Pavan Kadandale et al. (2004) West Coast Worm Meeting "Phenotypic characterization of egg-1, a gene required for fertilization."

Andrew Singson, Barth Grant, Pavan Kadandale, Allison Stewart

[West Coast Worm Meeting, 2004]

Standard forward genetic approaches have resulted in the isolation of a number of mutants of C. elegans that are defective in the process of fertilization. However, due to methodological limitations all of these mutants have been in sperm-specific molecules. Traditional mutagenesis screens have not yet identified any fertilization-defective mutants that affect molecules present in the oocyte. We have used a combination of bioinformatics and reverse genetics to obtain the first such mutant, which we have named egg-1 . We have obtained a deletion allele (from the Japanese Knock-out Consortium) of egg-1 , and this mutant has a temperature-sensitive fertility defect. egg-1 encodes a Type II transmembrane molecule that has multiple LDL-receptor repeats. We present our ongoing phenotypic analysis of egg-1 and characterization of its role in gamete interactions during fertilization.

Pavan Kadandale et al. (2004) East Coast Worm Meeting "Phenotypic characterization of egg-1, a molecule involved in fertilization"

Allison Stewart, Richard Klancer, Barth Grant, Andrew Singson, Pavan Kadandale

[East Coast Worm Meeting, 2004]

Standard forward genetic approaches have resulted in the isolation of a number of mutants of C. elegans that are defective in the process of fertilization. However, due to methodological limitations all of these mutants have been in sperm-specific molecules. Traditional mutagenesis screens have not yet identified any fertilization-defective mutants that affect molecules present in the oocyte . We have used a combination of bioinformatics and reverse genetics to obtain the first such mutant, which we have named egg-1 . We have obtained a deletion allele (from the Japanese Knock-out Consortium) of egg-1 , and this mutant has a temperature-sensitive fertility defect. At 16degC, egg-1 animals have brood sizes that are 91% of wild type. When shifted to 25degC, however, these worms have only 7% of wild type brood sizes. egg-1 encodes a Type II transmembrane molecule that has multiple LDL-receptor repeats. We will present our ongoing analysis of the phenotypic analysis of egg-1 and its role in gamete interactions during fertilization.

Pavan Kadandale et al. (2006) Development & Evolution Meeting "Identification Of The First Egg-Specific Molecule Required For Fertilization"

Scott Gordon, Peter Schweinsberg, Jacob Rubin, Allison Stewart-Michaelis, Pavan Kadandale, Andrew Singson, Barth Grant

[Development & Evolution Meeting, 2006]

The use of traditional forward genetics stratagems has resulted in the identification of numerous molecules required for sperm function during fertilization. However, a limitation of this mutagenesis approach is that we cannot generate lines of mutants that have defects in egg-specific molecules required for fertilization. Here, we report our use of reverse genetics to discover the first molecule in the egg (EGG-1) that is required for fertilization. Unexpectedly, the phenotypic characterization of the mutant phenotype led us to uncover the complex coordination of ovulation and sperm behavior with fertilization. Interestingly, while EGG-1 function appears to be conserved in C. briggsae and C. remanei, we found a semi-redundant molecule in C. elegans, EGG-2 that appears to be the result of a gene duplication event that occurred after the speciation event that led to the C. briggsae and C. remanei lineages. Further studies should shed light on not only the mechanism by which EGG-1 mediates its function, but also on how ovulation and sperm function is coupled to fertilization.

Rika Maruyama et al. (2006) Development & Evolution Meeting "The Anti-phosphatase EGG-3 Is Required For Egg Activation At Fertilization"

Asako Sugimoto, Barth Grant, Peter Schweinsberg, Richard Klancer, Pavan Kandandale, Jacob Rubin, Andrew Singson, Nobuko Uodome, Rika Maruyama, Scott Gordon, Allison Stewart

[Development & Evolution Meeting, 2006]

Upon being fertilized by a sperm, the egg undergoes activation and starts development as a new individual. However, in spite of intense study, the oocyte factors that detect sperm entry and trigger egg activation still remain largely unknown.We identified egg-3 in the large-scale phenotypic analysis by RNAi-by-soaking as a gene that caused eggs without eggshells. The egg-3 gene encodes as a tyrosine-phosphatase-like protein but some catalytic residues are not conserved. Therefore, EGG-3 is predicted to be an anti-phosphatase, which might function as a competitor to active phosphatases for substrate or as an adaptor/scaffold module that bind phosphorylated targets. The egg-3(tm1191) hermaphrodites produced no progeny and their eggs had no eggshells. The lack of progeny production was not rescued by wild-type sperm. A single sperm entered each egg-3 mutant oocyte, which indicated that fertilization and the block to polyspermy occurred normally. Furthermore, egg-3 embryos underwent meiosis I and meiosis II without polar body formation, which is similar to spe-11 phenotypes (McNally et al. Developmental Biology 282: 218-230 2005). Thus, egg-3 mutants were defective in egg activation following fertilization. GFP-EGG-3 driven by the pie-1 promoter was localized at the oocyte cortex, which is similar to the localization of GFP-MBK-2. MBK-2 is required for the transition from oocytes to embryos (Pellettieri et al. Developmental Cell 5: 451-462 2003). We found that GFP-MBK-2 was not localized at the oocyte cortex in egg-3 mutants. However, OMA-1-GFP, which is phosphorylated by MBK-2, was degraded in egg-3 mutants similar to wild-type. These data suggest that the oocyte cortex localization of MBK-2 that is dependent on egg-3 might not be essential to the MBK-2 activity. Taken together, we speculate that EGG-3 might be one of the essential oocyte factors that detect the signal of the sperm entry and trigger egg activation after fertilization and might also function as an scaffolding protein for MBK-2 at the oocyte cortex.

Francesca P et al. (1998) European Worm Meeting "HP1 homologues in C. elegans"

Francesca P, Fritz M

[European Worm Meeting, 1998]

The chromosomes of almost all higher eukaryotes contain regions, termed heterochromatin, that differ from the bulk of chromatin (euchromatin) in that they remain constitutively condensed throughout the cell-cycle. The DNA of heterochromatin consists almost entirely of repetitive sequences and encodes relatively few genes. Heterochromatin may play essential roles in mitosis, meiosis, gene expression and nuclear organization. The most well studied heterochromatin binding protein is the essential Drosophila heterochromatin binding protein 1 (HP1). HP1 is found in the heterochromatin of the chromocenter and in a limited number of other loci, including telomeres. HP1-like proteins have subsequently been identified in a number of species and share two regions with a high degree of amino acid conservation: an amino-terminal chromo domain (chromosome organization modifier), and a carboxy-terminal chromo shadow domain (Aasland and Stewart, 1995.) Both chromo and chromo shadow domains are able to target proteins to their site of action on chromatin. C. elegans mitotic chromosomes are holocentric and by definition do not have a localized centromeric region, nor do they have large tracts of heterochromatin. The C. elegans sequencing project, however, has identified two protein containing both chromo and chromo shadow domains, which we are now studying in our laboratory. hpl-1 maps on the right arm of the X chromosome, to the right of the unc-9 locus. Analysis of the genetic map of this region has not revealed the presence of any previously isolated candidate mutations for the hpl-1 locus. The other gene being studied hpl-2, maps to the middle of chromosome III. We are now testing whether the dominant maternal effect mutation mel-23, which maps to this region, corresponds to hpl-2 by microinjection rescue. We are also trying to isolate mutations in these two genes by a reverse genetic approach using Roberts Barstead method for isolating deletions in PCR screens of mutagenized populations. The study of HP1 homologues in C. elegans should shed light on the functions of this conserved family of proteins in an organism with vastly different chromosome organization and behaviour. Aasland and Stewart, N.A.R. 23:3168-3173

Schein JE et al. (1996) West Coast Worm Meeting "CONSTRUCTION AND UTILIZATION OF THE CHROMOSOME IV COSMID TRANSGENIC LIBRARY"

Schein JE, Rose AM, Baillie DL, Franz NW, Janke DL

[West Coast Worm Meeting, 1996]

Construction of a chromosome IV cosmid transgenic library of C. elegans strains carrying sequenced cosmids in extrachromosomal arrays has begun as part of our labs' collaboration with the Genome Sequencing Consortium (see abstract by D. Janke et al.). There are 20 chromosome IV cosmids available in transgenic strains at the time of this writing, and we estimate a total of 40-50 will be available to the community by the end of July. In order to enhance the utility of this library, we are also using these strains to localize approximately 50 essential gene mutations, previously identified in the Baillie lab, to cosmids by way of transgenic rescue experiments (for results of similar efforts on chromosomes III and V, see abstracts by Nigel O'Neil et al., Helen Stewart et al., and Brad Barbazuk et al.). Correlation of the genetic and physical maps in this manner will facilitate the selection of candidate cosmids for use in further rescue experiments, and thereby increase the speed with which the connection between genetic mutations and DNA sequence is made. Currently these rescue experiments are focused in the sDf2 region, in particular in the lin-3 - let-60 interval, which contains 13 essential gene mutations and 21 overlapping cosmids. The two genes bracketing this interval, lin-3 and let-60(ras), were previously placed on the physical map by others. To date, two additional essential genes have been positioned to cosmids on the physical map; let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido) and let-64. It is anticipated that by the time of the meeting several additional mutations will have been positioned to cosmids. This work is being funded by a grant from the Canadian Genome and Associated Technologies Program to Ann Rose and David Baillie.

Collins D et al. (1995) International C. elegans Meeting "CONSTRUCTION OF TRANSGENIC STRAINS USING SEQUENCED COSMIDS FROM CHROMOSOME III: A COMMUNITY RESOURCE"

Schein JE, Rose AM, Franz NW, Lundgren C, Baillie DL, Collins D

[International C. elegans Meeting, 1995]

We are systematically creating transgenic worms using DNA from chromosome III cosmids which have been sequenced by the Genome Sequencing Labs in St. Louis, MO and Hinxton, UK. While function can be assigned to some of this sequence based on similarities to genes with known functions, a significant percentage of sequence data cannot be deciphered in this manner. For those sequences which have no matches in the databanks, alternative approaches are required to determine the function of the gene product. In collaboration with the J. Sulston and R. Waterston Sequencing Consortium, we have embarked on a project to correlate mutant phenotypes with regions of DNA sequence. We are producing a large number of genetically marked extra-chromosomal arrays which can be used in rescue experiments to place genetic markers on the physical map. To date we have created over 80 transgenic strains from cosmids generated from chromosome III, representing approximately 2.8 Mb of DNA. These strains are all produced by co-injecting cosmid DNA with the plasmid pCes1943 into wild-type (N2) hermaphrodites. pCes1943 is a derivative of C. Mello's rol-6(su1006)- containing plasmid pRF4, modified by B.Barbazuk and S.Jones to contain a Kanamycin cassette. These extra-chromosomal arrays are being crossed into strains which carry mutations in essential genes (see poster by H. Stewart et al., this meeting). The correlation of lethal mutations with rescuing cosmids will eventually allow for the assignment of biological function to coding regions. As well, this will facilitate a good correlation between the genetic and physical maps in this region. This work is being funded by a grant from the Candadian Genome Analysis and Technology Program.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

Haag, E.S. et al. (2019) International Worm Meeting "Evolution of sex ratio through gene loss."

Haag, E.S., Yin, D.

[International Worm Meeting, 2019]

The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species of Caenorhabditis nematodes have evolved the androdioecious mating system, where selfing hermaphrodites and males coexist. They reproduce mostly through self-fertilization, but can also outcross. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio [1]. Here, we focus on the contribution of the male secreted short (mss) gene family to male mating success, sex ratio, and population growth. The mss family is essential for full sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species [2]. Using a transgene to restore mss function to the androdioecious C. briggsae, we examined how mating system and population subdivision influence the fitness of the mss+ genotype. Consistent with theoretical expectations, mss+ is sufficient to increase male frequency and depress population growth in genetically homogenous androdioecious populations. When mss+ and mss-null (i.e. wild-type) genotypes compete, mss+ is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness of mss+, it is insufficient to explain its parallel loss. We propose that the lack of inbreeding depression [3] and the strong subdivision that characterize natural Caenorhabditis populations [4] impose selection on sex ratio that makes loss of mss adaptive. By reducing, but not completely eliminating outcrossing, loss of the mss genes tunes the sex ratio to its new optimum after self-fertility is established. 1. Stewart AD, Phillips PC (2002) Genetics 160: 975 2. Yin et al. (2018) Science 359: 55 3. Dolgin et al. (2007) Evolution 61: 1339 4. Kiontke KC, et al. (2011) BMC Evol Biol 11: 339

Mihail Sarov et al. (2007) International Worm Meeting "Recombineering based high throughput protein tagging in C. elegans."

Mihail Sarov, Francis Stewart, Anthony Hyman

[International Worm Meeting, 2007]

The methods for protein tagging in C. elegans so far have been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. The recent generation of a genomic fosmid library[1] opened the possibility for direct modification of any gene of interest in its native genomic environment by recombineering in E. coli. We recently published a method for precise in vivo engineering of large genomic clones into GFP tagged transgenes for ballistic transformation[2]. Using such transgenes, we reproduce known and document new expression patterns. We further demonstrate that the tagged protein can complement the function of its endogenous counterpart. Applying the latest developments of the recombineering field and introducing several innovations of our own we have now streamlined the protocol to allow rapid 96 well format liquid culture processing in a continuous recombineering pipeline that takes just four days. This unprecedented throughput and the achieved economy of scale allow us to rapidly produce thousands of tagged transgenes. Our method has become the basis for a large-scale project aimed at comprehensive description of the expression pattern (through fluorescent reporters) and the DNA binding sites (through affinity tag based chromatin immunoprecipitation) of more than 400 C. elegans transcription factor genes (as part of the NIH funded ModENCODE program). At the meeting we will discus the advantages of a distributed, community based collaboration model that will bring this technology to any worm lab and will make the genome wide protein tagging a feasible goal. 1. Perkins, J., K. Wong, R. Warren, J.E. Schein, J. Stott, R. Holt, S. Jones, M.A. Marra, and D. Moerman. 2005. A Caenorhabditis elegans fosmid library. In 15th International C. elegans Conference, University of California, Los Angeles. 2. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A.A. Hyman, and A.F. Stewart. 2006. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods 3: 839-844.