Andersen, Erik C. et al. (2013) International Worm Meeting "Using natural variation to decipher the complex genetic causes of C. elegans drug sensitivities."

Andersen, Erik C., Shimko, Tyler

[International Worm Meeting, 2013]

C. elegans is an important model for a variety of biomedically relevant traits, including sensitivities to diverse compounds and pharmaceuticals. Sensitivities to these compounds vary in the human population, and we know little about the genetic causes of this variation. Using C. elegans, we hope to identify and characterize these variant genes exploiting the existing natural genetic and phenotypic variation of the species. Quantitative geneticists use the correlations between genetic and phenotypic differences to identify the variants that control complex traits. Species-wide variation in genome-wide association studies or pairwise strain variation in linkage mapping studies is used to identify variant genes. Using these two approaches, one needs to score the phenotypes of a large number of independent strains. In C. elegans, we must phenotype over one hundred wild strains or nearly six hundred N2xCB4856 recombinant inbred lines in drug susceptibility assays. In order to score these large collections of independent strains for drug susceptibility traits, we optimized highly reproducible and high-throughput growth, fecundity, and pharyngeal pumping assays to score 384 strains per day per researcher in the presence of any aqueous compound. These assays allow us to probe both the acute and chronic phenotypic effects of important drugs, like chemotherapeutics, pesticides, and anthelmintics. To determine the efficacy of these approaches, we scored susceptibility to the most widely applied herbicide paraquat. In one week, we phenotyped growth and fecundity of 97 wild strains and 574 recombinant inbred lines between N2 and CB4856. These data implicated genomic intervals as small as 274 kb with 60 genes that control paraquat sensitivity. To verify these loci, we constructed nearly isogenic lines, in which a genomic region from one strain is isolated in another strain background. These results recapitulate the predicted effects, and we will discuss methods to characterize small phenotypic effects. Additionally, we will discuss recent progress towards scaling up these techniques to a larger set of compounds and pharmaceuticals.

Andersen, Erik C. et al. (2011) International Worm Meeting "N2-specific differences in behavior, growth, and physiology are explained by a laboratory-derived allele of npr-1."

Andersen, Erik C., Kruglyak, Leonid, Bloom, Josh S., Gerke, Justin P., Ghosh, Raj

[International Worm Meeting, 2011]

Over the past forty years, an amazing amount of research has been performed on a single C. elegans strain: N2. This strain may have gone through as many as 1000 generations (over 16 years) before long-term storage was discovered (1). During that time, N2 likely accumulated many mutations through selection in the laboratory. We know of at least two genes that are affected by laboratory-derived mutations, npr-1 and glb-5, which regulate an oxygen avoidance behavior in the presence of food (1). Not only is the N2 strain easier to pick and propagate, as clumping, bordering, and burrowing are highly reduced, but we will present evidence that it grows larger, has more offspring, crawls faster, and is more resistant to pathogen infection than wild C. elegans strains because of the lab-derived allele of npr-1. We mapped quantitative trait loci (QTL) for each of these traits using an advanced intercross recombinant inbred line (RIAIL) collection between N2 and the wild isolate from Hawaii CB4856. Additionally, we mapped thousands of gene expression differences between these two strains to QTL overlapping npr-1. We will present data that suggest npr-1 is the causal gene for many of these traits, using nearly isogenic lines (NILs) and loss-of-function alleles, and the growth and physiological differences are caused by the npr-1-mediated oxygen avoidance behaviors. We are interested in identifying natural variants between C. elegans strains for a variety of phenotypic traits. Nevertheless, npr-1 makes complex trait mapping more difficult, as it explains the majority of the genetic trait variance for crosses involving N2. To circumvent the laboratory-derived npr-1 allele, we constructed a collection of 359 RIAILs using CB4856 and a nearly isogenic line that has the normal form of npr-1 in an otherwise N2 background. However, this NIL with corrected npr-1 has other N2-specific laboratory-derived mutations, so using high-throughput selective sequencing, we have identified the most divergent C. elegans strains for use in future quantitative genetic studies. Because assays on agar plates lead to differences in food exposure, oxygen concentration, and humidity, we have optimized high-throughput liquid assays of growth and fecundity using robotic liquid handling, worm sorting, and image analysis. (1) McGrath et al. Neuron 2009, (61)5: 692-699.

Andersen, Erik C. et al. (2011) International Worm Meeting "Recent chromosome-scale selective sweeps reshaped C. elegans genomic diversity."

Gerke, Justin P., Crissman, Jonathan R., Andersen, Erik C., Ghosh, Raj, Felix, Marie-Anne, Kruglyak, Leonid, Bloom, Joshua S., Shapiro, Joshua A.

[International Worm Meeting, 2011]

Even though C. elegans is central to research in molecular, cell, and developmental biology, nearly all of the research on this organism has been conducted on a single strain - N2. Comparatively little is known about the genomic and phenotypic variation of the species or its evolutionary history. I will report the characterization of C. elegans genetic variation using high-throughput selective sequencing of eight megabases from a worldwide collection of 200 wild strains representing nearly every known collection location. The identified set of 41,188 single nucleotide polymorphisms (SNPs) was used to define the most genotypically diverse strains for future quantitative genetic studies. We used these SNPs and strains to map loci controlling novel quantitative traits to haplotypes of less than 50 kb by genome-wide association studies. Unexpectedly, we found that the genome is dominated by a set of commonly shared haplotypes that each spans many megabases, suggesting recent shared ancestry. Furthermore, the distribution of polymorphism is strikingly non-uniform and correlates with variation in recombination rate found between the N2 and CB4856 strains. Simulations show that these patterns were generated by selective sweeps that have dramatically reduced variation worldwide and suggest that at least one of these sweeps occurred in the past few hundred years. Likely the result of human activity, these sweeps dramatically reshaped the global C. elegans population.

Andersen, Erik C. et al. (2009) International Worm Meeting "Quantitative genetic analyses of C. elegans intraspecies variation in responses to Pseudomonas aeruginosa and Staphylococcus aureus."

Reddy, Kirthi, Okhah, Zachary, Kruglyak, Leonid, Andersen, Erik C., Kim, Dennis H.

[International Worm Meeting, 2009]

The nematode Caenorhabditis elegans responds to pathogenic bacteria with conserved innate immune responses and pathogen avoidance behaviors. We investigated natural variation in C. elegans responses to different pathogen infections, using a collection of advanced intercross recombinant inbred lines (RILs) between the laboratory wild-type strain N2 and a wild isolate from Hawaii CB4856. Initially, we chose to focus on the opportunistic human pathogen Pseudomonas aeruginosa, a gram-negative, aerobic bacterium. Quantitative trait mapping of the susceptibilities of 126 RILs led to the identification of a polymorphism in the npr-1 gene as causal in the difference in susceptibility to P. aeruginosa between these two strains. We showed that the mechanism of NPR-1-mediated pathogen resistance is through oxygen-dependent behavioral avoidance rather than through direct regulation of innate immunity. Because the npr-1 allele present in the N2 strain is a lab-derived gain-of-function allele (1), we sought natural variants involved in pathogen resistance by scoring susceptibility to P. aeruginosa in only those RILs with the ancestral npr-1 allele (found in CB4856). Through quantitative genetic analysis of 89 additional strains, we identified a second quantitative trait locus (QTL) on LGV. We will discuss our progress in determining the gene underlying this novel QTL. Subsequently, we investigated differences in the susceptibilities of N2 and CB4856 to Bacillus thuringiensis, Candida albicans, Cryptococcus neoformans, Enterococcus faecalis, Serratia marcescens, and Staphylococcus aureus. Assays using the gram-positive bacterium S. aureus had the most robust survival difference between the two parental strains. Quantitative trait mapping of the susceptibilities of 73 RILs led to a causal polymorphism in the npr-1 gene, indicating that behavior determines the susceptibility to this pathogen as well. We will present our progress on the identification of additional QTL controlling susceptibility to S. aureus. (1) McGrath PT et al. Neuron 2009; 61(5):692-699.

Erik C Andersen et al. (2002) East Coast Worm Meeting "IDENTIFICATION AND CHARACTERIZATION OF SYNMUV SUPPRESSORS, INCLUDING A HOMOLOG OF THE CHROMATIN-REMODELING ATPASE ISWI"

Erik C Andersen, Scott Clark, Bob Horvitz, Xiaowei Lu

[East Coast Worm Meeting, 2002]

The receptor tyrosine kinase (RTK)/Ras, Notch, and Wnt signal transduction pathways play important roles in the formation of the C. elegans vulva by influencing developmental fates. Another group of genes that function in the specification of vulval cell fates by antagonizing the RTK/Ras pathway is the synthetic Multivulva (synMuv) genes. The synMuv genes act in two functionally redundant classes, A and B, to negatively regulate vulval development. Animals with a mutation in one or more genes within the same class are non-Muv. By contrast, animals with mutations in both class A and class B genes are Muv. Among some of the identified class B gene products are counterparts of Rb (lin-35), the Rb-associated protein RbAp48 (lin-53), the heterodimeric transcription factors DP (dpl-1) and E2F (efl-1), and the chromatin-associated proteins HDAC (hda-1), Mi-2 (let-418), and HP1 (hpl-2). The three molecularly characterized synMuv A genes encode novel proteins of uncharacterized function. To understand more about the interactions between the synMuv A and B pathways and their link(s) to the RTK/Ras pathway, two screens for synMuv suppressors were performed. In these screens, 80 suppressors of the synMuv phenotype of lin-15AB(n765ts) and 41 suppressors of the lin-53(n833); lin-15A(n767) synMuv phenotype were isolated. We will present the mapping, complementation, and genetic characterization of some of these suppressors. One suppressor of lin-53(n833); lin-15A(n767) was cloned and found to be a homolog of the chromatin-remodeling ATPase ISWI. RNAi of the ISWI homolog suppresses the Muv phenotype of most, if not all, synMuv combinations. Therefore, the ISWI homolog may be required for the ectopic induction of vulval fates observed in synMuv mutants. Genetic characterization is underway to confirm these observations. Additionally, we are seeking a deletion allele and producing an antibody.

Erik C Andersen et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "AN UNBIASED GENOME-WIDE INVESTIGATION OF WORLD-WIDE C. ELEGANS POPULATION STRUCTURE AND GENOMICS"

Leonid Kruglyak, Erik C Andersen, Justin P Gerke, Joshua A Shapiro, Marie-Anne FELIX

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

We leveraged next-generation sequencing technology to obtain a genome-wide and unbiased understanding of C. elegans population structure. Through collaboration and generous donations, we obtained a set of 202 wild isolates from throughout the world. In order to reduce the 100 Mb genome to a manageable size amenable to multiplexing, we employed Restriction-Assisted DNA marker sequencing (1) where the genome of each strain was cut using EcoRI and sequenced in both directions from each restriction site. This method allowed us to sequence the same eight megabases from each strain in two runs of an Illumina Genome Analyzer. We sequenced to an average of 12.6X coverage of each region, and SNPs were identified using SAMtools after mapping to the C. elegans genome using bwa. We will present the results of our analysis of population structure, linkage disequilibrium, and indications of genome-wide selection using the roughly 20,000 identified SNPs with minor allele frequencies greater than 5%. So far, we found the average pair-wise differences between strains is roughly 1/900 base pairs, as compared to the reference N2 genome. However, there is a wide range in the pair-wise differences with some strains being much more divergent from the reference N2 than the Hawaiian strain CB4856. These data will allow us to pursue genome-wide association studies and new recombinant inbred line crosses with maximally diverse wild isolates. (1) Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, et al. 2008 Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers. PLoS ONE 3(10): e3376.

Erik C Andersen et al. (2003) International Worm Meeting "Characterization of the Synthetic Multivulva Suppressor isw-1, the C. elegans Homolog of the Chromatin-Remodeling ATPase ISWI"

Bob Horvitz, Scott Clark, Xiaowei Lu, Erik C Andersen

[International Worm Meeting, 2003]

The synthetic Multivulva (synMuv) genes are grouped into two functionally redundant classes, A and B, that negatively regulate the induction of vulval cell fates. Animals with a mutation in one or more genes within the same class are non-Muv. By contrast, animals with mutations in both a class A and a class B gene are Muv. Among some of the identified class B gene products are counterparts of a transcriptional repression complex: Rb (LIN-35), the Rb-associated protein RbAp48 (LIN-53), the heterodimeric transcription factor DP/E2F (DPL-1/EFL-1), and the chromatin-regulating proteins HDAC (HDA-1), Mi-2 (LET-418), and HP1 (HPL-2).To understand better the synMuv A and B pathways, two screens for synMuv suppressors were performed. In these screens, 166 suppressors of the synMuv phenotype of lin-15AB(n765ts) and 43 suppressors of the lin-53(n833); lin-15A(n767) synMuv phenotype were isolated. One lin-53(n833); lin-15A(n767) synMuv suppressor was cloned and found to encode a homolog of the chromatin-remodeling ATPase ISWI. RNAi or a presumptive null allele of this gene, which we call isw-1, suppresses the Muv phenotype of most, if not all, synMuv mutant combinations. The loss-of-function Vul phenotype of mutants in the RTK/Ras pathway (except lin-3 EGF) is epistatic to the synMuv phenotype. Loss of isw-1 function cannot suppress the Muv phenotype of lin-1 mutants or a gain-of-function mutation in let-60. We conclude that isw-1 is acting downstream of or in parallel to one or both synMuv classes and upstream of or in parallel to the RTK/Ras pathway.ISWI has been biochemically purified from Drosophila embryo extracts as a member of multiple complexes, including the Nucleosome Remodeling Factor (NURF), the Chromatin Accessibility Complex (CHRAC), and the ATP-utilizing Chromatin assembly and remodeling Factor (ACF). RNAi of C. elegans homologs of NURF, CHRAC, and ACF complex members led us to identify a NURF301 homolog, F26H11.2, as a gene that likely acts with isw-1 to regulate vulval development. NURF301 is the largest subunit of the NURF complex and is implicated in complex formation, stability and interactions with transcription factors. We are seeking a deletion allele of F26H11.2 to further characterize the activity of the putative NURF complex in antagonism of the synMuv phenotype. Furthermore, we are mapping and cloning additional mutationally-defined suppressors of the synMuv phenotype in an effort to identify other factors that may act with ISW-1 or a putative C. elegans NURF complex.

Ding, Siyu Serena et al. (2021) International Worm Meeting "Using high-throughput behavioural assays to identify heritable natural genetic variants in three Caenorhabiditis species"

Andersen, Erik C, Ding, Siyu Serena, Brown, Andre EX, Shrestha, Priyanka, Stinson, Loraina

[International Worm Meeting, 2021]

The most standard behavioural assay involves imaging worms crawling on NGM agar. Recent developments in hardware and software increase the throughput of these experiments by running multiple experimental replicates in parallel and by automating post-acquisition worm tracking and feature extraction. We imaged and analysed the crawling behaviour of 197 C. elegans wild isolates in the presence of the OP50 food source, and used genome-wide association (GWA) analyses to identify several quantitative trait loci that are linked to specific behavioural features. Additionally, we explored multiple behavioural assays involving more "challenging" environments, and found that these assays reveal more pronounced behavioural differences at the species level (between C. elegans, C. briggsae, and C. tropicalis) compared to the standard crawling assay. We plan to apply these behavioural assays to hundreds of wild isolates from the three species, in order to identify natural genetic variants that underlie heritable behavioural traits across the Caenorhabiditis genus.

Lee, Daehan et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "TO RIDE OR NOT TO RIDE: GENETIC BASIS FOR NATURAL VARIATION OF C. elegans HITCHHIKING BEHAVIOR"

Kruglyak, Leonid, Andersen, Erik C., Lee, Daehan, Lee, Junho, Paik, Young-Ki, Kim, Heekyeong

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

In parasitic nematodes, cruising (widely foraging) and ambushing (sit-and-wait) are two main dispersal strategies for survival and host finding. Free living nematode use similar strategies. Given harsh conditions, Caenorhabditis elegans develops to a dauer larva, which can either cruise or display an ambushing behavior called nictation. This behavior may facilitate dispersal of dauers to new habitats in the wild environment through phoresy - using other animals as a transportation vehicle. Therefore, nictation may greatly affect the survivorship and reproduction of C. elegans. However, this ambushing strategy decreases the probability of finding new habitats by the cruising strategy. Thus, the decision making between cruising and nictating might be selected in nature. In this work, we characterized natural variation in nictation behavior among C. elegans wild isolates. By linkage mapping, we identified a major-effect quantitative trait locus (QTL) and a candidate gene for nictation behavior. This QTL might underlie natural variation of the C. elegans hitchhiking behavior in the wild. Our work clearly defines a specific QTL that may have an important evolutionary role by modulating decision making probability for a specific behavior.

Baer, Charles F. et al. (2013) International Worm Meeting "Revisiting the effects of spontaneous mutations on the (micro)environmental variance in Caenorhabditis."

Farhadifar, Reza, Baer, Charles F., Andersen, Erik C., Needleman, Daniel

[International Worm Meeting, 2013]

An obvious feature of living organisms is that their development is robust to variation in the environmental circumstances in which the organism finds itself - within limits. That is, the phenotype is more or less "canalized" (canalized "robust"). The "more or less" is of considerable interest to evolutionary biologists, for a variety of reasons, i.e., under what environmental or genetic circumstances does development become more or less canalized? Environmental canalization can be quantified as the phenotypic variation among genetically identical individuals raised in a uniform environment - the "(micro)environmental variance" in the lingo of quantitative genetics, V(E). Here we provide quantitative estimates of the effects of spontaneous mutations on V(E) for a variety of phenotypic traits subject to different selective regimes. Three general quantitative trends emerge. First, in almost all cases, mutation accumulation tends to de-canalize the phenotype (i.e., V(E) increases), typically at a rate similar to the rate of change of the trait itself. Second, there is a strong positive association between the rate of increase of V(E) for a trait and the mutational variance, V(M) for the trait itself. Third, and most intriguingly, mutations affecting V(E) for a trait appear to be usually under stronger selection than mutations affecting the trait itself.