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Griffiths M, Young JD, Baldwin SA, Yao SYM, MacGregor D, Cass CE, Hope IA, Appleford PJ, Chomey EG, Isaac RE, Coates D
[
European Worm Meeting,
2000]
Nucleoside transporters (NTs) are essential for nucleotide synthesis by salvage pathways in cells which lack de novo biosynthetic pathways and have important roles in adenosine-mediated processes in mammals (e.g. neurotransmission, platelet aggregation and coronary vasodilation). Most eukaryotes possess multiple NTs which can be grouped into two unrelated transporter families; the equilibrative nucleoside transporters (ENTs) and the concentrative transporters (CNTs). In order to understand the physiological functions of nucleoside transporters and the reasons underlying ther biogical diversity, we are using C. elegans as a model system. C. eleganspossesses two genes encoding putative CNTs and six encoding putative ENTs. To date, one of the genes, ZK809.4 has been expressed in Xenopus oocytes and shown to encode a genuine ENT. It exhibits broad substrate specificity for natural purine and pyrimidine nucleosides and also transports antiviral nucleosides such as 3'-azido-3'-deoxythymidine (AZT). In an attempt to elucidate the biological roles of this and other NTs, we have used Green Fluorescent Protein reporter constructs to investigate the temporo-spatial expression patterns of these genes. Additional clues to the biological roles of NTs have been obtained by double-stranded RNA interference (dsRNAi).
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[
International Worm Meeting,
2017]
In a recent article it was shown that the architecture of the tube of epithelial cells that comprise the stoma and pharynx of C. elegans is basically the same as for other nematodes: the sequential order and characteristics of the cell classes are conserved (Burr and Baldwin 2016). The row of radial cell classes
pm1-
pm5 are paired within each interradial sector and in this way are distinct from the single, unpaired radial cells
e1,
e3,
pm6 and
pm7. Immunofluorescence labeling (MH27) showed that marginal cells
e2 and
mc1 are not separated by the encircling
pm1 syncytium, but share a common circumferential apical junction. Here I will add TEM evidence that the encircling
pm1 cell syncytium passes peripheral to the adjoining
e2 and
mc1. Marginal cells
e2,
mc1,
mc2 and
mc3 form a continuous row and border with the same classes of radial cells in most nematodes. In this paper I will compare certain features of the pharyngeal cells in C. elegans and closely related taxa that are different from other nematodes. 1) The unpaired radial cells
e1 and
e3 are not muscles in C. elegans and other taxa within Rhabditina, but do express muscle cytoskeleton in most other nematode taxa. To provide a terminology consistent across Nematoda, the pharyngeal 'epithelial' and 'muscle' cells of the C. elegans pharynx would better be distinguished by topological terms such as 'unpaired radial' cells (
e1,
e3,
pm6-7) or 'paired radial' cells (
pm1-5), and 'marginal' cells (
e2,
mc1-3), rather than by the confusing functional terms. 2) In C. elegans the paired radial cells of the pharynx become fused shortly after hatching or molting - a segment of the apposed plasma membranes that normally separate the pairs is dissolved (Shemer et al. 2004). However the muscle cytoskeleton remains paired. Fusion of these cells occurs only in two clades within the suborder Rhabditina: in Eurhabditis, which includes C. elegans, and in a clade within Diplogasteromorpha. 3) In C. elegans the six
pm1 cells are fused within and between sectors to form a syncytium that encircles the stoma (Albertson and Thomson, 1976). Circumferentially fused radial cells have rarely been reported in other taxa. An interesting question: What are the functional advantages of these special features? Albertson and Thomson 1976. Philos Trans R Soc Lond B 275:299-325. Burr and Baldwin 2016. J Morphology 277:1168-86. Shemer, Suissa, Kolotuev and Hall 2004. Curr Biol 14:1587-91.
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[
International Worm Meeting,
2011]
Oxygen (O2) is essential for the growth and development nearly all metazoans. O2 concentrations in the environment fluctuate and C. elegans exhibits an aerotaxis behavior dependent on the activity of soluble guanylate cylcases,
npr-1 and
glb-5. We show that, in addition to the previously described aerotaxis pathway, C. elegans displays a genetically distinct acute hypoxia avoidance response (HAR). When worms are placed in hypoxic atmospheres containing less than 5% O2 they immediately switch from a dwelling to roaming behavior. Roaming speed is inversely correlated with %O2 down to 1% O2 and is positively correlated with O2 concentrations <1%.
In addition to its requirement for oxidative phosphorylation, molecular O2 is required for the synthesis of the monoamine neurotransmitters which regulate the switch from dwelling to roaming behaviors. The inhibition of roaming by monoamine neurotransmitters requires the activity of the heterotrimeric G-protein GOA-1. We find that loss of function mutations in
goa-1 eliminate HAR.
goa-1 animals also display a premature entry into suspended animation (SA) at O2 concentrations 0.1%. GOA-1 negatively regulates the activity of the PLC-b EGL-8 through EGL-30.
egl-8 animals display a severely attenuated HAR and also prematurely enter SA. EGL-8 signaling through diacylglycerol (DAG) is negatively regulated by the DAG kinase DGK-1. In moderate hypoxia (³5% O2)
dgk-1 worms, like
npr-1 worms, show a pronounced decrease in locomotion. However, unlike
npr-1 worms, at < 5% O2,
dgk-1 worms show an inverse HAR; locomotion is increased in room air but suppressed in hypoxia.
Hypoxia survival in C. elegans and other metazoans requires the transcription factor HIF-1.
hif-1 loss of function animals show no defects in HAR, however, animals that lack the function of the prolylhydroxylase EGL-9, the negative regulator of HIF-1, show an immediate arrest of locomotion when transferred to hypoxia. This arrest is transient and
egl-9 animals slowly increase speed during hypoxia eventually reaching wildtype levels of locomotion. Because
dgk-1 worms show a similar arrest immediately upon transition to hypoxia, we tested the affect of hypoxia on embryonic diapause, a phenotype which requires HIF-1. We found that
dgk-1 worms, like
hif-1 worms, exhibit a precocious diapause at 0.5% O2. This suggests that the GOA-1/DGK-1 pathway might influence HIF-1.
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[
West Coast Worm Meeting,
2002]
Faithful segregation of chromosomes during cell division is essential for the inheritance of equivalent genetic information by each daughter cell. One mechanism to ensure the fidelity of chromosome segregation during mitosis is to maintain the tight association of sister chromatids from DNA replication until chromatid separation at anaphase. A conserved mitosis-specific multimeric protein complex, termed cohesin, is largely responsible for maintaining sister chromatid cohesion. The core of this complex consists of a heterodimer of Smc1 (structural maintenance of chromosome) and Smc3. At least two additional proteins are found within this complex; these include the Scc3p/SA (stromal antigen) and Scc1p/Mcd1p/RAD21 protein families. In meiosis, chromosomes undergo two rounds of division following a single round of replication in order to generate haploid gamates. To accommodate this specialized and highly regulated dispersal of meiotic chromosomes, there is also a meiosis-specific cohesin complex. A conserved difference between the mitotic and meiotic cohesin complexes is the substitution of the mitotic Scc1p/RAD21 protein by the meiotic REC-8 protein. Although the worm Scc1p homologs and their meiotic ortholog have been identified, the larger cohesin complexes, as defined by the four cohesin proteins, had not been fully characterized. Here we report the characterization of the mitotic and meiotic cohesin complexes in order to examine 1) the composition of the worm complexes relative to their counterparts in other eukaryotes and 2) the regulated localization of these proteins to meiotic chromosomes.
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[
International Worm Meeting,
2017]
Imaging mass spectrometry (IMS) is a two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules. The free-living soil nematode C. elegans is a common model organism, extensively used in life science research. Though, various investigations have been performed for metabolomic profiling of worms, the information of a single worm has been lost by? the conventional mass spectrometry (MS) techniques. Thus, the development of a label-free, non-targeted MS technique for molecular mapping in C. elegans has been required. We have previously performed MALDI imaging of C. elegans. However, the resolution was not enough to analyze cellular or subcellular level of biomoleculer distribution. Thus, we next tried the application of TOF-SIMS (Time-of-Flight Secondary Mass Spectrometry) system for C. elegans, which enables us to obtain subcellular distribution of metabolites. By comparison of several sample preparation methods, the frozen sections of C. elegans fixed by paraformaldehyde (PFA) were suitable for TOF-SIMS analysis. By sputtering of Ar gas cluster ion beam (Ar-GCIB), the sensitivity to fatty acids (e.g. stearic acid (SA), oleic acid (OA), and eicosapentaenoic acid (EPA)) was significantly enhanced, and high-resolution images of biomolecules were acquired. Further modification to prepare C. elegans samples for TOF-SIMS imaging is in progress. This is promising to obtain the cellular and subcellular distributions of the various biomolecules easily and efficiently.
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[
European Worm Meeting,
2000]
We are interested in usage of C. elegans as system for expression of hookworm genes. Hookworms (Ancylostomatideae ) are economically and medically important blood feeding nematodes which cause intestinal infections in humans and domestic animals. It has been reported recently that above 1 billion of human beings are currently infected with these worms. The application of C. elegans as a heterologous host for hookworm genes is limited mainly by the lack of hookworm genomic data. For this purpose we have constructed cDNA and gDNA libraries from the Ancylostoma ceylanicum -important human/animal parasite related to C. elegans. The cDNA library is based on PCR mediated amplification of spliced leader of parasite cDNA reported previously ( Martin SA, Thompson FJ, Devaney E. Mol. Biochem. Parasitol. 1995 Mar;70(1-2):241-5.). PCR amplified cDNA has been gel fractionated and cloned. The most abundant library is available now from adult mixed sex A. ceylanicum (above 6000 clones), smaller library was constructed from infective L3 larvae. The large inserts gDNA libraries were constructed by ligation of PFGE fractionated Ancylostoma genomic DNA into the pBACe3.6 (provided by Pieter deJong), both digested with EcoRI. Ligation mixture was precipitated with ethanol in the presence of yeast tRNA prior electroporation into DH10B host. Both cDNA and gDNA libraries were arrayed in 96 well format, and stored at -70C. Randomly picked clones were sequenced from both ends, blastn and blastx searches were performed. For cDNA clones our sequencing results show clear similarities to C. elegans genes : gene F38E11.2
hsp-12.6; similar to Human Alpha crystallin B chain, gene C15C7.6; similar to syntaxin-6, gene T20F5.2; similar to the S25B family of peptidases. For BAC clones, end sequencing was also performed ( in collaboration with M. obocka, Institute of Biochemistry and Biophysics ) , however no significant similarities were found (yet!).
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[
East Coast Worm Meeting,
2002]
Most free living nematodes feed on bacteria. Within Diplogastridae, however, diet changed repeatedly, as did stoma morphology. The evolution of a large dorsal tooth probably allowed the evolution of carnivory and fungivory within this taxon. Our aim was to understand which changes occurred in the diplogastrid stoma at the ultrastructural level. Ultrastructure was already known for some bacterivorous species (e.g. De Ley, P et al. 1995, Nematologica 41: 153) and for the partly carnivorous Diplogaster halicti with its derived shortened stoma and large dorsal tooth (Baldwin, J et al. 1997, Can J Zool 75: 407). We used TEM to investigate Diplogasteroides nasuensis, a bacterivorous species with a tube-shaped stoma, probably representing an early branch of the diplogastrid clade. The stoma of D. nasuensis is formed by cell processes whose number and arrangement corresponds to that observed in Caenorhabditis elegans, cephalobids and panagrolaimids. Some novelties evolved early in Diplogastridae: (1) The dorsal tooth is formed by two sets of cell processes (instead of one). (2) The channel of the dorsal pharynx gland lies between these cell processes, allowing the evolution of the tooth functioning as injection device. (3) Pharyngeal cell processes in the diplogastrid stoma are shortened and interlaced. (4) Some myofilaments are arranged longitudinally instead of radially, possibly allowing the kind of complex mobility of stoma parts observed in carnivorous species. In the past it was debated how to homologize stoma parts in Secernentea. We reject the attempt to use cell lineage in C. elegans and Cephalobus cubanensis as a basis for homologization (Dolinski, C et al. 1998, Dev Genes Evol 208: 495), which disregards the importance of cell-cell signaling and positional information in cell fate determination. More importantly, this hypothesis leads to non-parsimonious assumptions for evolutionary events, involving a double gain and subsequent loss of cell processes in the lineage leading to C. elegans. However, stoma parts in Cephalobidae, Panagrolaimidae, C. elegans and D. nasuensis can be unambiguously homologized based on the conserved spatial arrangement of the cell processes by which they are formed. We are currently using sequences of small subunit ribosomal RNA genes to independently test the phylogenetic relationships among these species and to trace evolutionary changes in stoma characters. Supported by the Deutsche Forschungsgemeinschaft (Su 198/2-2)
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[
International Worm Meeting,
2011]
Previous studies have found that species in the sister group of Caenorhabditis, the Protorhabditis group (1), show early embryonic cellular patterns strikingly different from that in Caenorhabditis. In embryos at the four-cell stage, all four blastomeres are arranged in a row instead of in the rhomboid pattern (2). To better characterize these differences, we have undertaken a systematic study that combines phylogenetic analysis with time-lapse microscopy. First, we reconstructed a molecular phylogeny for nine species of the Protorhabditis group. We then compared early cellular events and cell lineage division timing in eight species from the Protorhabditis group, using C. elegans as a reference. Our molecular phylogeny confirms that the monophyletic Protorhabditis group contains species of the genera Protorhabditis, Prodontorhabditis and Diploscapter, the latter of which has traditionally been treated as a separate Family Diploscapteridae. We find two clades within the Protorhabditis group: clade A includes Diploscapter species as well as some Protorhabditis species, and clade B includes Prodontorhabditis species and some Protorhabditis species. Analysis of the time-lapse movies confirmed that early embryogenesis in the Protorhabditis group is quite different from that in Caenorhabditis. In both clades at the two-cell stage, the posterior blastomere P1 divides first, and the axis of division of the anterior blastomere AB is parallel to the antero-posterior axis (3,4). This is in contrast to C. elegans where AB divides first and its axis of division is transverse. However, we found distinct differences between the two clades within Protorhabditis at the four-cell stage. Clade A species show the "four-cell-in-a-row" phenotype that has been described previously (3,4). For clade B, we observed a novel cellular phenotype. In these species, AB divides much later than P1, giving rise to at least three descendants before AB begins to divide. This difference in timing prevents the four descendants of AB and P1 from being positioned in a row. Early development in the Protorhabditis group is much slower than in C. elegans, with clade B displaying an even slower development than clade A. In both clades, the germline divides faster relative to other lineages. 1. W. Sudhaus, D. Fitch, J Nematol 33, 1 (2001). 2. C. Dolinski, J. G. Baldwin, W. K. Thomas, Can J Zool 79, 82 (Jan, 2001). 3. V. Lahl, J. Schulze, E. Schierenberg, Int J Dev Biol 53, 507 (2009). 4. M. Brauchle, K. Kiontke, P. MacMenamin, D. H. Fitch, F. Piano, Dev Biol 335, 253 (Nov 1, 2009).
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[
International Worm Meeting,
2021]
Axonal regeneration is a promising approach to overcome impaired functionality due to axonal injury. In mammals, central nervous system has poor regenerative capacity due to both extrinsic and intrinsic factors. The regenerative capacity also declines significantly with ageing. Therefore, functional axon regeneration in adulthood is challenging and needs more understanding. The pharmacological manipulations are not very successful for functional restoration whereas rehabilitation and physical activity shows improvement. As physical exercise has complex systemic effects, understanding the downstream effectors of physical exercise that is relevant for axon regeneration might be useful. Studying this using simple model organism has several advantages. Using posterior gentle touch circuit neuron (PLM) of Caenorhabditis elegans, we are studying effect of swimming exercise on functional restoration after laser assisted axotomy. We found that a single swimming exercise session of 90 minutes, which is an established paradigm of exercise in worm (Laranjeiro et al., 2017; Laranjeiro et al., 2019) improves functional recovery irrespective of age. However multiple swimming session is required for older worms (A5 stage). Anatomical correlation showed that swimming session improves regrowth initiation, regrowth length and functional connections. We found that the energy sensor kinase AMPK/AAK-2 plays an essential role mediating swimming benefits. Characterizing tissue specific requirement, we found that it has both cell autonomous (PLM neuron) and non-autonomous (muscle) requirement. Pharmacological activation of AMPK/AAK-2 showed enhanced functional restoration similar to swimming. We are studying the downstream molecules and their specific roles in various tissues for swimming mediated functional enhancement which will be helpful for better implementation of this approach. References Laranjeiro R, Harinath G, Burke D, Braeckman BP, Driscoll M (2017) Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biology 15. Laranjeiro R, Harinath G, Hewitt JE, Hartman JH, Royal MA, Meyer JN, Vanapalli SA, Driscoll M (2019) Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc Natl Acad Sci U S A 116:23829-23839.
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[
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).