Natural variation in a single amino acid substitution underlies physiological responses to topoisomerase II poisons.

Many chemotherapeutic drugs are differentially effective from one patient to the next. Understanding the causes of this variability is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate sensitivity to a group of anti-neoplastic drugs that target topoisomerase II using the model organism Caenorhabditis elegans. We show that wild strains of C. elegans vary in their sensitivity to these drugs, and we use an unbiased genetic approach to demonstrate that this natural variation is explained by a methionine-to-glutamine substitution in topoisomerase II (TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and its poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms (methionine in hTOPIIα and glutamine in hTOPIIß). We go on to show that this amino acid difference between the two human topoisomerase isoforms influences cytotoxicity of topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIß are differentially affected by various poisons and demonstrate the utility of C. elegans in understanding the genetics of drug responses.

C. elegans germ cells divide and differentiate in a folded tissue.

Knowing how stem cells and their progeny are positioned within their tissues is essential for understanding their regulation. One paradigm for stem cell regulation is the C. elegans germline, which is maintained by a pool of germline stem cells in the distal gonad, in a region known as the 'progenitor zone'. The C. elegans germline is widely used as a stem cell model, but the cellular architecture of the progenitor zone has been unclear. Here we characterize this architecture by creating virtual 3D models of the progenitor zone in both sexes. We show that the progenitor zone in adult hermaphrodites is organized like a folded epithelium. The progenitor zone in males is not folded. Analysis of germ cell division shows that daughter cells are born side-by-side along the epithelial-like surface of the germline tissue. Analysis of a key regulator driving differentiation, GLD-1, shows that germ cells in hermaphrodites differentiate along a folded path, with previously described "steps" in GLD-1 expression corresponding to germline folds. Our study provides a three-dimensional view of how C. elegans germ cells progress from stem cell to overt differentiation, with critical implications for regulators driving this transition.

Population genomic analysis of outcrossing and recombination in yeast.

The budding yeast Saccharomyces cerevisiae has been used by humans for millennia to make wine, beer and bread. More recently, it became a key model organism for studies of eukaryotic biology and for genomic analysis. However, relatively little is known about the natural lifestyle and population genetics of yeast. One major question is whether genetically diverse yeast strains mate and recombine in the wild. We developed a method to infer the evolutionary history of a species from genome sequences of multiple individuals and applied it to whole-genome sequence data from three strains of Saccharomyces cerevisiae and the sister species Saccharomyces paradoxus. We observed a pattern of sequence variation among yeast strains in which ancestral recombination events lead to a mosaic of segments with shared genealogy. Based on sequence divergence and the inferred median size of shared segments (approximately 2,000 bp), we estimated that although any two strains have undergone approximately 16 million cell divisions since their last common ancestor, only 314 outcrossing events have occurred during this time (roughly one every 50,000 divisions). Local correlations in polymorphism rates indicate that linkage disequilibrium in yeast should extend over kilobases. Our results provide the initial foundation for population studies of association between genotype and phenotype in S. cerevisiae.

A novel sperm-delivered toxin causes late-stage embryo lethality and transmission ratio distortion in C. elegans.

The evolutionary fate of an allele ordinarily depends on its contribution to host fitness. Occasionally, however, genetic elements arise that are able to gain a transmission advantage while simultaneously imposing a fitness cost on their hosts. We previously discovered one such element in C. elegans that gains a transmission advantage through a combination of paternal-effect killing and zygotic self-rescue. Here we demonstrate that this element is composed of a sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-1 and zeel-1 are located adjacent to one another in the genome and co-occur in an insertion/deletion polymorphism. peel-1 encodes a novel four-pass transmembrane protein that is expressed in sperm and delivered to the embryo via specialized, sperm-specific vesicles. In the absence of zeel-1, sperm-delivered PEEL-1 causes lethal defects in muscle and epidermal tissue at the 2-fold stage of embryogenesis. zeel-1 is expressed transiently in the embryo and encodes a novel six-pass transmembrane domain fused to a domain with sequence similarity to zyg-11, a substrate-recognition subunit of an E3 ubiquitin ligase. zeel-1 appears to have arisen recently, during an expansion of the zyg-11 family, and the transmembrane domain of zeel-1 is required and partially sufficient for antidote activity. Although PEEL-1 and ZEEL-1 normally function in embryos, these proteins can act at other stages as well. When expressed ectopically in adults, PEEL-1 kills a variety of cell types, and ectopic expression of ZEEL-1 rescues these effects. Our results demonstrate that the tight physical linkage between two novel transmembrane proteins has facilitated their co-evolution into an element capable of promoting its own transmission to the detriment of organisms carrying it.

Analysis of the C. elegans Germline Stem Cell Pool.

The Caenorhabditis elegans germline is an excellent model for studying the genetic and molecular regulation of stem cell self-renewal and progression of cells from a stem cell state to a differentiated state. The germline tissue is organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated gametes at the other. A simple mesenchymal niche caps the GSC pool and maintains GSCs in an undifferentiated state by signaling through the conserved Notch pathway. Notch signaling activates transcription of the key GSC regulators lst-1 and sygl-1 proteins in a gradient through the GSC pool. LST-1 and SYGL-1 proteins work with PUF RNA regulators in a self-renewal hub to maintain the GSC pool. In this chapter, we present methods for characterizing the C. elegans GSC pool and early stages of germ cell differentiation. The methods include examination of germlines in living and fixed worms, cell cycle analysis, and analysis of markers. We also discuss assays to separate mutant phenotypes that affect the stem cell vs. differentiation decision from those that affect germ cell processes more generally.

Stripes and loss of color in ball pythons (Python regius) are associated with variants affecting endothelin signaling.

Color patterns in nonavian reptiles are beautifully diverse, but little is known about the genetics and development of these patterns. Here, we investigated color patterning in pet ball pythons (Python regius), which have been bred to show color phenotypes that differ dramatically from the wildtype form. We report that several color phenotypes in pet animals are associated with putative loss-of-function variants in the gene encoding endothelin receptor EDNRB1: (1) frameshift variants in EDNRB1 are associated with conversion of the normal mottled color pattern to skin that is almost fully white, (2) missense variants affecting conserved sites of the EDNRB1 protein are associated with dorsal, longitudinal stripes, and (3) substitutions at EDNRB1 splice donors are associated with subtle changes in patterning compared to wildtype. We propose that these phenotypes are caused by loss of specialized color cells (chromatophores), with loss ranging from severe (fully white) to moderate (dorsal striping) to mild (subtle changes in patterning). Our study is the first to describe variants affecting endothelin signaling in a nonavian reptile and suggests that reductions in endothelin signaling in ball pythons can produce a variety of color phenotypes, depending on the degree of color cell loss.

A community-science approach identifies genetic variants associated with three color morphs in ball pythons (Python regius).

Color morphs in ball pythons (Python regius) provide a unique and largely untapped resource for understanding the genetics of coloration in reptiles. Here we use a community-science approach to investigate the genetics of three color morphs affecting production of the pigment melanin. These morphs-Albino, Lavender Albino, and Ultramel-show a loss of melanin in the skin and eyes, ranging from severe (Albino) to moderate (Lavender Albino) to mild (Ultramel). To identify genetic variants causing each morph, we recruited shed skins of pet ball pythons via social media, extracted DNA from the skins, and searched for putative loss-of-function variants in homologs of genes controlling melanin production in other vertebrates. We report that the Albino morph is associated with missense and non-coding variants in the gene TYR. The Lavender Albino morph is associated with a deletion in the gene OCA2. The Ultramel morph is associated with a missense variant and a putative deletion in the gene TYRP1. Our study is one of the first to identify genetic variants associated with color morphs in ball pythons and shows that pet samples recruited from the community can provide a resource for genetic studies in this species.

Selfing is the safest sex for Caenorhabditis tropicalis.

Mating systems have profound effects on genetic diversity and compatibility. The convergent evolution of self-fertilization in three Caenorhabditis species provides a powerful lens to examine causes and consequences of mating system transitions. Among the selfers, Caenorhabditis tropicalis is the least genetically diverse and most afflicted by outbreeding depression. We generated a chromosomal-scale genome for C. tropicalis and surveyed global diversity. Population structure is very strong, and islands of extreme divergence punctuate a genomic background that is highly homogeneous around the globe. Outbreeding depression in the laboratory is caused largely by multiple Medea-like elements, genetically consistent with maternal toxin/zygotic antidote systems. Loci with Medea activity harbor novel and duplicated genes, and their activity is modified by mito-nuclear background. Segregating Medea elements dramatically reduce fitness, and simulations show that selfing limits their spread. Frequent selfing in C. tropicalis may therefore be a strategy to avoid Medea-mediated outbreeding depression.

Septins have a dual role in controlling mitotic exit in budding yeast.

In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus [1-6]. Mitotic exit is controlled by the small G protein Tem1p. Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body. The GEF Lte1p is located in the bud. This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck. However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck [7-9]. To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck [10]. We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint. When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother. In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother. In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p. These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor.

Analysis of the C. elegans Germline Stem Cell Pool.

The Caenorhabditis elegans germline is an excellent model for studying the regulation of a pool of stem cells and progression of cells from a stem cell state to a differentiated state. At the tissue level, the germline is organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated cells at the other. A simple mesenchymal niche caps the GSC region of the germline and maintains GSCs in an undifferentiated state by signaling through the conserved Notch pathway. Downstream of Notch signaling, key regulators include novel LST-1 and SYGL-1 proteins and a network of RNA regulatory proteins. In this chapter we present methods for characterizing the C. elegans GSC pool and early germ cell differentiation. The methods include examination of the germline in living and fixed worms, cell cycle analysis, and analysis of markers. We also discuss assays to separate mutants that affect the stem cell vs. differentiation decision from those that affect germ cell processes more generally.

Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans.

Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj et al., 2008 ; Lee et al., 2016a ). We adapted this technique to visualize RNAs in the C. elegans whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee et al., 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for C. elegans extruded gonads.

Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch.

Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for facultative stem cell quiescence. We find that mitotically dividing germ cells--including germline stem cells--become quiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a block to M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, we demonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance: The signaling pathway required for stem cell maintenance under fed conditions--GLP-1/Notch signaling--becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence can itself maintain stem cells, independent of the signaling pathway otherwise essential for such maintenance.

A Powerful New Quantitative Genetics Platform, Combining Caenorhabditis elegans High-Throughput Fitness Assays with a Large Collection of Recombinant Strains.

The genetic variants underlying complex traits are often elusive even in powerful model organisms such as Caenorhabditis elegans with controlled genetic backgrounds and environmental conditions. Two major contributing factors are: (1) the lack of statistical power from measuring the phenotypes of small numbers of individuals, and (2) the use of phenotyping platforms that do not scale to hundreds of individuals and are prone to noisy measurements. Here, we generated a new resource of 359 recombinant inbred strains that augments the existing C. elegans N2xCB4856 recombinant inbred advanced intercross line population. This new strain collection removes variation in the neuropeptide receptor gene npr-1, known to have large physiological and behavioral effects on C. elegans and mitigates the hybrid strain incompatibility caused by zeel-1 and peel-1, allowing for identification of quantitative trait loci that otherwise would have been masked by those effects. Additionally, we optimized highly scalable and accurate high-throughput assays of fecundity and body size using the COPAS BIOSORT large particle nematode sorter. Using these assays, we identified quantitative trait loci involved in fecundity and growth under normal growth conditions and after exposure to the herbicide paraquat, including independent genetic loci that regulate different stages of larval growth. Our results offer a powerful platform for the discovery of the genetic variants that control differences in responses to drugs, other aqueous compounds, bacterial foods, and pathogenic stresses.

Strategies from UW-Madison for rescuing biomedical research in the US.

A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US.

The oogenic germline starvation response in C. elegans.

Many animals alter their reproductive strategies in response to environmental stress. Here we have investigated how L4 hermaphrodites of Caenorhabditis elegans respond to starvation. To induce starvation, we removed food at 2 h intervals from very early- to very late-stage L4 animals. The starved L4s molted into adulthood, initiated oogenesis, and began producing embryos; however, all three processes were severely delayed, and embryo viability was reduced. Most animals died via 'bagging,' because egg-laying was inhibited, and embryos hatched in utero, consuming their parent hermaphrodites from within. Some animals, however, avoided bagging and survived long term. Long-term survival did not rely on embryonic arrest but instead upon the failure of some animals to produce viable progeny during starvation. Regardless of the bagging fate, starved animals showed two major changes in germline morphology: All oogenic germlines were dramatically reduced in size, and these germlines formed only a single oocyte at a time, separated from the remainder of the germline by a tight constriction. Both changes in germline morphology were reversible: Upon re-feeding, the shrunken germlines regenerated, and multiple oocytes formed concurrently. The capacity for germline regeneration upon re-feeding was not limited to the small subset of animals that normally survive starvation: When bagging was prevented ectopically by par-2 RNAi, virtually all germlines still regenerated. In addition, germline shrinkage strongly correlated with oogenesis, suggesting that during starvation, germline shrinkage may provide material for oocyte production. Finally, germline shrinkage and regeneration did not depend upon crowding. Our study confirms previous findings that starvation uncouples germ cell proliferation from germline stem cell maintenance. Our study also suggests that when nutrients are limited, hermaphrodites scavenge material from their germlines to reproduce. We discuss our findings in light of the recently proposed state of dormancy, termed Adult Reproductive Diapause.

Widespread genetic incompatibility in C. elegans maintained by balancing selection.

Natural selection is expected to eliminate genetic incompatibilities from interbreeding populations. We have discovered a globally distributed incompatibility in the primarily selfing species Caenorhabditis elegans that has been maintained despite its negative consequences for fitness. Embryos homozygous for a naturally occurring deletion of the zygotically acting gene zeel-1 arrest if their sperm parent carries an incompatible allele of a second, paternal-effect locus, peel-1. The two interacting loci are tightly linked, with incompatible alleles occurring in linkage disequilibrium in two common haplotypes. These haplotypes exhibit elevated sequence divergence, and population genetic analyses of this region indicate that natural selection is preserving both haplotypes in the population. Our data suggest that long-term maintenance of a balanced polymorphism has permitted the incompatibility to persist despite gene flow across the rest of the genome.