et al. (2001) International C. elegans Meeting "The C. elegans G-protein gamma subunit gpc-1 is involved in adaptation to water-soluble attractants."

David Weinkove, Gert Jansen, Ronald H A Plasterk

[International C. elegans Meeting, 2001]

Heterotrimeric G proteins form a first intracellular step in many signal transduction cascades. C. elegans has 21 Galpha, 2 Gbeta and 2 Ggamma subunits. Based on functional analyses the Galpha subunits can be divided into 2 groups. The conserved alpha subunits are ubiquitously expressed and regulate muscle and neuron activity. At least 14 other Galpha's play a role in sensory transduction. A similar discrimination can be made between the 2 G-protein gamma subunits, gpc-1 and gpc-2 . This latter gamma subunit is quite ubiquitously expressed. By contrast, gpc-1 is specifically expressed in only 6 pairs of amphid neurons and 1 pair of phasmid neurons, suggesting a function in sensory signaling. However, gpc-1 is not essential for the detection of various environmental cues, such as odorants or salts. To test if GPC-1 is involved in other types of sensory plasticity we developed a water-soluble compound adaptation assay. The behavior of wild-type animals in this assay confirms the idea that prolonged exposure to water-soluble compounds can abolish chemo-attraction to these same water-soluble compounds. This decrease in chemotaxis is time and concentration dependent, salt specific and reversible. gpc-1 mutant animals show clear deficits in their ability to adapt to three water-soluble compounds, NaAc, NaCl and NH 4 Cl. Also adp-1 and osm-9 , two loci previously implicated in odorant adaptation, are involved in adaptation to these salts. Our findings clearly indicate that G proteins are involved in the perception of salts. It is still unclear where in the signaling cascade these molecules function.

Jens Schulze et al. (2007) International Worm Meeting "Cytoplasmic segregation, interphase MT and establishment of cell polarity in the embryo of the basal nematode R. culicivorax."

Einhard Schierenberg, Jens Schulze

[International Worm Meeting, 2007]

One of the central questions for developmental biologists is how cellular polarity is established and leads to blastomeres with different fate. We study nematodes from various phylogenetic positions in comparison to C. elegans in order to assess variations in the pattern of embryonic development. One of our study objects, the more basal species Romanomermis culicivorax, shows peculiarities with respect to cell polarity not observed in any other nematode. Due to differences in the orientation of cleavage spindles spatial pattern formation in R. culicivorax differs markedly from that in C. elegans. Blastomeres which in C. elegans perform a transverse, equal cleavage divide unequally with an a-p spindle orientation and vice versa. In addition, we find - so far unique among nematodes - that colored cytoplasm is segregated into a single blastomere (EMS) which inherits it to all of its descendants. Furthermore, as another phenomenon, to our knowledge not described in other animal systems, during interphase microtubule caps form in specific regions of the cortex which disappear prior to mitosis. We find these caps predominantly in cells destined to execute polar divisions indicating their involvement in cell polarisation. The EMS cell containing the brown pigment, will divide into equal left and right daughters and is the only 4-cell blastomere without interphase MT caps. Our data demonstrate that developmental variations among nematodes are more prominent and abundant than anticipated so far.

Y Kimura et al. (1999) International C. elegans Meeting "Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans"

K Eto, H Tokumitsu, Y Kimura, M Muramatsu

[International C. elegans Meeting, 1999]

In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

Schlosser, A. et al. (2015) International Worm Meeting "Chronic exposure to pesticide residues exacerbates alpha-synuclein pathology in a Caenorhabditis elegans Parkinson's disease model."

Flaherty, D., Schlosser, A., Davis, M.

[International Worm Meeting, 2015]

Human health issues and disease have long been linked to environmental exposures which may be the greatest risk factors for the development of neurodegenerative diseases (Franco et al. 2010). Epidemiological studies show that exposure to environmental agents such as pesticides is a key contributor to the development and exacerbation of Parkinson's Disease (PD) (Franco et al. 2010). Intraneuronal inclusions, known as Lewy bodies, composed primarily of aggregated alpha-synuclein, are the primary causative formations associated with the loss of dopaminergic neurons and the development of PD (Baltazar et al. 2014; Brown et al. 2006). The loss of these neurons causes dysfunction of the basal ganglia and the subsequent inhibition of motor control, the defining characteristic of the disease (Baltazar et al. 2014). This study looks to assess the possible toxicological effects of chronic exposure to three common pesticides, chlorpyrifos, carbaryl and indoxacarb, at maximum tolerated residue levels set by the Environmental Protection Agency (EPA) in a Caenorhabditis elegans PD model. While the EPA employs strict regulations on individual pesticides, there are no regulations governing the mixing of pesticides. In this study, pesticides are tested both individually and as binary mixtures in order to investigate alpha-synuclein toxicity. Results from the study indicate that individually, these pesticides, with the exception of indoxacarb, have a noticeable effect on alpha-synuclein pathology. When combined to form binary mixtures, all pesticides have a drastic effect on alpha-synuclein pathology and mortality, despite being within EPA limits.

H Qadota et al. (1999) International C. elegans Meeting "Functional analysis of C. elegans PKN homologue"

T Miyauchi, H Qadota, K Kasuya, K Kaibuchi

[International C. elegans Meeting, 1999]

The Rho family GTPases (Rho, Rac, Cdc42) are involved in the regulation of actin reorganization, cell polarity, cell growth, and cell-cell adhesion. The Rho family GTPases have two interconvertible forms; the GTP-bound active and GDP-bound inactive forms. The GTP-bound form of Rho family GTPases interacts with their specific effectors. The GTP-bound form of RhoA binds preferentially to Protein Kinase N (PKN), Rho-kinase (also called ROK), myosin binding subunit of myosin phosphatase (MBS). Besides proceeding of functional analysis of Rho-kinase and MBS, the physiological functions of other effectors of Rho GTPase including PKN remain to be clarified. Here we examined physiological functions of PKN using C. elegans as a genetic model system. We have identified the C. elegans homologue of mammalian PKN in genome sequencing database and Yuji Kohara's EST sequence library. The cDNA clone, yk345c10, which we call pkn-1 ( C. elegans PKN), maps to cosmid F46F6 on the right arm of LGX. The predicted protein shows significant homology to mammalian PKN. E. coli expressed NH 2 -terminus of PKN-1 fused with maltose binding protein binds preferentially to GTP g S •GST-CeRhoA, but not to GDP•GST-CeRhoA, suggesting that the C. elegans PKN homologue also interacts with Rho GTPase in C. elegans . Based on expression studies using the pkn-1::gfp fusion construct, pkn-1 is preferentially expressed in the muscle cells. Overexpression of the catalytic domain of PKN-1 under the control of heat shock promoter caused abnormal movement, suggesting that CeRhoA and PKN-1 are involved in the regulation of the muscle contraction.

Brockett, Mary et al. (2015) International Worm Meeting "Virulence of a Novel Melanin Producing Pseudomonas Species UC17F4 on Caenorhabditis elegans."

Brockett, Mary, Morreale, Meghan, Aaronson, Lawrence, Shinn-Thomas, Jessica

[International Worm Meeting, 2015]

Pseudomonas sp. UC17F4 is a novel species of Pseudomonas isolated in our lab from the cutaneous microbial flora of the red-backed salamander, Plethodon cinereus. UC17F4 produces intracellular melanin, a brown pigment, in the presence of tyrosine-rich media. Melanin has been shown to serve as a virulence factor for several organisms, including bacteria and fungi. Our lab generated several mutant strains of UC17F4: UC17F4 (MM1) produces less melanin than the wild-type strain, UC17F4 (MM7, 8, and 9) hypersecrete extracellular melanin, and UC17F4 (PV 21 and 22) produce no melanin. We demonstrated that UC17F4 exhibits virulence by using C. elegans as a model host. Worms were transferred to lawns of UC17F4 bacteria on Nematode Growth Media (NGM) supplemented with tyrosine and lethality was assessed over time using touch assays. Our studies show that C. elegans exposed to the wild-type strain (UC17F4) have the highest mortality rate and worms exposed to UC17F4 (PV21) have the lowest mortality. Worms do not die after exposure to the hypersecreting strains or the strains without melanin. We investigated the effect of UC17F4 exposure on the different larval stages of C. elegans (L1, L2, L3, L4, and adult worms). Our results show that L1 and L2 worms are most vulnerable to the virulence of UC17F4 compared to later developmental stages. L1 and L2 worms die in less than 24 hours of exposure, whereas later developmental stages are viable and reproduce. Our current studies include determining the kinetics of L1 and L2 lethality using both touch assays and in vivo fluorescent cell death assays (SYTOX®) in a microplate reader, and determining the mode of pathogenesis using high-magnification light microscopy to examine the anatomical areas of melanin and UC17F4 biofilm accumulation. .