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[
European Worm Meeting,
2004]
Vulval development has been extensivly studied and is a good model system to study signalling in a metazoan. There are six vulval precursor cells that develop to form the 22 cells of the final vulva. This requires complex signalling involving the EGFR-ras-raf-MAPK, LIN-12/NOTCH and Wnt pathways. We wished to use RNAi to identify novel components of signalling pathways regulating vulval development and morphogenesis. Since any one screen is unlikely to capture all of the genes involved, we are carrying out multiple large-scale RNAi screens and combining the data from all of them to obtain as comprehensive a picture as possible of the components. These include screens in the
rrf-3 background, screens with a variety of GFP reporters as well as screens in sensitised backgrounds such as in strains with gain-of-function
let-60-ras or loss-of-function
lin-31. We will present the results of several of these screens.
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[
European Worm Meeting,
2006]
Catriona Crombie and Andrew Fraser. In C. elegans, genome-wide RNAi screens are a powerful way to identify genes involved in any given process. We are seeking to use RNAi screens in the worm to find as comprehensively as possible the genes involved in different signalling pathways and the points of cross talk between the pathways.. C. elegans vulval development is well characterised: the 22 cells of the fully developed vulva are derived from 6 precursor cells and this is already known to require the EGFR-ras-raf-MAPK, NOTCH, and Wnt signalling pathways. Thus in this simple tissue we can examine multiple conserved signalling pathways, all of which can play major roles in cancer when deregulated. We have carried out multiple RNAi screens to identify new genes that modulate signalling in the vulva. These include screens in the
let-60 (
n1046) strain, which has a gain of function mutation in the ras ortholog
let-60. ~50% of
let-60 (
n1046) worms are Muv and we have used RNAi to screen for both enhancers and suppressors of this phenotype. We have also screened for suppressors in strains that are 100% Muv such as
lin-15 (
n765) and
lin-1 (
e1026). Finally we have screened for genes that have a Muv RNAi phenotype in wild type strains. We have found several novel genes that appear to repress ras signalling along with several downstream effectors of signalling including transcription factors. The human orthologues of genes identified in these screens may play a role in cancer. We will present these results.
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[
International Worm Meeting,
2019]
Caenorhabditis elegans is an androdioecious nematode with hermaphrodites and males. A self-fertilizing hermaphrodite rarely produces males, with frequencies of approximately 0.1% in the laboratory strain N2. By contrast, a hermaphrodites fertilized by males have 50% male progeny. Many studies have suggested that self-fertilization is the primary reproduction mode of C. elegans in nature, and the transition from obligately outcrossing might be recent (Cutter et al., 2008). Natural variation of male frequencies were also observed in C. elegans, ranging from 0.1% to 35% (Teotonio et al., 2006; Anderson et al., 2010). In a recent sampling of Caenorhabditis in Hawaii (see abstract by Crombie et al.), we isolated a C. elegans strain, named ECA701, which exhibited extremely high male frequencies of 34% - 57% and low brood sizes of 80 - 170 by selfing of hermaphrodites. Mating of ECA701 between hermaphrodites and males showed male frequencies of 47% - 70% and brood sizes of 150 - 290. We further found that ECA701 produced embryos with high levels of embryonic lethality (30% in young adults), which is exacerbated in older adults (60% in two-days old adults). Investigations on meiotic prophase I demonstrated that ECA701 is defective in the meiotic processes (see abstract by Adler et al.). Notably, using whole genome sequencing and phylogenetic analysis, we also found that ECA701 is one of the most divergent C. elegans strains that have yet been described. Taken together, ECA701 provides a great opportunity to improve our understanding on the possible ancestral state and the evolution of reproduction mode in C. elegans.
-
[
International Worm Meeting,
2005]
In C. elegans, genome-wide RNAi screens are a powerful way to identify genes involved in any given process. We are seeking to use RNAi screens in the worm to find as comprehensively as possible the genes involved in different signalling pathways and the points of cross talk between the pathways. We are beginning our analysis with RNAi screens to find genes that affect vulval development. C. elegans vulval development is well characterised: the 22 cells of the fully developed vulva are derived from 6 precursor cells and this is already known to involve the EGFR-ras-raf-MAPK, NOTCH, and Wnt signalling pathways. Thus, in this simple tissue we can examine multiple conserved signalling pathways, all of which can play major roles in cancer when deregulated. We have carried out multiple RNAi screens to identify new genes that modulate signalling in the vulva. We have found over 60 genes that appear to modulate ras signalling along with several downstream effectors of signalling including transcription factors. We will present these results.
-
[
European Worm Meeting,
2006]
Ben Lehner, Catriona Crombie, Julia Tischler, Angelo Fortunato and Andrew G. Fraser Most heritable traits, including disease susceptibility, are affected by the interactions between multiple genes. However, we still understand very little about how genes interact since only a minute fraction of possible genetic interactions have been explored experimentally. To begin to address this, we are using RNA interference to identify genetic interactions in C. elegans, focussing on genes in signalling pathways that are mutated in human diseases. We tested ~65,000 pairs of genes for possible interactions and identify ~350 genetic interactions. This is the first systematically constructed genetic interaction map for any animal. We successfully rediscover most components of previously known signalling pathways; furthermore, we verify 9 novel modulators of EGF signalling. Crucially, our dataset also provides the first insight into the global structure of animal genetic interaction maps. Most strikingly, we identify a class of highly connected ''hub'' genes: inactivation of these genes greatly enhances phenotypes resulting from mutations in many different pathways. These hub genes all encode chromatin regulators, and their activity as genetic hubs appears conserved across metazoans. We propose that these genes function as general buffers of genetic variation and that these hub genes will act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.
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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.
-
[
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.
-
[
International Worm Meeting,
2009]
Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven 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 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. 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 seven 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. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. 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 few years. There is no indication that we are even close to knowing all species in this genus.