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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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[
Dev Cell,
2005]
The functional module is fast becoming the operational unit of the postgenomics era. A new report in Nature by Gunsalus and colleagues describes, using a multiply supported network, functional modules within early C. elegans embryos and identifies several new components of known molecular machines ().
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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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Gutwein, Michelle, Mecenas, Desirea, Piano, Fabio, Scheid, Paul, Ahmed, Rina, Gunsalus, Kris
[
International Worm Meeting,
2013]
Post-transcriptional regulation of gene expression is largely mediated through sequence elements in the 3'UTR of protein-coding genes. One of our major interests is to understand post-transcriptional regulatory mechanisms during development. The C. elegans germline provides an excellent model to study post-transcriptional regulation because it is the primary determinant of gene expression in this organ (Merritt et al., Curr Biol 2008). We would like to characterize differences in 3'UTR isoform usage throughout gametogenesis by profiling 3'UTR ends in mitotic and meiotic regions of the gonad as well as in oocytes as a first step toward analyzing the contribution of putative regulatory elements in different regions of 3'UTRs.
Next-generation sequencing has been useful to provide a deep sampling of transcriptional landscapes. However, transcript 3' termini are under-represented using standard RNA-seq library preparation protocols, rendering targeted analysis challenging. A number of specialized protocols to characterize the exact position of 3'end cleavage and polyadenylation have been developed, but they generally require larger sample sizes than are practical to extract from very specific tissues or cells in model organisms like C. elegans.
Our aim is to develop new protocols that optimize 3'UTR endpoint analysis using small sample sizes for tissue-specific profiling with RNA-seq analysis. We have designed and tested a library preparation protocol for linear amplification and 3' end capture followed by deep sequencing on the Illumina HiSeq. We perform paired-end sequencing using a non-standard protocol designed specifically to avoid issues with sequencing the low-complexity polyA tail of transcripts. Here we present our progress in using this protocol with samples of RNA extracted from whole male and hermaphrodite N2 gonads, as well as dissected mitotic and meiotic regions and oocytes from hermaphrodite gonads in order to characterize 3'UTR diversity during germline development.
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[
International Worm Meeting,
2013]
To thoroughly explore the genetic architecture of early embryogenesis in C. elegans, we are searching for conditional relationships between embryonic genes. Genome-wide screens have identified genes that affect embryogenesis in a single wild-type background, providing a lot of information about the genetics underlying the process; we are leveraging this information to probe natural genetic variation across many wild C. elegans isolates. We have silenced a suite of 43 critical embryonic genes in 50 wild strains and characterized differences in lethality across strains. We observe pervasive "cryptic genetic variation" (CGV) for embryogenesis---that is, variation within the networks controlling early cell divisions that has functional consequences only under particular conditions. We find that disrupting genes responsible for polarizing the embryo in the first two cell divisions (PAR family members) uncover particularly high levels of CGV. Using association and linkage mapping, we find that very rarely are cryptic variants uncovered by different silenced genes, even among genes that interact closely and produce the same mutant phenotype. These results imply that CGV for embryogenesis has low pleiotropy, and likely point to new components in the embryogenesis gene network.
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Piano, Fabio, Paul, Florian E., Gunsalus, Kris, Chen, Jia-Xuan, Selbach, Matthias, Mana, Miyeko
[
International Worm Meeting,
2013]
Protein-protein interactions (PPIs) are crucial for most biological processes. Many proteins exist as components of dynamic protein complexes which execute a plethora of cellular functions. Most studies on PPIs have so far used cell culture methods. Studying PPIs in an in vivo system provides the chance to reveal important information in a specific and functionally relevant biological context. To gain insights into the complex biological processes during early development of C. elegans, we developed an in vivo quantitative proteomics approach to study PPIs using C. elegans embryos. We combined label-free quantitative mass spectrometry with co-immunoprecipitation plus transgenic techniques to screen for the interaction partners of some key player proteins during early embryogenesis. In a proof-of-principle experiment using a P-granule component protein (CAR-1) as bait, we identified a number of novel putative interactions along with known interaction partners of CAR-1 and many other previously known P-granule components. A benchmark analysis confirmed the accuracy of our label-free quantification, although false positives remain a potential problem. Further bioinformatic analyses are consistent with previous findings that CAR-1 is involved in embryonic cell division, germ cell development and P-granule related processes and also suggest novel roles of CAR-1 during embryogenesis. These results provide interesting candidates for follow-up experiments to explore new functional roles of CAR-1 during embryogenesis and suggest our approach can be adapted to studying interaction partners of a wider range of proteins in vivo.
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[
Development & Evolution Meeting,
2006]
Using combined network analysis of large-scale functional genomic data we mapped multi-protein modules required for distinct processes during early embryogenesis (Gunsalus et al. 2005). A basic question is how these molecular modules are coordinated through the mitotic cycle to ensure the proper unfolding of early developmental events. To identify proteins that could coordinate different modules we searched for proteins that bridged different modules. One such protein, C38D4.3, could be placed in either the nuclear pore complex module or in the chromosome maintenance module by a network clustering algorithm M-CODE (Bader et al. 2003). Consistent with its predicted roles at the nuclear pore and in chromosome segregation, GFP fusions and anti-C38D4.3 immmunolocalizations show that C38D4.3 shuttles between the nuclear envelope and the kinetochore during the cell cycle. Functionally, C38D4.3 is required for proper nuclear envelope and chromatin maintenance. C38D4.3 (RNAi) embryos, like embryos without nuclear pore components, are incapable of completely separating cytoplasm from nucleoplasm, failing to exclude microtubules and affecting the nuclear localization of PIE-1, a protein normally enriched in the P1 nucleus (Mello, 1996). Additionally, pronuclei fail to meet, and centrosomes do not remain attached to the paternal pronucleus and segregate prematurely. In these embryos, metaphase spindles are not established and chromatin neither condenses, congresses, nor segregates properly. These phenotypes are reminiscent of RNAi phenotypes of genes from the Ran GTPase cycle (Askjaer 2002). Looking for C38D4.3 (RNAi) phenotypic neighbors using PhenoBlast (Gunsalus et al 2004) or phenoclusters from large-scale RNAi analyses (Sonnichsen et al 2005; Gunsalus et al 2005) we identified ~25 other genes with similar defects when analyzed by time-lapse Nomarski microscopy. Of these, genes that are part of the RanGTPase pathway (
ran-1,
ran-2, and
npp-9) were required for proper C38D4.3 localization. Thus C38D4.3 is critical for both mitotic and interphase cell functions and is a likely target of the Ran GTPase pathway.
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Munarriz, Eliana, Amelia, White, Cipriani, Patricia, Julie, Young, Huey-Ling, Kao, Erickson, Katherine, Piano, Fabio, Gunsalus, Kris C
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
To uncover the structure of the underlying genetic networks during early embryogenesis and to identify new components and pathways that participate in these processes, we set up a system to systematically test for enhancing and suppressing interactions using RNAi. We are using 24 available temperature sensitive (ts) alleles whose strong loss-of-function phenotype affects the early embryo and genome-wide RNAi. We conducted a pilot study with an RNAi test set of about 2000 genes. Of this set ~ 400 genes composed a constant set that was tested against all strains, and ~100 genes were specific for each mutant. Several bioinformatics criteria were used in the selection process of this group of genes to try to increase the likelihood of finding positive interactions. From this initial screen, we identified 463 enhancing and 133 suppressing high confidence genetic interactions. Most of these interactions were not previously predicted or known. We are currently expanding the number of interactions in the second stage of this project and we will report on the progress of this screen as well as on the automatic scoring of the images produced.
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[
International Worm Meeting,
2007]
Untranslated regions (UTRs) are found at the 5‘ and 3 flanking ends of transcribed RNAs and contain elements important for the post-transcriptional regulation of the RNA. UTRs are implicated in the control of gene expression through interaction with regulatory proteins and with small non-coding RNAs, such as miRNAs. These interactions can inhibit translation or alter the stability of the messenger RNA resulting in a decrease of protein levels. Computational predictions suggest that each miRNA controls a network of proteins through interaction with consensus sequences in their respective 3 UTRs; thus collectively miRNAs likely regulate the expression of thousands of transcripts. Here we aim to begin to study the biology of 3 UTRs. We developed a high throughput approach to clone all 3‘ UTRs present C. elegans into a vector that can easily be used for downstream in vivo testing. Using a 3 RACE strategy, we amplify 3‘ UTRs from total RNA obtained from mixed developmental stages. We began this project by cloning the 3‘ UTRs found in both the ORFeome and the PROMOTERome databases. Initial results suggest that our strategy will lead to over 80% of 3UTRs in this set cloned and sequenced verified. Preliminary analyses also suggest that for over 20% of the mRNAs there are bona fide alternative 3UTRs. Given the strategy we are using to identify 3UTRs we will have in C. elegans a collection of clones that can be used to assemble a gene locus in the three componenent parts: the promoter, the ORF and 3UTR in a modular way which we can use in a variety of downstream analyses.
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Stoeckius, Marlon, Selbach, Matthias, Dieterich, Christoph, Kirchner, Marieluise, Thierfelder, Nadine, Chen, Wei, Maaskola, Jonas, Rajewsky, Nikolaus, Gunsalus, Kris C
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Development can be interpreted as the regulated expression of genes over lifetime of an organism. Transcription profiling has been extensively used to measure changes in mRNA levels during animal development. However, the final products of most genes are proteins and it is not clear how informative mRNA expression patterns are for the protein level. Another fundamental open problem for understanding development is the question how mRNA and protein levels are conserved during evolution and how conservation of these levels relate to each other. These questions have been impossible to address on a genome-wide level since accurate and yet global quantification of proteins in vivo is technically challenging. Thus, a developmental proteome has not yet been obtained for any multicellular organism. To overcome these problems, we developed a new method which allowed us to accurately measure changes in abundance of thousands of proteins in C. elegans and C. briggsae. Similar to SILAC (Stable Isotope Labelling by Amino acids in Cell culture), our method is based on the metabolic incorporation of stable isotope containing amino acids. We generated heavy SILAC worms by cultivating them on a diet of SILAC labelled E. coli. A mixture of heavy SILAC worms from different developmental stages contained a wide range of isotopically labeled proteins that served as an internal standard for accurate mass spectrometry-based quantification. This approach enabled us to identify and quantify changes in expression levels of thousands of proteins (FDR<1%) across six major developmental stages (mixed embryos, larval stages L1 through L4, adult hermaphrodites) for C. elegans and C. briggsae. In parallel, we quantified changes in mRNA levels by massive paired-end next generation sequencing in the same samples. We will report on the currently ongoing analyses of our proteome and transcriptome profiling, including cross-species comparisons of developmental regulation of gene expression