Lowry, Josh et al. (2011) International Worm Meeting "A Genetic Screen for Temperature-Sensitive, Embryonic Lethal Mutations in C. elegans."

Lowry, Josh, Bowerman, Bruce, Yochem, John, Connolly, Amy

[International Worm Meeting, 2011]

We are conducting a genetic screen for temperature-sensitive, embryonic lethal mutants, with a goal of identifying 2000 new mutants. Initial characterization of these mutants involves using Differential Interference Contrast (DIC) microscopy to quickly examine early embryonic cell divisions. New mutants are then sorted into one of four broad categories: (i) wild-type early divisions, (ii) meiotic and mitotic cell division defective, (iii) eggshell defective, or (iv) delayed P1 division. We are most interested in the cell division defective and eggshell defective classes. For cell division defective mutants we use DIC videomicroscopy to make time-lapse movies of the first two mitotic divisions. Thus far we have found several mutants with novel phenotypes. For example, 1/3 of or1180ts embryos were missing a paternal pronucleus. The or1190ts mutation results in both a loss of AP polarity in the one-cell zygote, and in P0 mitotic spindle assembly and chromosome segregation defects. Lastly, or1167ts mutants appear to have a monopolar spindle in the first division, and a bipolar spindle in the second division. From the eggshell defective class we have defined a new sub-class based on gene requirements at multiple stages of development. When shifted to the restrictive temperature as L4 larvae, adults produce embryos that lyse in water, while upshifting L1 larvae results in adult sterility. This mutant class may provide a model for investigating the role of membrane trafficking and its regulation in gonad morphogenesis. We are also exploring next generation DNA sequencing methods to rapidly identify affected loci. To this end we employ Restriction-site Associated DNA (RAD) mapping. RAD mapping takes advantage of single nucleotide polymorphisms (SNPs) that exist between our parental N2 strain and the Hawaiian strain CB4856. Mutant strains are crossed to Hawaiian males. F2 outcross progeny that are homozygous for the mutation are pooled, and genomic DNA extracted and digested with a restriction enzyme (EcoRI), and short barcoded adaptors are ligated to the fragments. The barcodes allow us to collect data for ~100 mutants per Illumina Hi Seq lane. Plotting the ratio of Hawaiian SNP reads to total reads for all sequenced SNPs defines roughly one megabase genome intervals that include the ts mutations.

Lowry, Josh et al. (2015) International Worm Meeting "A genetic screen for mutants with defects in the regulation of gonad membrane dynamics during adult oogenesis."

Lowry, Josh, Bowerman, Bruce

[International Worm Meeting, 2015]

C. elegans hermaphrodites produce oocytes in an assembly line fashion with syncytial nuclei located at the periphery of the distal arm sharing a common cytoplasm called the rachis. As oocyte nuclei move proximally and approach the bend of the gonad they begin to cellularize and then enlarge as they move through the proximal arm toward the spermathecum and uterus. This process results in the production of oocytes equivalent in volume to the entire gonad every six hours1. Thus oogenesis must involve a dynamic regulation of the complex membrane architecture of the gonad as oocyte nuclei move proximally and undergo cellularization. To identify genes that mediate this regulation of membrane dynamics, we have screened for temperature-sensitive mutants that share two phenotypes: osmotically sensitive eggshell-defective (Osm) after up-shifts of L4 larvae to the restrictive temperature, and adult sterility (Ste) after up-shifts of L1 larvae. Because eggshell formation and gonad development both require membrane trafficking, we hypothesized that Osm/Ste mutants might be defective in membrane-trafficking functions important for the dynamic regulation of the adult gonad membrane architecture during oogenesis. From a collection of 102 Osm mutants, we identified a subset of 25 that are recessive and also are highly penetrant Ste mutants. To determine if these Osm/Ste mutants have defects in adult oogenesis, we applied a high-throughput positional cloning approach that uses Illumina-based whole-genome sequencing and SNP mapping2,3. After identifying candidate mutations, we then used genetic complementation tests with available deletion alleles to determine gene identity for 10 Osm/Ste mutants. With one exception, all of the genes we identified are highly conserved and a subset-vps-15 (yeast vacuolar protein sorting), drp-1 (dynamin-related protein), ippk-1 (homolog of vertebrate inositol polyphosphate kinase 1)-have orthologs in other organisms that are known to be involved in membrane trafficking or membrane metabolism. We also identified an allele of sqv-8, which is required for the glycosylation of extracellular matrix proteins, and alleles of genes that are likely to be required for more general processes of gene expression and thus are unlikely to be specifically required for membrane regulation: abtm-1 (ABC-family iron transport protein, mitochondrial), crn-3 (cell death-related nuclease), and rpl-7 (large ribosomal subunit protein). To explore the requirements for vps-15, drp-1, and ippk-1, we have examined gonad membrane architecture after up-shifting mutants to the restrictive temperature at the L1, L4 and early adult stages, using confocal microscopy and a fluorescent marker labeling the plasma membrane.

Lowry, Josh et al. (2013) International Worm Meeting "Using Next-Generation Sequencing to Determine Gene Identity in Temperature-Sensitive, Embryonic Lethal Mutants."

Connolly, Amy, Lowry, Josh, Bowerman, Bruce

[International Worm Meeting, 2013]

While ~2500 genes in C. elegans are essential, temperature-sensitive alleles are available for only 100-200 of these genes. A major hurdle following any genetic screen has been the positional cloning of causal mutations. This process can be difficult and time consuming, and in the past most research has focused on narrow sub-classes of mutant phenotypes. To more rapidly determine gene identity in conditionally lethal mutants, we are employing simultaneous whole-genome sequencing and SNP mapping (Doitsidou et al, 2010). Briefly, the mutant of interest is outcrossed to the polymorphic strain CB4856.Then populations from ~50 F2 homozygous mutant progeny are pooled and genomic DNA extracted and sequenced on an Illumina HiSeq 2000. SNP mapping data is extracted from the resulting sequencing data using the CloudMap tool (Minevich et al, 2012), defining a chromosomal region in which the mutation lies. The same sequencing data is then used to generate a list of candidate mutations in that region. Our goal is to map and identify the causal mutations in hundreds of conditionally lethal mutants, sampling a wide range of mutant phenotypes in an effort to explore new areas of essential gene function research. One class of mutants we are now exploring exhibit lethal defects at multiple stages of development. Mutants shifted to the restrictive temperature at the L4 stage produce embryos with eggshell defects. Shifting up at the L1 stage produces sterile adults with gonad development defects. We hypothesize that this class of mutants, the Osm/Ste class, may prove useful for studying the role of membrane trafficking during gonad morphogenesis. To date, we have obtained WGS-SNP mapping data for 16 mutants, with another set of 8 soon to follow. A short list of candidate mutations has been determined for each of them, with complementation tests and transgenic rescue to follow where appropriate. Current candidates include rpl-7, gip-2, atx-2, aco-2, abtm-1 and drp-1. This high-throughput approach will allow us to clone more mutants far more quickly than has previously been possible.

Connolly, Amy et al. (2011) International Worm Meeting "A screen for C. elegans oocyte meiotic spindle-defective mutants."

Connolly, Amy, Yochem, John, Bowerman, Bruce, Lowry, Josh

[International Worm Meeting, 2011]

We are interested in identifying and investigating essential genes involved in oocyte meiotic spindle assembly, as part of a large-scale screen for temperature-sensitive, embryonic lethal-mutants. We initially identify mutants based on temperature-sensitive embryonic lethality, and then examine their early embryonic cell divisions using time-lapse DIC imaging. In a small number of these mutants, we detect an abnormal number of maternal pronuclei in the one-cell zygote, suggestive of oocyte meiotic spindle assembly or function defects. This far we have found several mutants with either multiple or no maternal pronuclei. One new mutant, or1129ts, has multiple maternal pronuclei, while another or974ts sometimes lacks a maternal pronucleus. We have narrowed the locations of or1129ts and or974ts to linkage group IV and V, respectively, by using Restricted-site Associated DNA polymorphism (RAD) mapping. RAD mapping relies on crossing the mutant into the polymorphic Hawaiian strain (CB4856), and then isolating and pooling F2s that are again homozygous for the mutation. The genomic DNA is purified then cut with a restriction enzyme (EcoRI) and ligated to barcoded adaptors. Finally, Illumina DNA sequencing is used to sample SNPs throughout the genome and narrow the mutation to a 1-3 Mb region. The or1129ts and or974ts mutations complement the kinesin mutants klp-18(or447ts) IV and bmk-1(or627ts) V, respectively, the only other meiotic spindle mutants we know of that map to these linkage groups (see abstract by Osterberg et al), suggesting that these mutations are alleles of other genes. We are now using whole genome sequencing to determine the identity of the affected gene in each mutant. We will characterize their mutant phenotypes using GFP and mCherry fusions to microtubules, histones, and other proteins for live cell imaging, as part of an ongoing effort to define the genetic pathways and networks that control C. elegans meiotic spindle assembly.

Keikhaee, Reza et al. (2013) International Worm Meeting "Systematic characterization and positional cloning of temperature-sensitive, embryonic-lethal C. elegans mutants."

Chiang, Chien-Hui, Keikhaee, Reza, Connolly, Amy, Yochem, John, Bowerman, Bruce, Lowry, Josh

[International Worm Meeting, 2013]

While most genetic screens for embryonic-lethal C. elegans mutants have focused on a few narrow sub-classes of mutant phenotypes, we now seek to isolate and systematically categorize by mutant phenotype 2000 new temperature-sensitive, embryonic lethal C. elegans mutants. For every new mutant we find, we initially use Differential Interference Contrast (DIC) microscopy to examine the early embryonic cell divisions in live mutant embryos. We broadly classify all new mutants in four categories: (i) meiotic and mitotic cell division defective, (ii) eggshell defective, (iii) delayed P1 division, and (iv) wild-type early embryogenesis but penetrant embryonic lethality. To date 626 mutants from the 890 new mutants that we have found are categorized in above categories as follows: ~28% exhibit cell division defects, ~16% are eggshell defective, ~9% exhibit delays in P1 cell division, and ~44% appear to have wild type early cell divisions. We also are categorizing each mutant using two additional criteria. First, our mutants are isolated by scoring embryonic lethality after shifting L4 worms to the restrictive temperature. We therefore also have shifted mutant worms to the restrictive temperature as arrested L1s, and then scored for larval lethality and sterility. Finally, to further classify embryos with normal eggshells and early cell divisions, we examine terminal mutant phenotypes to classify their development with respect to proper morphogenesis. Thus far we have found several mutants with abnormal phenotypes. For example, ~40% of or867ts and or1687ts resemble vab (variable abnormal morphology) mutants with mis-shapen but elongated two- to three-fold embryos developing but failing to hatch. We will use next generation DNA sequencing and single nucleotide polymorphism (SNP)-based mapping strategy to rapidly map and identify affected loci in mutants with penetrant and consistent phenotypes. Once we have identified causal mutations, we will further explore the requirements for genes that are conserved in humans but have not been extensively characterized in C. elegans.

O'Rourke, Sean et al. (2011) International Worm Meeting "RAD mapping and genomic interval pulldown sequencing for rapid mutation identification."

Johnson, Eric, Miller, Michael, Turnbull, Doug, Connelly, Amy, Stiffler, Nick, Lowry, Josh, Yochem, John, O'Rourke, Sean, Bowerman, Bruce

[International Worm Meeting, 2011]

Forward genetic screens in C. elegans and other model organisms represent a powerful approach to explore gene function by assigning phenotypes to mutated genes. However, determining the causal mutation by traditional mapping is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in C. elegans strains. First, we report our use of restriction site associated DNA (RAD) markers for rapidly mapping mutations identified after chemical mutagenesis. We have used the N2 background to isolate mutants and crossed them to the polymorphic Hawaiian CB4856 strain for mapping. Approximately 200 F2 animals are used for the RAD mapping which simultaneously samples SNPs throughout the genome and can resolve genetic intervals smaller than one megabase. Second, we present our use of genomic interval pull-down sequencing to selectively capture and sequence portions of a genome up to several megabases in size. Many samples for both RAD mapping and genomic interval pull-down sequencing can be multiplexed into one Illumina sequencing run saving resources. Our methods should greatly decrease the time required to identify the causal mutation in C. elegans and other organisms.

Jud, Molly et al. (2019) International Worm Meeting "A screen for temperature-sensitive morphogenesis-defective mutants."

Fennell, Francesca, Yang, Yuqi, Bowerman, Bruce, Jud, Molly, Miller, Alex, Lowry, Josh, Bao, Zhirong, Tran, Nhah, Padilla, Thalia, Shao, Hong

[International Worm Meeting, 2019]

Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs. Cell adhesion and the cytoskeleton are important for morphogenesis, but the signaling pathways involved remain poorly understood. As genes required for morphogenesis often have earlier roles in development, temperature-sensitive embryonic lethal (TS-EL) alleles are useful tools for investigating morphogenesis. From a collection of ~1,000 TS-EL mutants, we have classified the terminal phenotypes of 191 with normal early embryogenesis, after upshifts from the permissive (15 deg C) to the restrictive temperature (26 deg C) at the L4 stage, to identify 79 with highly penetrant elongation defects. We identify causal mutations using whole genome sequencing (WGS) and genetic complementation tests. So far, we have identified 30 alleles representing 21 genes. Many of these genes likely regulate gene expression globally with roles in cell fate specification (e.g. glp-1, which encodes a Notch receptor involved in blastomere specification) or differentiation (e.g. chaf-1, which encodes a chromatin assembly factor subunit), while others appear to be weak alleles of cell division genes (e.g. zwl-1, which encodes a kinetochore protein). To identify mutants that are specifically defective in morphogenesis, we upshifted mutant embryos just prior to the bean stage, after most cell division, cell fate specification and differentiation has occurred, and identified 12 with penetrant elongation defects. These include two with causal mutations in rib-1 and rib-2, which encode heparin sulfate synthesis proteins that regulate morphogenesis in other organisms, although how post-translational sugar modifications influence morphogenesis is still unclear. We are now using WGS to identify genes for 6 more of these 12 late-upshift mutants. We also are characterizing morphogenetic defects in fixed mutant embryos, using antibodies against epidermal cell membrane proteins to assess cell shape dynamics. Our long term goal is to advance our understanding of morphogenesis by identifying more genes that influence this process.

Jorgensen EM et al. (1995) International C. elegans Meeting "CLONING AND CHARACTERIZATION OF THE SYNAPTIC FUNCTION MUTANT RIC-1."

Harris TW, Jorgensen EM

[International C. elegans Meeting, 1995]

We have taken a genetic approach to identify molecules involved in neurotransmission. We identified several previously unidentified genes in a behavioral screen for mutants defective in synaptic function. Among these was ric-5(n1337). When touched on the head, ric-5(n1337) mutants move backwards in a jerky, uncoordinated fashion, and frequently coil. ric-5 maps to the cluster of LG III. To obtain putative null alleles of ric-5, a noncomplementation screen was conducted to generate additional alleles. Approximately 7000 EMS mutagenized genomes were screened, and we recovered 7 new alleles of ric-5. We mapped the position of ric-5 to the right of mec-14 and to the left of lin-39. Several other mutations resembling the ric-5 phenotype mapped to this same region. osm-13(n1602) (Josh Kaplan and Anne Hart, personal communication) mapped to this interval and we demonstrated that n1337 and n1602 failed to complement. Carl Johnson mapped ric-1(e239) to this interval, and we demonstrated that e239 and n1337 failed to complement. In light of these data, ric-5 has now been renamed ric-1. Jim Rand's laboratory has isolated 19 mutations in ric-1, bringing the total number of ric-1 alleles to 28. Microinjection of cosmid pools spanning this region weakly rescue the coiling phenotype of ric-1.

Boeck, Max E. et al. (2009) International Worm Meeting "Predicted 3D Structures of the DNA-Binding Domain for the Ten C. elegans GATA Transcription Factors and Their Preferred DNA Sequence."

Waterston, Robert, Thompson, James, Bradley, Phil, Boeck, Max E.

[International Worm Meeting, 2009]

Based on the paradigm that structure dictates function we used a computational approach to determine the preferred DNA motif bound by each of the GATA transcription factors found in C. elegans. GATA transcription factors have been shown to be important for organogenesis and cell fate specification in every metazoan from C. elegans to humans. The ability of GATA factors to specify cell-fate decisions is dictated by their ability to bind a DNA motif, WGATAR, with high fidelity. GATA factors have been shown to bind the motif through the actions of a conserved DNA-binding structure, CXXC(X)17-19CXXC [1]. While the basic DNA-binding structure is conserved between members of the GATA family,numerous specificity-determining residues vary between GATA genes. Using a maximum-likelihood phylogenetic approach [2] we estimated a phylogenetic tree in order to model the evolutionary relationships between GATA factors in the sequenced Caenorhabditis species. We then used Rosetta homology modeling [3] to construct structural models of the DNA-binding structures of all the GATA factors found within C. elegans. By tracking the specificity-determining residues that have changed along the phylogenetic tree of these GATA factors, we have defined a prediction of which GATA factors are likely to bind very similar segments of DNA. [1] Lowry, Atchley J Mol Evol. 2000 50:103-115 [2] Guindon S, Gascuel O. Syst Bio. 2003 52(5):696-704. [3] Qian et al, Nature. 2007 Nov 8;450(7167):259-64.

Jacqueline K Rose et al. (2003) International Worm Meeting "Activity dependent plasticity during development: Early isolation alters behavior, rate of development and gene expression"

Kenneth R Norman, Susan Rai, Susan Sangha, Jacqueline K Rose, Catharine H Rankin

[International Worm Meeting, 2003]

A basic feature of mammalian nervous systems is that during development activity dependent processes sculpt the final patterns and strengths of synaptic connections. Changes in synapses have been reported as a result of altered early experience. Recent evidence of this same phenomenon in invertebrates suggests that this may be a basic feature of nervous systems that is conserved across phylogeny. In these experiments we investigate the effects of early experience on the nervous system and behavior of C. elegans. We have shown that depriving C. elegans of sensory stimulation by raising them in isolation during larval stages of development, leads to adults that show: 1) decreased responsiveness to mechanical stimuli (a tap) 2) slower rates of physical development (worms raised in isolation are smaller and slower to begin laying eggs than age matched worms raised in groups) and 3) altered morphology of synapses between the mechanosensory neurons and the interneurons of the tap withdrawal circuit. The anatomical differences between worms raised in isolation and worms raised in groups were seen using two different transgenic strains of worms. One strain, developed by Michael Nonet of Washington University was pMEC-7::SNB-1::GFP (a marker of vesicles in the mechanosensory neurons). The other strain was GLR-1::GFP developed by Josh Kaplan of UC Berkeley (marker for glr-1 receptors on the command interneurons). Using these strains we found that worms raised in isolation have lower levels of both transgenes compared to worms raised in groups. Brief mechanical stimulation during larval development rescues the effects of isolation on behavior and on GLR-1::GFP expression, but not on size or on pMEC-7::SNB-1::GFP expression.