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[
WormBook,
2006]
The architecture and dynamics of molecular networks can provide an understanding of complex biological processes complementary to that obtained from the in-depth study of single genes and proteins. With a completely sequenced and well-annotated genome, a fully characterized cell lineage, and powerful tools available to dissect development, Caenorhabditis elegans, among metazoans, provides an optimal system to bridge cellular and organismal biology with the global properties of macromolecular networks. This chapter considers omic technologies available for C. elegans to describe molecular networks - encompassing transcriptional and phenotypic profiling as well as physical interaction mapping - and discusses how their individual and integrated applications are paving the way for a network-level understanding of C. elegans biology.
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[
Worm Breeder's Gazette,
2001]
RNAi is being used routinely to determine loss-of-function phenotypes and recently large-scale RNAi analyses have been reported (1,2,3). Although there is no question about the value of this approach in functional genomics, there has been little opportunity to evaluate reproducibility of these results. We are engaged in RNAi analysis of a set of 762 genes that are differentially expressed in the germline as compared to the soma (4 -- "Germline"), and have reached a point in our analysis that allows us to look at the issue of reproducibility. We have compared the RNAi results of genes in our set that were also analyzed by either Fraser et al. (1 -- Chromosome 1 set "C1") or Gonczy et al. (2 -- Chromosome 3 set "C3"). In making the comparison we have taken into account the different operational definition of "embryonic lethal" used by the three groups. In the C3 study, lethal was scored only if there were fewer than 10 surviving larva on the test plate, or roughly 90% lethal. In our screen and the C1 screen the percent survival was determined for each test. To minimize the contribution of false positives from our set, in our comparison with the C1 set we defined our genes as "embryonic lethal" if at least 30% of the embryos did not hatch, but included all lethals defined by Fraser et al. (> 10%). For our comparison with the C3 set, we used a more restrictive definition of "embryonic lethal" that required that 90% of the embryos did not hatch. (This means that in Table 1, five genes from our screen that gave lethality between 30-90% were included in the not lethal category; one of these was scored as lethal by Gonczy et al.). We have analyzed 149 genes from the germline set that overlap with the C1 set and 132 genes that overlap with the C3 set. The table below shows the number of genes scored as embryonic lethal (EL) or not embryonic lethal (NL) in each study. (Note that these comparisons do not include data from our published collection of ovary-expressed cDNAs.) Table 1. Comparing RNAi analysis of the same genes in different studies. Germline Chromosome 1 Germline Chromosome 3 NL (117) EL (32) NL (97) EL (35) NL (104) 100 4 NL (89) 87 2 EL (45) 17 28 EL (43) 10 33 Overall, the degree of reproducibility is high. The concordance between our results and the published results was 86% with C1 (128/149 genes) and 90% with C3 (120/132). However, we scored a larger number of genes as giving rise to embryonic lethal phenotypes than the other studies did. What does this mean? One possibility is that we are generating a large number of false positives (God forbid!). The other interpretation is that there is a fairly high frequency of false negatives in each screen (4-8% in our screen (2/45; 4/49); 22% in the C3 screen (10/45); and 35% (17/49) in the C1 screen). It is no surprise that the different methods used by the three groups resulted in slightly different outcomes and we can only speculate on which methodological variation contributed most. In comparing our methods to those used in the C3 study we note that our two groups used different primer pairs for each gene; that we tested genes individually while they tested genes in pairs; and that the operational definition of "embryonic lethal" differed. Considering the latter two differences, we speculate that even with pools of two, the competition noted by Gonczy et al. in dsRNA pools could reduce levels of lethality below the 90% cutoff. The major difference between our approach and the C1 approach is feeding vs. injection, raising the possibility that for some genes feeding may be a less effective means of administering dsRNA. Whatever the basis for the difference, these comparisons indicate that genes scored as "non-lethal" in any single study may show an embryonic lethal RNAi phenotype when reanalyzed. It therefore seems useful to have more than one pass at analyzing C. elegans genes via RNAi. We are indebted to P. Gonczy for very useful comments. Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408 , 325-330. Gonczy, P., Echeverri, G., Oegema, K., Coulson, A., Jones, S. J., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E. et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408 , 331-336. Piano, F., Schetter, A. J., Mangone, M., Stein, L. and Kemphues, K. J. (2000). RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Curr Biol 10 , 1619-1622. Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C., Begley, R., Jones, S. J., Davis, E. B., Scherer, S., Ward, S. et al. (2000). A global profile of germline gene expression in C. elegans. Mol Cell 6 , 605-616.
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[
International C. elegans Meeting,
2001]
We are interested in identifying genes that play essential roles in early embryogenesis. Toward this goal, we have been studying the function of ovary expressed genes using RNAi. Here we report on the RNAi analysis of 762 genes selected by microarray studies to have high expression in the ovary. We inject dsRNA representing each gene into 9 worms and follow each worm and its progeny separately. We score for both embryonic and post-embryonic defects. For each gene giving embryonic lethality above 80%, we obtain time-lapse movies of the first 50 minutes of embryogenesis from embryos from three separate affected worms. Our protocol and results differ slightly from other large scale RNAi studies and we will present a comparison of the results. We have found that 31% of the germline enriched genes give rise to embryonic lethal phenotypes. Interestingly, although there is a strong correlation between a high degree of sequence conservation and embryonic lethality, the major lethal class is composed of proteins that are not annotated, thus these RNAi data provide the first functional data for these genes from any genome. Using the microarray data we are exploring the connection between expression patterns and phenotypes. Our analysis thus far allows us to extend a previous observation that genes required for embryogeneis are less likely to map on the X-chromosome. We are categorizing genes using defects seen in early embryogenesis. We expect that genes giving similar embryonic defects will identify components of biochemical pathways and protein complexes allowing us to predict functions for previously uncharacterized genes.
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[
International Worm Meeting,
2003]
Complete genome sequences of model organisms lead to large numbers of predicted genes, of which many are poorly or un-characterized functionally. In recent years various functional genomic projects have been carried out to characterize large numbers of genes at a time, and a large data sets emerging from these projects have been deposited into publicly available databases. However, due to the volume and error-prone property of such data sets, it is not obvious how they can help in elucidating biological processes of interest. Here we demonstrate an application of combined experimental and computational functional genomic approaches to C. elegans early embryogenesis. Proteins required for early embryogenesis were subjected to yeast two-hybrid analysis and putative protein-protein interactions were identified. For a significant number of these putative interactions, both proteins correspond to genes that gave rise to embryonic lethality phenotypes in high-throughput RNAi assays. In addition, gene pairs corresponding to some interactions have been found to exhibit very similar RNAi phenotypes during early embryogenesis, as recorded by differential interference contrast microscopy. In addition, we computationally generated a network of functional associations for these proteins by searching through publicly available data for genes that are either co-expressed across many different conditions, or share similar RNAi phenotypes, or both. Gene products that are specifically linked to proteins that are required for early embryogenesis were identified as potentially involved in this process. By examining the neighborhood cohesiveness of this network, we identified protein pairs of functional similarity for early embryogenesis. We propose that such integration of functional genomic information can be applied to other biological processes and to other organisms as well.
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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]
The eukaryotic innovation of genetic diversification through recombination confers survival advantages by providing the ability to rapidly purge deleterious mutations and fix beneficial mutations in a population. In animals, asexual lineages have evolved independently many times from sexual ancestors, but they are usually short-lived "evolutionary dead ends". Surprisingly, then, some rare asexual lineages are exceptionally long-lived and thus appear to enjoy unusual evolutionary success. Very little is known about the genetic mechanisms that drive transitions from sexual to asexual reproduction, but two common features are modified meiotic programs and altered genome organization. We have previously published the genome and transcriptome of Diploscapter pachys, a parthenogenetic nematode from a long-lived (est. ~18M years) asexual lineage with abridged meiosis and a karyotype of 2n = 2. Our analyses revealed that the D. pachys genome is highly heterozygous and resulted from end-to-end fusions of ancestral chromosomes. D. pachys appears to lack clusters of ancestral telomeric repeats (TTAGGC) and canonical telomere maintenance proteins found in yeast, mammals and C. elegans - suggesting that its chromosomes may have atypical ends. D. pachys also appears to skip meiotic recombination and the reductional meiotic division, and several genes for homologous pairing and recombination are not detected in the current assembly. To better understand the evolutionary trajectory of D. pachys from sexual reproduction to parthenogenesis, we are undertaking a comparative analysis of genome evolution across the clade of parthenogenetic Diploscapter/Protorhabditis species, as well as a related sexual outgroup. For each species, we are in the process of generating phased diploid chromosome-level assemblies using long-read DNA and RNA sequencing complemented with chromatin conformation capture. This will allow us to establish the patterns of chromosomal fusions and heterozygosity, which will inform hypotheses on their evolutionary history and potential crossover suppression through genomic rearrangements. With our new assemblies and transcriptomes, we will also define the nature of the chromosome ends as well as the complete repertoire of telomeric and meiotic genes in this clade. Finally, we plan to determine how the expression patterns of heterozygous alleles have evolved to adapt to non-recombining diploid genomes of the parthenogens in the Diploscapter/Protorhabditis clade.
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[
International C. elegans Meeting,
2001]
Since the complete genome sequence of C. elegans became available at the end of 1998, several functional genomics projects have been initiated to functionally annotate the many predicted genes that had remained previously uncharacterized. The data generated by these projects can be viewed as hypotheses until validated further. We have proposed that the likelihood of such hypotheses to be biologically meaningful might increase as the data from different projects are integrated. For example, genes that cluster in expression profiling, protein interaction maps and phenotypic analysis might have a relatively high likelihood to functionally interact in the same biological process. Recently, microarray analysis has led to the identification of a set of genes that show increased expression in the germline (Reinke et al, Mol. Cell. 6, 605-616, 2000). We have cloned these ~750 open reading frames and transferred them into yeast two-hybrid vectors using Gateway cloning. Subsequently, we have generated a 750 X 750 protein interaction map. The data obtained and its integration with the RNAi data obtained by Fabio Piano and colleagues will be discussed.
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[
International Worm Meeting,
2009]
MEL-28/ELYS is a large AT-hook protein required for nuclear envelope integrity and chromosome segregation in metazoans. As expected by its fundamental function, MEL-28 is ubiquitously expressed in all cells analyzed. However, mutations in the
mel-28 gene are maternal-effect, causing embryonic lethality in the progeny of homozygous mutant mothers that are otherwise wild-type. Therefore, the function of MEL-28 in most cells is predicted to be buffered by other molecules. To identify additional proteins working with MEL-28 we looked for synthetic phenotypes in
mel-28 homozygous animals using RNAi. The challenge in this type of screen, as with any modifier screen of mel mutants, is to collect large numbers of homozygous animals. To accomplish this in high throughput, we generated a strain with the
mel-28(
t1684) mutation balanced by a GFP-marked chromosome and used a fluorescence-activated cell sorter (FACS) to isolate non-fluorescent
mel-28 homozygous L1 larvae from the rest of the population. Using this approach we have collected, in one sitting, as many as 100,000 larvae, over 99% of which are
mel-28 homozygous. We subjected the
mel-28 larvae to RNAi in 96-well plates and recorded results by high-throughput digital imaging. We present here the results from screening essentially all the genes encoded on Chromosome I. Of the 2260 clones tested, we found 14 that are synthetic sterile with
mel-28. Among these genes we found members of expected molecular complexes. For example, most the nucleoporins known to be on Chromosome I were uncovered. In addition, we find other classes of proteins, like histones, as genetic interactors of
mel-28 suggesting new molecular connections between MEL-28 and chromatin organization. Our results also show that FACS-RNAi screening is a powerful way to uncover tissue-specific roles for pleiotropic genes.
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[
International Worm Meeting,
2011]
Cryptic genetic variation (CGV) is allelic variation that affects phenotype, but only under certain conditions: when the system is "perturbed" by changes in the environment or genomic background. Such conditional effects are probably common in biological systems, but they pose barriers to the identification of causal alleles that underlie complex traits.
In an to effort understand the nature of CGV, we are exploring the genetic architecture of early embryogenesis in C. elegans. Genome-wide screens have identified genes that affect embryogenesis in a single wild-type background (N2), providing a high degree of resolution in our understanding of the genetics underlying this process. We are utilizing this information to knock down embryonic genes in wild isolates, in order to identify natural allelic variants that affect early embryogenesis in perturbed animals. Embryogenesis is normally invariant, but using RNAi to silence critical embryonic genes reveals differences in embryonic lethality across strains.
We have used high-throughput phenotyping methods to evaluate differences in hatching across 64 wild C. elegans strains, silenced at 43 different genes. The patterns of lethality indicate significant levels of CGV for embryogenesis. Some genes reveal high variance in lethality, suggesting that these loci are particularly good perturbation targets for revealing CGV elsewhere across the genome. We also observe significant variation in sensitivity to germline RNAi in these worms.
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[
International Worm Meeting,
2015]
Even a well-studied gene might have important functions in development that are masked by contributions of other genes. MEL-28/ELYS plays key roles in post-mitotic rebuilding of the nuclear envelope and in chromosome segregation. In C. elegans
mel-28 mutants show strict maternal-effect lethality, suggesting that MEL-28 is required in the embryo but is dispensable post-embryonically. To identify post-embryonic roles for MEL-28, we performed an RNAi-based genetic interaction screen, seeking genes that cause novel phenotypes in the
mel-28 mutant background. We identified multiple interactors, including genes that encode components of dynein, a minus-end directed motor responsible for the movements of multiple intracellular cargoes, and dynactin, a complex that helps couple dynein to its cargo. To characterize the interactions, we generated double mutants between
mel-28 and
dhc-1 (which encodes the heavy chain of dynein) or
dnc-1 (which encodes the
p150 subunit of dynactin).
dhc-1;
mel-28 double mutants produce a drastically reduced brood size compared to each single mutant. In addition,
dhc-1;
mel-28 males are infertile, and hermaphrodites have a disorganized proximal gonad and ovulation defects. None of these defects are present in either of the single mutants, suggesting that
mel-28 and
dhc-1 redundantly contribute to germ-line function. The
dnc-1 single mutant has a decreased brood size compared to the wild type and lays many unfertilized oocytes, suggesting defects with fertilization. Disruption of
mel-28 rescues the brood size and fertilization defects of
dnc-1 animals. This suggests that the dynactin complex and MEL-28 act antagonistically in C. elegans fertilization. In conclusion, we have shown that MEL-28 activities intersect with dynein and dynactin in the gonad, and in so doing we have revealed a novel role for MEL-28 in C. elegans fertility.