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[
International C. elegans Meeting,
2001]
An immediate challenge of the post-genomic era is to quantitatively determine the precise temporal and spatial expression patterns of every gene in a model organism during its embryonic development. The relationship between genotype and phenotype can then be predictively modeled in terms of the regulation and consequences of these precise expression patterns. In addition to being quantitative, microarrays have the benefit of making measurements in parallel, thus enabling global observations of the transcriptome during development. The C. elegans embryo is ideally suited for genomic analysis: it is experimentally accessible, well characterized genetically, has a compact genome, and the requirement for specific genes can be directly assessed using RNAi. To measure absolute abundance of transcripts during early development, we developed a sensitive and representative RNA amplification technique that enables quantitative analysis of mRNA levels from as few as 10 embryos. We then collected (in triplicate) pools of 10-15 embryos precisely staged (+/- ~3 min)by morphology at the 4-cell stage and allowed them to develop for proscribed amounts of time. mRNA from each of five time points spanning the 4 to ~102 cell stage was amplified and hybridized to oligonucleotide arrrays generating absolute measurements of transcript abundance for the entire genome. Approximately 7,500 genes are reproducibly detected in the time course, many of which are not represented in EST collections, and about 2,600 of them are significantly modulated over time (ANOVA p<10 -2 ). Transition from maternal to zygotic control of development is evident in the observed degradation of over 1,100 maternal transcripts along with the induction of approximately 1,500 zygotic transcripts. Among these, both simple and complex temporal expression patterns are detected. Intriguingly, reproducibility among replicates and similarity between adjacent timepoints increases towards the end of the time course. This result is consistent with the rate of molecular development decreasing and/or the stability of regulatory networks increasing as embryos approach the 100 cell stage. In either case, the observation suggests convergence onto a regulatory steady state. Statistical, computational and bioinformatic analysis of the data will be presented.
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[
Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI,
2010]
Animals have evolved great ability to adapt to fluctuating nutrient availability by balancing growth and survival. In C. elegans, newly hatched L1 stage larvae remain developmentally arrested until feeding. This phenomenon, called L1 arrest, provides an opportunity to study regulatory mechanisms mediating nutritional control of growth and development. Insulin-like signaling is a key regulator of L1 arrest (Baugh and Sternberg, 2006; Kao et al, 2007). In spite of great interest in the insulin-like pathway given its function in dauer formation, aging and L1 arrest, the function of specific insulin-like peptide ligands is generally not understood. The C. elegans genome encodes 40 insulin-like peptides, and they are thought to function as either agonists or antagonists of the insulin-like receptor DAF-2 and growth (Pierce et al., 2001). We are using a combination of expression and phenotypic analysis to characterize their regulation and function during L1 arrest. Although several insulin-like genes may be broadly redundant, we propose that there is specificity to their function in timing and site of action, and that they comprise regulatory network that responds to nutrient availability and controls growth and development. From our expression data we identify several putative agonists, including
ins-4,
ins-5,
ins-6, and
ins-7, which are up-regulated by feeding, and several putative antagonists, including
ins-17,
ins-18 and
ins-24, that are up-regulated by starvation. Transcriptional YFP reporters of them show that the agonists and antagonists are expressed in different subsets of amphid sensory neurons in L1 worms, and emphasize the intestine as a site of nutrient-dependent transcriptional regulation. Moreover, phenotypic analysis of growth rate, starvation survival and early postembryonic cell division on single and multiple deletion mutants will help determine their role in promoting growth and development.
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[
Zootaxa,
2022]
Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.
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[
International Worm Meeting,
2019]
Chemical exposure during development can have dramatic and sustained impacts, especially if the chemicals target the mitochondrial electron transport chain. Altered mitochondria function can contribute to the initiation of metabolic and degenerative diseases. We hypothesized that developmental (pre-conception) exposure to rotenone, a widely-used pesticide and piscicide that inhibits mitochondrial complex I, would result in lifelong and intergenerational effects. For experiments, Caenorhabditis elegans N2 strain (parental generation, P0) were exposed to rotenone (0.03 and 0.5 M) or vehicle (0.25% DMSO; control) in liquid with HB101 bacteria for 52 hours or until they reached the L4 larval stage. Animals were then transferred to OP50-agar plates for 48 hours for chemical depuration, and gravid adults were bleached to obtain synchronized eggs (F1 generation). F1 eggs were grown in liquid for 52 hours in the same conditions as P0 but with no chemical exposure, and then transferred to OP50-agar plates. The P0 generation had a dose-dependent decrease in growth, which was associated to a developmental delay. Since mitochondria metabolism is intrinsically different throughout worm development, we stage matched all treatments so that all assays were performed on mid-L4 worms. Stage-matched P0 animals still had a significant decrease in size; however, rotenone exposures did not significantly affect basal, ATP-linked, or maximal oxygen consumption. Furthermore, we did not observe a difference in mitochondrial copy number in the P0 generation post exposure or in later life. In the F1 generation, growth, respiration, and mitochondrial copy number at the L4 stage showed no difference between offspring from exposed and control P0 animals. Since development is known to be affected by mitochondrial dysfunction, these preliminary data suggest that mitochondria may be impaired in the P0 (exposed) animals but recovers in the next generation based on the parameters analyzed under ideal conditions. However, future experiments will include challenging the F1 generation with rotenone exposure, looking at other phenotypes later in life, and transgenerational effects.
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[
Southeast Asian J Trop Med Public Health,
1979]
A total of 2,337 rodents trapped from various parts of Peninsular Malaysia were dissected and studied for the distribution and prevalence of parasitic infections. Four new rodent hosts for Sarcocystis in Malaysia are reported (Bandicota indica, Rattus sabanus Rattus argentiventer and Rattus norvegicus). Sarcocystis was found in 17.2 percent of the rodents examined. Rattus annandalei, Rattus tiomanicus and Rattus norvegicus are new hosts of Syphacia muris in Peninsular Malsysia. Rattus sabanus was found to be infected with Zonorchis borneonenis. Brachylaima ratti Baugh, 1962 was recovered from the small intestine of Rattus rattus diardii for the first time in Malaysia. The prevalence and distribution of other parasites are also discussed.
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[
J Biol Chem,
1990]
The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
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[
Worm Breeder's Gazette,
1994]
R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with
bc1-2, the human homolog to
ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and
bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar
lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with
ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.
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[
Nat Commun,
2021]
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.
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[
Dev Biol,
2024]
While the nervous system of bilaterian animals is mainly left-right (L-R) symmetric at the anatomical level, some molecular and functional L-R asymmetries exist. However, the extent of these molecular asymmetries and their functional consequences remain poorly characterized. C. elegans allows to study L-R asymmetries in the nervous system with single-neuron resolution. We have previously shown that a neural bHLH transcription factor, HLH-16/Olig, is L-R asymmetrically expressed in the AIY neuron lineage and regulates AIY axon projections in a L-R asymmetric manner. Here, by combining a candidate approach and single-cell RNA sequencing data analysis, we identify the ephrin protein EFN-2 and the Flamingo protein FMI-1 as downstream targets of HLH-16 that are L-R asymmetrically expressed in the AIY lineage. We show that EFN-2 and FMI-1 collaborate in the L-R asymmetric regulation of axonal growth. EFN-2 may act via a non-canonical receptor of the L1CAM family, SAX-7. Our study reveals novel molecular L-R asymmetries in the C. elegans nervous system and their functional consequences.
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[
Commun Integr Biol,
2011]
The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.