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[
International Worm Meeting,
2021]
In cases where genetic mutants are not readily available, RNA interference has been used for many years for loss-of-function analyses. Recently, the genome-editing technology has made it possible to fuse a degron tag to endogenous proteins of interest, allowing us to induce their degradation for loss-of-function analyses . The auxin-inducible degron (AID) system is one of the major degron-based technologies that has been used in many research communities, including the C. elegans filed. The pair of a 44-amino acid degron tag (namely AID*) and the TIR1 E3 ligase subunit derived from Arabidopsis thaliana (AtTIR1) has been used in studies using C. elegans. While the AID system is getting popular, two major drawbacks have been reported: 1) leaky degradation of AID-fused proteins in the absence of auxin and 2) the requirement for a high concentration of auxin. Recently, we developed an improved version, namely AID2, that worked in yeast, mammalian cells, and mice. In the AID2 system, we employed a TIR1 mutant derived from rice (OsTIR1(F74G)) and a new ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). In this study, we applied AID2 to C. elegans and compared it with the original AID system. To this end, we inserted a transgene encoding AtTIR1(F79G) into the genome. First, we found that strain expressing AtTIR1(F79G) showed no significant basal degradation. Then, we found 5-Ph-IAA for AID2 induced rapid target degradation with a concentration 1,300 times lower than IAA for AID. Moreover, we developed a modified 5-Ph-IAA that has better permeability. Using this new ligand, we showed that AID2 worked effectively in the embryos surrounded by the eggshell. Our results indicate that the AID2 system is a better option for loss-of-function analyses of C. elegans in the future.
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[
International Worm Meeting,
2021]
Mutation or loss of six collagen genes (
dpy-2, 3, 7, 8, 9, and 10) disrupts annular furrows in adult cuticles and causes a short and wide 'Dumpy' body morphology. Loss of these collagens also activates osmotic, detoxification, and antimicrobial defense genes, but not other stress responses. High environmental osmolarity reduces internal turgor pressure, physically distorts the epidermis, and activates the same stress responses, but non-furrow dpy mutations and other disruptions of the epidermis (e.g., Lon, Rol, Mlt, and Sqt) do not. These results are consistent with a damage sensor associated with furrows in the adult cuticle that regulates responses to environmental stress. Several cuticle characteristics change between molts, but all stages have annular furrows and express furrow-associated collagen genes raising the possibility that this signaling mechanism functions throughout development. We find high variation in stress gene responses to
dpy-7 mutation with the largest induction in adults and little or no induction in early larvae. Alternatively, stress responses are induced by osmotic stress at all stages demonstrating that environmental response mechanisms are functional in early larvae. These results suggest that furrows can develop despite
dpy-7 mutation or that furrows are not essential for stress response regulation in early larvae.
dpy-7 mutants are not Dpy until the L3 stage suggesting that cuticles of early larvae may be able to compensate. We are currently using fluorescent collagen reporters to visualize annular furrows at all stages in
dpy-7 mutants and investigating deletion of a furrow collagen reported to cause Dpy at all stages. This work was supported by NSF grant IOS-1452948 to KPC.
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Kolyvanov, Klim, Harrington, Kyle I S, Preibisch, Stephan, Epstein, Leo, Ercan, Sevinc, Lionnet, Timothee, Breimann, Laura, Bahry, Ella
[
International Worm Meeting,
2021]
Precise quantification of mRNA transcripts in space and time throughout embryogenesis is essential for understanding gene regulation, a process critical for embryogenesis in all animals, including C. elegans. We developed an imaging approach using 3D widefield microscopy-based single-molecule RNA fluorescence in situ hybridization (smFISH) to quantify mRNA transcripts. To count individual single-molecule mRNA spots, we developed RS-FISH, a fast 3D spot detection method that we implemented in Fiji that combines radial symmetry and RANSAC outlier removal. To assign each fixed, imaged C. elegans embryo to its developmental stage, we used advanced machine learning-based image classification that relies on the concept of auto-encoders. Currently, we are applying our methods to understand the role of condensins in chromosome compaction and transcription regulation. In C. elegans, an X-specific condensin binds to and represses X chromosomes in XX hermaphrodites by 2-fold for dosage compensation. In our study, we want to understand condensin DC's effect on transcript numbers and dynamics in single embryos across development. We obtained thousands of smFISH images for a set of condensin DC-regulated and control genes and extracted mature and nascent RNA counts in 3D, which we use to determine transcription burst characteristics throughout embryonic development. The distribution of total transcripts in wild-type and condensin DC-depleted embryos shows that single genes on the X chromosome are downregulated ~2-fold. Our machine learning approach to separate embryo images by development stage allowed us to observe the timing of condensin DC-mediated transcription repression, which occurs from the 100-cell stage on. RS-FISH is freely available as a Fiji plugin, and details for installation can be found at https://github.com/PreibischLab/RadialSymmetryLocalization and described at https://doi.org/10.1101/2021.03.09.434205
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[
International Worm Meeting,
2021]
A-to-I RNA editing is a conserved and prevalent type of RNA modification in which adenosines (A) are converted into inosines (I) by Adenosine Deaminases Acting on RNA (ADAR) enzymes. ADR-2, the only enzymatically active protein in C. elegans, is thought to mainly localize and function in the nucleus. However, it is still not clear whether ADR-2 can shuttle in and out of the nucleus and in which tissues it is expressed. To elucidate the localization of ADR-2, we performed immunofluorescence and RNA-seq experiments. Our results show that ADR-2 is localized to the nuclei and adjacent to the chromosomes during all stages of the cell cycle. We also found that ADR-2 is expressed in all stages and cells during embryo development. Moreover, we examined whether the nuclear localization of ADR-2 would be affected if one of its known regulators, ADR-1 or ADBP-1, were absent. Interestingly, ADR-1 does not affect the localization of ADR-2; however, we validated previous research and show ADBP-1 facilitates the nuclear localization of ADR-2. We also found that ADR-2 mislocalization strongly affects the expression of edited genes in the embryo and to a lesser extent at L4 developmental stage. Taken together, our results suggest that the localization of ADR-2 is highly regulated and likely affects the function of ADR-2.
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[
International Worm Meeting,
2005]
The gene
gei-16 was originally identified as the C. elegans homolog of OvB20, an antigen expressed by the parasitic nematode Onchocerca volvulus. It has since been shown to have a role in embryonic elongation . This gene appears to be nematode-specific, with no clear homolog in humans, mice or flies but clear matches to a range of parasitic nematodes.
gei-16 appears to have at least 19 different splice variants in C. elegans. These can be divided into three major types: long variants, short variants and the d variant. Knockdown by RNAi of all variants in N2 shows a severe phenotype with a high penetrance of Adl, Prz, Unc, Clr and Lva additional to the published Emb, Lva , Gro and Lva phenotypes. RNAi was directed against unique regions of the short and long variants. No obvious phenotype was seen for the short variants, however, knockdown of the long variants produced a severe phenotype identical to that found for knockdown of all variants. This may suggest that the long variants is most important for development. Semi-quantitative RT-PCR shows the long transcript is expressed through all stages of development, but is most highly expressed in the embryonic and early larval stages. Long variant expression levels appear higher than short variant levels in all stages. A GFP reporter construct is currently being made to elucidate the spatial and temporal expression of
gei-16.
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[
European Worm Meeting,
1996]
Glutathione-s-transferases (GSTs) are a large family of multifunctional enzymes involved in the detoxification and excretion of many physiological and xenobiotic substances in the cell. We are investigating the roles members of the sigma class of these enzymes may have in C.elegans in relation to their elimination of oxygen free radical damage which might contribute to cellular ageing. Initially we are examining expression pattern data in C.elegans lines transformed with LacZ reporter gene constructs containing predicted promoter regions of GST gene homologues. The predicted GST gene sequences were obtained from the genome sequencing project. All stages of development are being studied under a variety of environmental contditions to investigate GST gene expression inducibility, however particular attention will be paid to dauer stage larvae.
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[
International Worm Meeting,
2019]
Steinernema Carpocapsae (S. carpocapsae) is a entomopathogenic nematode (EPN) that is widely known to be a "natural insecticide". The infective juvenile (IJ) infect and reproduce in their insect hosts and kill them at their larvae stage together with specific symbiotic bacteria. We are interested in understanding the changes in chromatin openness of S. carpocapsae during development using ATAC-seq and comparing the results to matching stages in C. elegans. We are currently optimizing our protocol in L1 worms and hope to apply to other stages. After successfully replicating ATAC-seq on L1 at arrest of C. elegans and the matching stage of S. Carpocapsae, further comparative genomic in all stages can be studied to identify the conserved regulatory elements between the two species.
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[
International C. elegans Meeting,
2001]
The technique of RNA interference (RNAi) has the potential to expedite the search for new drug targets in parasitic nematodes, and to assist in their validation. The sugar trehalose is claimed to have important functions in the physiology of nematodes whereas it is considered to be unimportant in mammals. In this study we are assessing trehalose metabolism in nematodes, using C. elegans as a model. We have shown that trehalose is synthesised by the nematode and is present at all life cycle stages; highest concentrations were observed in dauer larvae and eggs. The enzyme trehalase, which converts trehalose to glucose, is active at all stages of the life cycle; we have identified three biochemically distinct trehalase activities, each with different properties, in preparations from C. elegans . Four genes encode putative trehalases: F57B10.7, T05A12.2, W05E10.4 and F15A2.2. Using Northern analysis and RT-PCR we have shown that each of these genes is expressed at all life cycle stages. The synthesis of trehalose is catalysed by two enzymes, trehalose 6-phosphate synthase (TPS) and trehalose 6-phosphate phosphatase (TPP). Two genes putatively encoding TPS are present in C. elegans : ZK54.2 and F19H8.1. We have shown by RT-PCR analysis that both genes are expressed at all stages of the life cycle. The gene(s) putatively encoding TPP have not yet been identified. We are using RNAi to examine the effects of temporarily knocking down the expression of these genes, individually and in combination. This investigation has received financial assistance from the Australian Research Council and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.
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Bessereau, Jean-Louis, Tafelmeyer, Petra, Masson, Maryline, Boulin, Thomas, Pintard, Lionel, Formstecher, Etienne
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Yeast two-hybrid (Y2H) protein interaction screening has proven instrumental for the analysis of the C. elegans interactome, thanks to dedicated resources such as ORF collections and oligo dT-primed cDNA libraries. However, map completeness has been limited so far by the use of full-length proteins (ORFeome) or C-terminal polypeptide fragments (oligo dT-primed cDNA libraries) that resulted in significant false negative rates. To circumvent these limitations, we have used a domain-based strategy to construct two highly complex, random-primed C. elegans cDNA libraries. The first library has been constructed by combining equimolar amounts of mRNA from 4 different N2 samples to increase transcript diversity: (1) males and hermaphrodites (all stages including embryos), (2) starved mixed stage culture, (3) heat-shocked mixed-stage culture, (4) dauer stage. The second library was prepared exclusively from C. elegans embryos of all stages. The complexity of both libraries is greater than 10 million independent fragments in yeast, with an average fragment size of 800 bp. To ensure reproducible and exhaustive Y2H results, these libraries are screened at saturation using an optimized mating procedure. This allows to test on average 100 million interactions per screen, corresponding to a 10-fold coverage of the library. As a consequence, multiple, independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score. These two C. elegans libraries have been integrated into our high-throughput yeast two-hybrid platform and are available for screening on a fee-for-service basis. The results of representative screens performed on both libraries will be presented at the meeting.
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[
International Worm Meeting,
2007]
Yeast two-hybrid (Y2H) protein interaction screening has proven instrumental for the analysis of C. elegans interactome, thanks to dedicated resources such as ORF collections and oligo dT-primed cDNA libraries1,2. However, map completeness has been limited so far by the use of full-length proteins (ORFeome) or C-terminal polypeptide fragments (oligo dT-primed cDNA libraries) that resulted in significant false negative rates3. To circumvent these limitations, we have used a domain-based strategy to construct two highly complex, random-primed cDNA libraries. The first library has been constructed by combining equimolar amounts of mRNA from 4 different N2 samples to increase transcript diversity: (1) males and hermaphrodites (all stages including embryos), (2) starved mixed stage culture, (3) heat-shocked mixed-stage culture, (4) dauer stage. The second library was prepared exclusively from C. elegans embryos of all stages. The complexity of both libraries is greater than 10 million independent fragments, with an average fragment size of 800 bp. To ensure reproducible and exhaustive Y2H results, these libraries are screened at saturation using an optimized mating procedure. This allows to test on average 100 million interactions per screen, corresponding to a 10-fold coverage of the library. As a consequence, multiple, independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score4. These two C. elegans libraries have been integrated into our high-throughput yeast two-hybrid platform and are available for screening on a fee-for-service basis. The results of representative screens performed on both libraries will be presented at the meeting. Please send inquiries to: Etienne Formstecher, eformstecher@hybrigenics.com 1. Zhang et al., 2004, Nature Genetics, 36:507 2. Li et al., 2004, Science, 303:540 3. Han et al., 2005, Nature Biotechnology, 23:839 4. Formstecher et al., 2005, Genome Research, 15:376.