Roy PJ et al. (2005) Dev Cell "Network news: functional modules revealed during early embryogenesis in C. elegans."

Roy PJ, Morris Q

[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 ().

Chung, George et al. (2021) International Worm Meeting "Genomic mechanisms of asexual reproduction"

Piano, Fabio, Gunsalus, Kris, Chung, George

[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.

Paaby, Annalise et al. (2011) International Worm Meeting "Uncovering cryptic genetic variation for C. elegans embryogenesis."

Paaby, Annalise, Piano, Fabio, Gunsalus, Kris, White, Amelia, Rockman, Matt

[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.

Paaby, Annalise B. et al. (2013) International Worm Meeting "C. elegans harbors pervasive cryptic genetic variation for embryogenesis."

Gunsalus, Kris, Paaby, Annalise B., Piano, Fabio, Rockman, Matt, White, Amelia

[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.

Gutwein, Michelle et al. (2013) International Worm Meeting "3' End Profiling of Gametogenesis in C. elegans Using RNA-Seq."

Ahmed, Rina, Gutwein, Michelle, Scheid, Paul, Mecenas, Desirea, Gunsalus, Kris, Piano, Fabio

[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.

Anita Fernandez et al. (2006) Development & Evolution Meeting "A Network Model of C. Elegans Early Embryogenesis Identifies a Novel AT-Hook Protein that Coordinates Nuclear Envelope Function and Chromatin Maintenance"

Fabio Piano, Anita Fernandez

[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.

Chen, Jia-Xuan et al. (2013) International Worm Meeting "Analysis of protein-protein interaction by in vivo quantitative proteomics during C. elegans embryogenesis."

Mana, Miyeko, Piano, Fabio, Chen, Jia-Xuan, Selbach, Matthias, Gunsalus, Kris, Paul, Florian E.

[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.

Marco Mangone et al. (2007) International Worm Meeting "The 3'UTRome project: A detailed genomic annotation of 3'UTRs of C. elegans."

Marco Mangone, Kourosh Salehi-Ashtiani, Hill David, Nikolaus Rajewsky, Philip MacMenamin, Gunsalus Kris, Piano Fabio, Vidal Marc

[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.

Mangone, Marco et al. (2009) International Worm Meeting "The UTRome project: A resource to study 3'UTR biology in C.elegans."

Mangone, Marco, Vidal, Marc, Piano, Fabio, Zegar, Charles, Gunsalus, Kris, MacMenamin, Philip, Attie, Oliver, Mis, Emily, Salehi-Ashtiani, Kourosh

[International Worm Meeting, 2009]

Three-prime untranslated regions (3''UTRs) are widely recognized as important post-transcriptional regulatory portions of mRNAs. RNA-binding proteins and small non-coding RNAs such as microRNAs (miRNAs) bind to functional elements within 3''UTRs to influence mRNA stability, translation and localization. To characterize and clone C.elegans 3''UTRs, we have developed a high throughput 3''RACE strategy and have characterized an initial target set of 7,014 genes. For each gene, we use a transcript-specific forward primer and a universal polydT(23) anchored reverse primer. The primers are designed to generate products compatible with the Promoterome and ORFeome resource. We cloned the 3''UTRs into an entry vector ready to be used for the third position in the multi-site Gateway system suitable for downstream functional analysis. In our first cycle, we cloned 3''UTRs of ~ 6,000 genes and sequence verified these as mixed bacterial transformants (or "minipools"). For half of these genes, the analysis of their 3''ends identified new 3''UTRs compared to data present in Wormbase WS190 (http://wormbase.org). We subsequently separated these minipools into ~56,000 isolated colonies and re-sequenced our library by deep sequencing (Illumina/Solexa and 454/Roche). We obtained unique cloned 3''UTR isoforms for over 90% of our target set, and observed multiple 3''UTR isoforms for over half. Most of the alternative isoforms are produced by differential PAS site usage. Our data and annotations are being deposited into the modENCODE website (http://www.modencode.org), and are also viewable in our 3''UTR-centric website (http://www.utrome.org). We will present the status of this project and its applications to study 3''UTR biology in C.elegans.

Cipriani, Patricia et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A network of suppressors and enhancers of early embryogenesis in C. elegans"

Erickson, Katherine, Piano, Fabio, Cipriani, Patricia, Munarriz, Eliana, Gunsalus, Kris C, Huey-Ling, Kao, Amelia, White, Julie, Young

[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.