Seydoux GC et al. (1994) Worm Breeder's Gazette "pes-10 RNA and Protein Are Expressed Transiently in Each Somatic Lineage in the Pre-Gastrulation Embryo"

Seydoux GC, Fire A

[Worm Breeder's Gazette, 1994]

pes-10 RNA and protein are expressed transiently in each somatic lineage in the pre-gastrulation embryo Geraldine Seydoux and Andy Fire. Carnegie Instituhon, Baltimore MD 21210.

(2024) Development "The people behind the papers - Alyshia Scholl, Yihong Liu and Geraldine Seydoux."

[Development, 2024]

Germ granules have been hypothesized to deliver mRNAs of germ cell fate determinants to primordial germ cells. Now, a new study in Development finds that many mRNAs enriched in germ granules are not involved in germline development in Caenorhabditis elegans. To find out more about the story behind the paper, we caught up with first author Alyshia Scholl, second author Yihong Liu and corresponding author Geraldine Seydoux, Professor at Johns Hopkins University School of Medicine.

(2022) Development "The people behind the papers - Madeline Cassani and Geraldine Seydoux."

[Development, 2022]

Specification of germ cell fate depends on the asymmetric segregation of germ granules in early embryos. Now, a new paper in Development describes 'germline P-bodies', germ granules in Caenorhabditis elegans embryos, which function cooperatively with another condensate, P granules, in germline specification. To find out more, we caught up with first author Madeline Cassani and corresponding author Geraldine Seydoux, Professor at Johns Hopkins University School of Medicine.

Wu, Edlyn et al. (2009) International Worm Meeting "MicroRNA-mediated translation repression and deadenylation in C.elegans embryos."

Flamand, Mathieu, Sonenberg, Nahum, Mathonnet, Geraldine, Wu, Edlyn, Fabian, Marc, Duchaine, Thomas, Thivierge, Caroline

[International Worm Meeting, 2009]

MicroRNAs (miRNAs) are small RNAs that play a pivotal role in post-transcriptional gene regulation. Most commonly in animals, these regulatory RNAs target mRNAs by imperfectly binding to complementary sites in 3'' untranslated regions (3''UTR), thereby affecting the translation of the targets, or reducing their stability. Despite the significant roles miRNAs play in various biological processes, the mechanistic details of how they regulate gene expression remain unclear. We present here the first in vitro translation system derived from C.elegans embryos that recapitulates cap- and polyA-dependent translation as well as miRNA-dependent silencing. Using luciferase reporters fused to an artificial 3''UTR containing sites complementary to the maternally contributed mir-35 miRNA family, we observed up to 70% inhibition by mir-35 over a 3-hour translation time-course. Upon addition of 2''-O-methyl oligonucleotide inhibitors for the endogenous mir-35 family, translation of our reporters was restored. To understand how this inhibition occurred, we examined the fate of our reporter mRNAs. Inhibited mRNA reporters became fully deadenylated within the first 60 minutes of incubation with our cell-free extract, while a reporter bearing a mutation in the region complementary to the miRNA seed was not susceptible to inhibition or deadenylation. MiRNA-mediated deadenylation is independent of the translation of the targets as neither ApppN capping of the transcripts, nor the presence of the translation inhibitor cycloheximide inhibited deadenylation. We will describe our efforts to screen for endogenous miR-35 targets via deadenylation and translation inhibition assays. We will further define the genetic requirements for translation repression and processing of target mRNAs by testing extracts that are RNAi-depleted. Our findings indicate that deadenylation plays a prominent role in miRNA-mediated control of gene expression in the C.elegans embryo.

Wu, Edlyn et al. (2009) International Worm Meeting "A cell-free C. elegans embryonic system reveals maternal translation control by the miR-35-42 microRNA family through deadenylation."

Thivierge, Caroline, Flamand, Mathieu, Wu, Edlyn, Sonenberg, Nahum, Wohschlegel, James, Duchaine, Thomas F., Fabian, Marc, Mathonnet, Geraldine

[International Worm Meeting, 2009]

Powerful genetic methods in C. elegans have unraveled numerous translational control phenomena, including microRNA-mediated silencing. Better resolution on the mechanisms of microRNA-mediated silencing requires in vitro translation systems that are currently missing from the C. elegans experimental paradigm. We report here the first C. elegans embryonic extract able to sustain potent 5''-Cap- and 3''-poly(A)- synergic translation initiation of exogenous transcripts. Using a reporter system based on the abundant and maternally loaded miR-35-42 family we recapitulated efficient microRNA-mediated silencing in vitro. We found mRNA reporters to be rapidly and completely deadenylated in a microRNA-dependent manner. Unexpectedly, complete deadenylation did not result in the destabilization of the targeted transcripts up until at least 2 hours post-deadenylation. The stability of the deadenylated intermediate prompted us to propose a model wherein miRNA-mediated silencing of maternally contributed transcripts is reversible via the regulated control of poly(A) tail length. Finally, functional proteomic analysis of the embryonic microRNA-mediated silencing machinery, and the systematic validation in this system is underway and progress will be presented. This work is supported by the Canadian Institute of Health and Research and by the National Sciences and Engineering Research Council of Canada..

Han M et al. (2000) West Coast Worm Meeting "Genetic and phenotypic characterization of evl-14 and evl-20, genes involved in C. elegans vulva development"

Han M, Antoshechkin I

[West Coast Worm Meeting, 2000]

In an attempt to identify new genes that participate in C. elegans vulva development, we carried out a screen for sterile mutants with protruding vulva. The mutants were then examined using Nomarski optics for abnormal vulva morphology at the "Christmas tree" stage. Several of isolated mutations were alleles of evl genes previously identified by Geraldine Seydoux . Here we report genetic and phenotypic characterization of two such genes, evl-14 and evl-20. Both mutations are completely recessive and result in a 100 % sterile phenotype. Their vulva structures are variably abnormal and are missing anywhere from 0 to 10 cells suggesting either under induction or cell cycle/division defect. In order to distinguish between these possibilities we are making doubles with members of Ras signaling pathway and GFP reporters for vulval cell fates. We are also performing detailed lineage analysis of the mutants. evl-20 also displays gonad migration defect not seen in evl-14. evl-14 maps to linkage group III just left of pal-1. evl-20 is located on chromosome II next to rol-6. We have injected cosmids from these regions and identified cosmid pools that rescue both evl-14 and evl-20. We are in the process of injecting single cosmids and making subclones to identify single open reading frames that will rescue the mutants.

RV Aroian et al. (1999) International C. elegans Meeting "Using C. elegans to Teach Embryonic Development to Undergraduates"

G Wienhausen, RV Aroian, D Johnson

[International C. elegans Meeting, 1999]

We have begun to use C. elegans as a model system in our undergraduate embryology laboratory class at the University of California, San Diego. In the past, this 10 week class has used Sea Urchin, Xenopus, Chick, and other non-genetic systems to demonstrate to students embryonic development. In this poster we will outline the 6 new weeks of C. elegans modules. These modules allow the students to use advanced techniques to explore widely applicable principals of embryonic development. These techniques include wild-type lineage analysis, mutant lineage analysis, use of specific inhibitors to alter embryonic development, immunofuorescence analysis of wild type and mutants using antibodies to various cell biology and developmental markers, genome searching, and knocking out genes using RNA-mediated interference. Our goal is to one day publish an article on our results in the Worm Breeder's Gazette. The embryonic development of more advanced model organisms such as Xenopus and chick are later used to compare and contrast to C. elegans. This work is greatly enhanced by the cooperativity of the C. elegans community in the sharing of antibodies, mutants, and other reagents such as GFP strains. Last year we used protocols, stains, and antibodies from to Geraldine Seydoux, Craig Mello, Pete Okkema, Michel Labouesse, Bruce Bowerman and the CGC. Thanks to them and others that have generously shared their reagents.

Evans TC et al. (1994) Worm Breeder's Gazette "An Update on in situ Hybridization"

Kimble JE, Evans TC, Henderson ST

[Worm Breeder's Gazette, 1994]

We have used an in situ hybridization protocol to explore the distribution of mRNAs in germlines and embryos (Evans et al., manuscript submitted). This protocol yielded strong staining for glp-1 mRNA with very little background; glp-1 mRNA was detected throughout the distal arm of the germline, in oocytes, and in all cells of 1-8 cell embryos. After the 8-cell stage, glp-1 mRNA disappears from all cells but persists in P3 and P4 ,consistent with observations made by Geraldine Seydoux (WBG 13(1): 25). We have also detected endogenous mRNAs for fem-3 and lag-2 ,and exogenous lacZ RNA that had been injected into the gonad. For the sex-determination gene fem-3 ,mRNA distribution was similar to glp-1 ;RNA was detected in the germline (distal arm and oocytes) and in 1-8 cell embryos. For lag-2 ,mRNA was detected in the distal tip cell, but not in the germline (see Henderson et al. abstract in this WBG). Following the last worm meeting, we received several requests for the protocol. Since then, we've made some modifications. In some cases, decreasing the hybridization time to 10-12 h may significantly improve sensitivity in addition to making the procedure shorter. Anyone wanting further information should call or e-mail tomevans@macc.wisc.edu (note that this is a different e-mail address from the last directory). Also, if anyone has made any improvements on the procedure or any interesting discoveries with it, we would love to hear from you.

JA Waddle et al. (1999) International C. elegans Meeting "Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis"

JA Waddle, RH Waterston

[International C. elegans Meeting, 1999]

A powerful feature of C. elegans research is the ability to determine mutant phenotypes at the resolution of single cells. The advent of green fluorescent protein (GFP) provides means to carry the single cell analysis one step further, that is, to characterize critical events that take place during a short interval within a cell cycle. Improvements in computers and imaging devices motivated us to develop a 3-dimensional image acquisition system capable of merging traditional embryonic cell lineage analysis with the latest GFP technology. To gauge the utility of our microscope setup, we imaged a transgenic line carrying a fusion between GFP and an isoform of histone H1 (kindly provided by Melanie Dunn and Geraldine Seydoux). The most important outcome of our efforts is the ability to non-destructively (e.g the embryos hatch and are fertile) collect a 3-dimensional time lapse fluorescence image sequence starting at first cleavage and spanning more than 8 hours of development. The histone H1::GFP images greatly facilitate the tracking of nuclear movements and cell divisions in the bottom half of later stage embryos. The application of computational methods, to reduce the effects of out of focus light, greatly improves image detail and the signal-to-noise ratio. Current efforts are focused on custom software capable of tracking the 3-dimensional distribution and behavior of the histone H1::GFP labeled nuclei, that is, automated embryonic cell lineage analysis. In summary, several technologies have coalesced to provide non-damaging means to track the 3-dimensional distribution of important molecules during embryogenesis. Such technology will complement existing methods for phenotypic analysis in an era when both the embryonic cell lineage and the genome descriptions are complete.

Laura Gallo et al. (2003) International Worm Meeting "A genetic approach to identify meiotic-specific chromosome segregation factors"

Kathleen St. John, Diane C Shakes, Laura Gallo, Erica Stewart, Ken Donohue

[International Worm Meeting, 2003]

During the normal process of eukaryotic cell division, chromosomes replicate during S-phase, undergo ordered compaction during prophase, and align in a central plate during metaphase. As cells subsequently transition from metaphase to anaphase, the replicated and aligned chromosomes separate and segregate into each of the two daughter cells. During anaphase, defects in any step of this multi-step process result in chromosome breakage and/or mis-segregation, problems that ultimately result in birth defects, cell death, and/or malignant transformations. Recent studies in a variety of eukaryotes suggest that the basic molecular mechanisms of chromosome segregation have been conserved both across species and between the related but distinctive processes of mitosis and meiosis. At the same time, the meiotic-specific process of homolog separation requires at least a few meiotic specific components, and chromosome segregation on anastral, acentriolar spindles is similarly expected to require at least a few oocyte-specific components. To identify the combination of unique and cell-type specific genes required for proper anaphase chromosome segregation in C. elegans, we are screening through a large collection of temperature-sensitive maternal mutants originally isolated by Matt Wallenfang and Geraldine Seydoux. Our overall strategy is to identify those with obvious defects in oocyte meiotic chromosome segregation and then to secondarily determine which of these also have defects in spermatocyte meiosis and/or germline mitosis. Using this approach, we expect to not only identify genes required during different stages of chromosome segregation but also those that differ in their cell-type specificity including those with general (mitosis and meiosis); meiosis-specific; and oocyte-specific defects. To date, subclasses of oocyte defects include problems in meiotic metaphase alignment, homolog separation, and meiotic cell cycle progression. Genetic mapping studies of these mutants as well as a comparative analysis of their oocyte, spermatocyte, and germline defects are currently in progress.