Flibotte, Stephane et al. (2021) MicroPubl Biol

Edgley, Mark, Au, Vinci, Flibotte, Stephane, Moerman, Donald G

[MicroPubl Biol, 2021]

We used whole-genome sequencing (WGS) data from a Caenorhabditis elegans strain homozygous for the reciprocal translocation hT2(I;III) to identify its breakpoints molecularly. The translocation structure is fairly straightforward, with only minor secondary rearrangement in addition to the primary breakpoints. The graphical representation below depicts the two hT2 half-translocations for ease of conceptualization.

Flibotte, Stephane et al. (2009) International Worm Meeting "Rapid high resolution SNP-CGH mapping in C. elegans."

Waterston, Robert, Chaudhry, Iasha, Edgley, Mark, Moerman, Donald, Flibotte, Stephane, Taylor, Jon

[International Worm Meeting, 2009]

Recently we published a manuscript on a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of Single Nucleotide Polymorphisms (SNPs) and oligonucleotide array Comparative Genome Hybridization (array CGH) (Genetics 181:33-37 2009). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. SNPs were selected from the published set in WormBase and expanded using new SNPs identified from the whole-genome sequence of CB4856. Using this method a mutation can be mapped to a resolution of about 200 kb in a single genetic cross for a few hundred dollars in reagent costs. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. We will discuss refinements we have made in microarray design, data analysis procedures and animal husbandry since the publication of the original paper. For example, we have evaluated the effects of varying the oligonucleotide length on data quality. The oligonucleotide length was varied between 35 and 50 and as expected the SNP signal tends to be larger but noisier for shorter oligonucleotides, which translates into only small differences in mapping accuracy and we are therefore still recommending our usual length of 50. Furthermore, as expected the magnitude of the signal differs slightly between various transition/transversion types and it can be useful take such information into account both at the design and analysis stages. Our current chip design interrogates nearly 13,000 SNPs scattered across the genome with an average of about thirty 50-mer probes per SNP. Our mapping method will be particularly powerful when applied to difficult or hard to map low penetrance phenotypes. It should also be possible to map polygenic traits using this method. We will illustrate the method with several examples.

Delaney, Colin et al. (2015) International Worm Meeting "The histone H4K20 methyltransferase SET-4 promotes dauer arrest in C. elegans insulin signaling mutants."

Moerman, Donald, Hu, Patrick, Graniel, Jacqueline, Delaney, Colin, Dumas, Kathleen, Flibotte, Stephan

[International Worm Meeting, 2015]

Insulin and insulin-like growth factor signaling (IIS) is highly conserved across metazoans and plays pivotal roles in development and aging. In C. elegans, the DAF-2/IIS pathway influences dauer diapause, a state of larval developmental arrest that occurs in response to stressful environmental conditions. Mutants that have reduced DAF-2/IIS pathway activity enter dauer constitutively in a manner requiring the FOXO transcription factor DAF-16/FOXO. DAF-16/FOXO function is antagonized by DAF-2/IIS signaling through AKT-mediated phosphorylation, leading to its nuclear export. When AKT activity is reduced, DAF-16/FOXO translocates to the nucleus, where it is inhibited by EAK-7/TLDC1, a conserved protein of unknown function. While eak-7 and akt-1 single mutants do not have strong dauer-constitutive phenotypes, eak-7;akt-1 double mutants always arrest as dauers.We designed a forward genetic screen to discover novel regulators of DAF-16/FOXO by mutagenizing eak-7;akt-1 worms and looking for suppressors of eak-7;akt-1 dauer arrest (seak mutants). Mapping and whole genome sequencing revealed that one seak mutant strain contained a mutation in set-4, which encodes a conserved histone H4 lysine 20 (H4K20) methyltransferase. Three independent set-4 mutants suppressed dauer arrest in an eak-7;akt-1 background, indicating that SET-4 functions to promote dauer arrest. SET-4 promotes dauer specifically through the IIS pathway, as set-4 mutation did not alter arrest in other conserved dauer pathways. Consistent with a role in DAF-16/FoxO regulation, set-4 mutants are short-lived. Furthermore, we found that expressing SET-4 under the control of the neuron-specific rab-3 promoter rescued dauer in set-4 mutants to a similar extent as the set-4 native promoter. We hypothesize that SET-4 acts in neurons to control dauer arrest by promoting DAF-16/FOXO activity. As both SET-4 and DAF-16/FoxO are conserved in mammals, our findings hint at a general role for histone H4K20 methylation in the regulation of FOXO transcription factor activity.

Gengyo-Ando K et al. (1986) Worm Breeder's Gazette "progress in sequencing of the unc-15 paramyosin gene."

Gengyo-Ando K, Kagawa H

[Worm Breeder's Gazette, 1986]

DNAfragments from -HK2 clone which contained paramyosin gene were processed and get results as follows. Nucleotide sequences of the HindIII 5kb were and HindIII 1.4kb fragments were nearly completed. There many ORFs with predicted-helix protein structure. Polarity of ORF was determined from the amino acid sequence predicted with the nucleotide sequence. Many fusion proteins(Miller et al, Pro. N. A. S.(1986) 83, 2305-2309) were isolated and were characterized by immunoassay. One of them which was corresponded to the 3'end part of the HindIII 5kb fragment crossreacted with a monoclonal antibody which only crossreacted with full sized paramyosin. Our results and the information from Stephan D. Rioux(personal communication and the worm gazette, Vol. 9, No.2, p39), Tc1 insertion site of TR605 munant located at the 3' region of the gene. There were three polyA site in the HindIII 1.4 kb fragment. Although we are not sure which site is a real polyA site the result suggests that Tc1 insertion into 3' noncording region might decrease the termination of the message. C. a fashionable organism to study in Japan. I do hope you could have a time to attend a workshop in Japan near future.

Jacques Pecreaux et al. (2006) European Worm Meeting "Mechanical Oscillations during Asymmetric Spindle Positioning Require a Threshold Number of Active Cortical Force Generators"

Jonathon Howard, Frank Julicher, Stephan W. Grill, Anthony A. Hyman, Karsten Kruse, Jens-Christian Roper, Jacques Pecreaux

[European Worm Meeting, 2006]

Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.

Edgley, Mark et al. (2009) International Worm Meeting "C. elegans Gene Knockout Consortium Report: Progress, New Technologies and Funding."

Rummage, John, Edgley, Mark, Barstead, Bob, Flibotte, Stephane, Moerman, Don, Holmes, Jeff

[International Worm Meeting, 2009]

The C. elegans Gene Knockout Consortium (http://www.celeganskoconsortium.omrf.org/) has for many years pursued systematic targeted gene disruption in the nematode, both upon request from the research community and in the larger context of the whole set of genes for which human orthologs exist (more than 7,000 genes). Our primary approach uses a PCR detection method to generate single-gene deletions. More recently we have incorporated array Comparative Genome Hybridization (array-CGH; Genome Research 17: 337-347) and whole-genome sequencing for efficient detection of mutations. CGH has proven valuable and cost-effective for deletion discovery and SNP mapping (Genetics 181: 33-37; see abstract by Flibotte et al., this meeting). With deep sequencing we have generated the first comprehensive catalog of all mutations in a genetic background after EMS mutagenesis. The combined strategies of directed and non-directed approaches improve throughput, broaden the types of mutations we can identify, and in the long term should lower the cost per allele. All Consortium deletions are stabilized as homozygous or balanced heterozygous strains, breakpoints are sequenced, and the strains and data are placed immediately into the public domain through our collaborators at the Caenorhabditis Genetics Center and WormBase. Distribution of Consortium strains accounts for a significant proportion of distributions from the CGC and has stood at 20% for the past two calendar years. To date the Consortium has generated deletions in more than 3,300 genes (not including about 700 genes deleted in natural isolates), and most of these have been stabilized and archived at the CGC. Together with those from the Mitani lab, deletion alleles represent over 5,000 genes. In the last two years we have been concentrating on genes encoding transcription factors and kinases, and we hope to complete the knockout coverage for these gene families in the next year. Of 934 total transcription factor genes, 720 are currently identified by gk, ok or tm deletions, with approximately another 50 identified by point mutation. Of 438 total kinase genes, 42 are represented by such deletions with another 150 identified by point mutation. Both our labs are currently in the process of renewing funding to continue this work. We plan to stay with the targeted PCR approach as funding allows, while continuing to develop CGH and deep sequencing and shifting resources to those methods as the technologies improve.

Rodriguez, Josana et al. (2011) International Worm Meeting "Novel players of cell polarity and asymmetric cell division in C. elegans identified through analysis of a polarity genetic network."

Fievet, Bruno, Ahringer, Julie, Rodriguez, Josana

[International Worm Meeting, 2011]

Cell polarity and asymmetric cell divisions are essential for the generation of cellular diversity. The importance of these processes during development has been emphasized by recent findings, which show that defects lead to the formation of tumors. Towards identifying most of the factors involved in cell polarity and asymmetric cell division, we carried out 17 large scale RNAi suppressor screens of temperature sensitive mutants of genes involved in actomyosin contraction, par polarity and microtubule pulling forces governing spindle positioning. This allowed us to generate a polarity genetic interaction network of 186 genes with 229 genetic interactions (see Fievet et al., abstract). Of these genes, 23 have been previously shown to have a role in the asymmetric first cell division, validating our approach. For the rest of the candidates (87% of our network), no early polarity phenotype has been reported for knock-down in a wt background. To identify functions of novel candidates, we have been studying effects of their knockdown in sensitized backgrounds. This strategy has successfully identified specific functions for genes in the network. For example, out of the 24 identified suppressors of nmy-2(ts) (non-muscle myosin II), we found that eight enhance embryonic lethality when knocked-down in a gain of function mutant of act-2 (actin) and seven out of these eight enhancers show defects in the actomyosin cytoskeleton during the first cell division. In collaboration with Stephan Grill we are using biophysical methods to characterize actomyosin dynamics upon loss of function of these candidates. We are using a similar approach to characterize the suppressors of par-2(ts) and pkc-3(ts). Functional antagonism between PAR-2 and PKC-3 is critical for C. elegans asymmetric first cell division and robustly found in our screens. Therefore, we are studying the enhancement phenotype of par-2 suppressors in pkc-3(ts) and vice versa. In another strategy, we have been using patterns of genetic interactions to predict functions for novel genes in the network. This analysis identified several potential new components and/or regulators of the anaphase-promoting complex (APC). In addition, the network functionally implicates different signalling pathways in polarity. For example, we have identified a role for PKA signalling in the regulation of the asymmetric first cell division, which we are currently studying. Due to the conservation of cell polarity mechanisms, we expect that the C. elegans polarity network we have built will be relevant for cell polarity events in other model organisms.