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[
International C. elegans Meeting,
1999]
Nuclear hormone receptors (NHRs) are known to be essential for developmental processes in a wide variety of multicellular organisms, including C. elegans . One extensively studied example of the developmental role of NHRs is in the control of molting and metamorphosis in Drosophila . Molting and metamorphosis in Drosophila are regulated by the steroid hormone 20-OH ecdysone, hereafter referred to as ecdysone. The ecdysone signal is transduced by NHRs. Nematodes also undergo a series of molts during development. Molting from the third to the fourth larval stage of the filarial parasite Dirofilaria immitis (the causative agent of dog heartworm disease) can be prematurely induced in vitro by ecdysone, suggesting that ecdysone, or a related compound, may play a role in the development of this nematode. Putative homologs of the EcR and usp genes, which encode NHRs that form the functional ecdysone receptor in Drosophila , have been identified in D. immitis . The ecdysone signal in Drosophila is modulated by a number of transcription factors that are induced as a primary response to ecdysone. We have identified a putative D. immitis homolog of the Drosophila E75 ecdysone primary response gene. E75 encodes multiple isoforms and is essential for metamorphosis in Drosophila . The protein encoded by the D. immitis E75 homolog, dinhr-6, is 83% identical to E75A in the DNA binding domain. We cloned the 5' end of dinhr-6 and determined that the transcript uses the SL1 spliced leader. We are currently using a variety of techniques to clone the 3' end of the gene and have identified the complete DNA and ligand binding domains. Northern blot analysis suggests that dinhr-6 encodes multiple isoforms and is female-specific in adults. A putative E75 homolog,
nhr-85 , was recently identified in C. elegans (Ann Sluder, personal communication). We discuss the potential of using C. elegans to help determine the function of parasite proteins.
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[
International Worm Meeting,
2007]
The Wnt signaling pathway is highly conserved throughout evolution and plays an integral part in both development and homeostasis of a diverse range of metazoan organisms. The pathway has been shown to regulate such processes as cell polarity, migration and fate determination. Mis-activation of the pathway has been implicated in both birth defects and disease in humans. We are interested in the role Wnt signaling plays in development, specifically in the regulation of cell fate determination. In C. elegans, homologs for most of the core vertebrate Wnt pathway components have been identified. However, only a handful of the worms Wnt target genes have been found. Wnt signaling is required for both embryonic and larval development. And our lab has shown that a beta-catenin dependent Wnt signaling pathway is involved in fate specification of the epithelial Vulva Precursor Cells (VPCs) and certain Ventral Cord Neurons (VCNs). The aim of my research is to identify Wnt pathway gene targets in the VPCs, VCNs and other cells. I used Affymetrix microarray analysis to identify differentially expressed genes in worms subjected to Wnt pathway wild-type activation vs. hyper-activation. The hyper-activated state was induced using a stable variant of the transcriptional activator BAR-1/beta-catenin. 117 up- and 72 down-regulated genes were identified (2-fold greater or lesser that wild-type expression levels). qRT-PCR analysis of these gene candidates, comparing expression levels under conditions of wild-type Wnt pathway activation to both pathway hyper- or hypo-activation, showed that 30% of the up-regulated microarray identified targets to be bona fide Wnt responsive genes. Of these, 18% are cuticular collagen genes and 9% are dauer related genes 41% are novel genes. I am currently characterizing the spatial and temporal expression patterns of these gene targets and analyzing them for altered expression when subjected to conditions of pathway hyper- or hypo-activation. In order to determine their biological function, future work will include analysis of phenotypic effects caused by RNA interference and/or mutation or deletion of these genes. Characterization of the Wnt responsive genes identified in this study will expand our knowledge of the genetic controls involved in cell fate determination.
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Reboul J, Jackson C, Brasch MA, Vidal MI, Thierry-Mieg J, Temple GF, Matthews LR, Hartley JL, Vaglio P, Tzellas N, Moore T
[
European Worm Meeting,
2000]
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Two basic strategies have been developed for the characterization of (nearly) complete sets of proteins (the proteome). In the "bottom-up" approach, endogeneous proteins expressed in vivo are analyzed using high-throughput techniques such as mass spectrometry (MS) or matrix-laser desorption/ionization (MALDI). In the "top-down" approach, (nearly) complete sets of ORFs (ORFeomes) are expressed exogeneously to perform various protein function assays such as large-scale two-hybrid analysis, biochemical assays, protein production and purification for structural analyses, etc... The "top-down" approach relies upon the availability of ORFeomes cloned into various expression vectors (i.e., for each ORF: the sequence between the start and the stop codons, in the absence of 5 and 3 untranslated sequences and introns). To clone the C. elegansORFeome into various expression vectors, we use the Recombination Cloning technique GatewayTM . This system, first developed by Life Technologies Inc. (Hartley et al., submitted; Walhout et al., 2000, Science, 287, 166-122; Walhout et al., 2000, Methods in Enzymology, in press), allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro . The features of RC make it amenable to automation and 96-well (or 384-) plates settings, which is crucial for large-scale ORFeome cloning. So far we have cloned 1500 C. elegans ORFs. With our current throughput, 3,000 ORFs (15% of the ORFeome) should be cloned by the time of the European C. elegansmeeting and ~70% of the ORFeome should be cloned by the end of year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a description of the cloning quality, iv) how this resource will be made available to the community, and v) how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et al.). The C. elegansORFeome project is likely to illustrate how to undertake ORFeome cloning projects for more complex multicellular organisms.
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Vidal MI, Tzellas N, Moore T, Vaglio P, Thierry-Mieg D, Jackson C, Temple GF, Thierry-Mieg J, Brasch MA, Hartley JL, Reboul J
[
East Coast Worm Meeting,
2000]
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Most of protein-based approaches rely upon the availability of near complete set of open reading frames ("ORFeomes") cloned into various expression vectors (i.e., for each ORF: the sequence between the start and the stop codons, in the absence of 5 and 3 untranslated sequences and introns). To clone the C. elegans ORFeome into various expression vectors, we use a Recombination Cloning technique (RC) referred to as GatewayTM (Walhout et al., 2000, Science, 287, 166-122). RC allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. In addition, RC is amenable to automation in 96-well (or 384-) plate settings, which is crucial for large-scale ORFeome cloning. So far we have cloned 2,000 C. elegans ORFs. At the current throughput (~400 ORFs/week), ~70% of the C. elegans ORFeome should be cloned by the end of the year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a description of the cloning quality, iv) how this resource will be made available to the community, and v) how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et al).
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Hartley JL, Moore T, Temple GF, Thierry-Mieg D, Vaglio P, Brasch MA, Tzellas N, Thierry-Mieg J, Reboul J, Jackson C, Vidal MI
[
West Coast Worm Meeting,
2000]
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Most of protein-based approaches rely upon the availability of near complete set of open reading frames ("ORFeomes") cloned into various expression vectors (i.e., for each ORF: the sequence between the start and the stop codons, in the absence of 5 and 3 untranslated sequences and introns). To clone the C. elegans ORFeome into various expression vectors, we use a Recombination Cloning technique (RC) referred to as GatewayTM (Walhout et al., 2000, Science, 287, 166-122). RC allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. In addition, RC is amenable to automation in 96-well (or 384-) plate settings, which is crucial for large-scale ORFeome cloning. So far we have cloned 2,000 C. elegans ORFs. At the current throughput (~400 ORFs/week), ~70% of the C. elegans ORFeome should be cloned by the end of the year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a description of the cloning quality, iv) how this resource will be made available to the community, and v) how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et al).
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[
Development & Evolution Meeting,
2006]
Wnt signaling functions in multiple developmental processes in the model organism C. elegans. Homologs of β-catenin and TCF/LEF are BAR-1 and POP-1, respectively. POP-1 functions in endoderm induction, Q-neuroblast migration, P12 and vulva precursor cell fate specification, and T cell and somatic gonad precursor cell polarity. We are taking two approaches to identify Wnt target genes in these developmental processes. First, by performing microarray analysis on worms that have been induced to over-express key Wnt pathway components (BAR-1 and POP-1) we will reveal target genes that are up or down regulated by Wnt signaling (see poster by Belinda Jackson). Second, I am using a selection and amplification method with the DNA-binding domain of POP-1 to identify genomic binding sequences. Fragments bound by POP-1 utilizing this technique will be subjected to alignment and sequence comparison with the C. elegans genome to identify putative gene targets. Candidate target genes regulated by POP-1 will be verified by inserting the promoter of genes in question upstream of the
pes-10 basal promoter::GFP vector and injected into worms. Lines expressing GFP will be subjected to
pop-1 RNAi to identify sequences regulated by POP-1 in vivo. Additionally, reporter constructs will be made containing mutated POP-1 consensus binding sites to verify that the consensus site(s) are needed.
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[
West Coast Worm Meeting,
2002]
To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.
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[
International Worm Meeting,
2019]
C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.
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[
Development & Evolution Meeting,
2008]
Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.
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[
International Worm Meeting,
2015]
Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.